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Retrospective Theses and Dissertations 1995 Sequence variation characteristics of D-loop, rRNA, tRNA, and polypeptide coding region of bovine mitochondrial DNA Jianming Wu Iowa State University Follow this and additional works at: http://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, Animal Sciences Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Wu, Jianming, "Sequence variation characteristics of D-loop, rRNA, tRNA, and polypeptide coding region of bovine mitochondrial DNA " (1995). Retrospective Theses and Dissertations. Paper 10994. This Dissertation is brought to you for free and open access by Digital Repository @ Iowa State University. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Digital Repository @ Iowa State University. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI the text directly from the original or copy submitted- Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and in::^roper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be remove4 a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overl^s. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for aity photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. A Bell & Howell tnformaiion Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1345 USA 313.'761-4700 800/521-0600 Sequence variation characteristics of D-loop, rRNA, tRNA, and polypeptide coding region of bovine mitochondrial DNA by Jianming Wu A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Interdepartmental Major: Animal Science Molecular, Cellular, and Developmental Biology Approved: Signature was redacted for privacy. Signature was redacted for privacy. In Chargb-bfjVIajpr Work Signature was redacted for privacy. For the Interdepartmental Major Signature was redacted for privacy. F Major Departnient Signature was redacted for privacy. For the Gra^ate^ollege Iowa State University Ames, Iowa 1995 UMI Number: 9540955 OMI Microform 9540955 Copyright 1995, by OMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ii TABLE OF CONTENTS ABBREVIATIONS iv GENERAL INTRODUCTION 1 Dissertation Organization 1 General Bactcground 1 LITERATURE REVIEW 4 General Information about Mitochondria 4 Mammalian mtDNA 7 Replication of mtDNA 11 Transcription and RNA Processing in Mitochondria 16 Protein Translation in Mitochondria 21 Import of Mitochondrial Proteins 23 Genetics of Mammalian mtDNA 25 Mutation and Sequence Variations of mtDNA 28 Mitochondrial DNA Heteropiasmy in Animals 31 Phenotypic Effects of mtDNA Variations 35 Mitochodrial DNA Genetics in Cattle 37 References 40 iii SEQUENCE HETEROPLASMY OF D-LOOP AND rRNA CODING REGIONS IN MITOCHONDRIAL DNA FROM HOLSTEIN COWS OF INDEPENDENT MATERNAL LINEAGES 56 Abstract 56 Introduction 57 Materials and Methods 60 Results 62 Discussion 72 References 77 SEQUENCE VARIATIONS IN tRNA AND PROTEIN CODING GENES OF BOVINE mtDNA 80 Abstract 80 Introduction 81 Materials and Methods 84 Results 88 Discussion 93 References 100 GENERAL SUMMARY 104 ACKNOWLEDGMENT 107 iv ABBREVIATIONS USED A Adenine Amino Acid Abbreviations: A Ala Alanine R Arg Arginine N Asn Asparagine D Asp Aspartate C Cys Cysteine Q Gin Glutamine E Glu Glutamate G Gly Glycine H His Histidine I He Isoleucine L Leu Leucine K Lys Lysine M iVIet Methionine F Phe Phenylalanine P Pro Proline S Ser Serine T Thr Threonine W Trp Tryptophan Y Tyr Tyrosine V Val Valine ADP Adenosine diphosphate ATP Adenosine triphosphate ATPase Adenosine triphosphatase ATPase 6 Adenosine triphosphatase subunit 6 ATPase 8 Adenosine triphosphatase subunit 8 bp Base pair C Cytosine COI Cytochrome c oxidase subunit I con com Cytochrome c oxidase subunit II CSB Conserved sequence block Cyt b Cytochrome b molecule in the electron transport chain D-ioop Displacement loop DNA Deoxyribonucleic acid G Guanine H-strand Heavy strand Kb Kilobase pairs L-strand Light strand MOM Mitochondrial outer membrane mRNA Messenger RNA mtDNA Mitochondrial DNA mtRNA Mitochondrial RNA mtTFl Mitochondrial transcription factor 1 N Any nucleotide (A, C, G, T, or U) NADH Nicotinamide adenine dinucleotide ND NADH dehydrogenase subunit nt Nucleotide PCR Polymerase chain reaction RFLP Restriction fragment length polymorphism RNA Ribonucleic acid RNase Ribonuclease RNase MPR. Ribonuclease involved in mitochondrial RNA processing rRNA Ribosomal RNA Cytochrome c oxidase subunit III SSB Single strand DNA binding protein ssDNA Single strand DNA tRNA Transfer RNA U Uracil 1 GENERAL INTRODUCTION Dissertation Organization The dissertation starts with the Literature Review, which provides the readers with general background about mitochondria and recent developments in mitochondrial genetics and mitochondrial DNA (mtDNA). Following the Literature Review, two separate manuscripts suitable for publication in scientific journals are presented, A general summary follows which is intended to conclude the major findings of the study. The data were gathered between 1991 and 1995 in partial fijlfillment of the requirements for the Doctor of Philosophy degree. A portion of data related to the sequence variations of D-loop region of bovine mtDNA, which were obtained collectively by the author and Renotta K. Smith, has been reported in Renotta K. Smith's thesis "Nucleotide sequence variations in the displacement-loop regions of bovine mitochondrial DNA". General Background Mitochondria are the major sites of cellular respiration in eucaryotic organisms. Mitochondria possess their own DNA (mtDNA) that replicates independently from the nuclear DNA and is inherited maternally. Mutations caused by changes in the nucleotide sequence of mtDNA can cause differences in productivity of dairy cattle and other domestic animals. Several human genetic diseases reportedly are caused by mutations of mtDNA. 2 The genes of mtDNA are essential for organelle function. It has been documented that the maternal lineage effects account significantly for differences in milk yield, fat yield , fat percentage, fat corrected milk volume, milk net economic returns, days open, and days to first breeding in dairy cattle (Schutz el al., 1992; 1993; 1994). Maternal lineages are defined by tracing an animal's geneology backward through the maternal pedigrees as many generations as possible, often to the beginning of a herd book registry. Because mtDNA in mammals is the only known genetic element in the cytoplasm, cytoplasmic inheritance of different mtDNA genotypes is thought to be the origin of the cytoplasmic effect. Maternal lineages frequently are considered to be indicative of cytoplasmic inheritance. Mitochondrial DNA is transmitted along with maternal cytosol and, being the only known extra-nuclear genetic component in mammals, is a logical explanation for cytoplasmic inheritance. Therefore, the relationship between mtDNA and the cytoplasmic effect is established. The sequence variability in the displacement loop (D-loop) region of bovine mtDNA affects specific traits including milk yield, fat yield, and total energy present in milk (Schutz et ai, 1992; 1993; 1994). However, the mechanism of transmission and segregation of different mtDNA genomes within an individual animal is not fully understood. It is not a consensus concept that mtDNA of mammals is exclusively maternally inherited (Gyllensten et al., 1991). There is ample evidence that human genetic diseases can be caused by mutations of mtDNA. Nucleotide sequence variations leading to the functional differences of protein products of the mtDNA coding genes are expected to explain the different rates of oxidative phosphorylation, and thus the differences on production, reproduction, and health traits of animals. Although 3 the complete nucleotide sequence of bovine mtDNA sequence has been determined (Anderson et al, 19S2), the extent and type of sequence variations in the protein coding region of mtDNA are not documented in dairy cattle. Our hypothesis is that the inheritance of mtDNA is the result of random passing from ancestor to progeny instead of the selection effect and that any nucleotide changes that atlect the function of gene products of mtDNA would have effects on the production traits of animals. Therefore, the first study was designed to determine the nucleotide sequence variation sites in the D-loop and rRNA coding regions of mtDNA from 18 maternal lineages of dairy cattle on which extensive records of relationship of the cows within the lineages are available and to analyze the phenomena of heteroplasmy (different types of mtDNA exist within an individual animal) and try to investigate the characteristics of bovine mtDNA inheritance. The DNA sequence variations that change amino acid sequence within protein coding regions of mtDNA would have direct impact on the enzyme activities. The second study was carried out to obtain the information about the type and extent of sequence variance within tRNA and protein coding regions of mtDNA and to predict the effect of the nucleotide sequence variations on the function of the mtDNA gene products. 4 LITERATURE REVIEW General Inrorniation about Mitochondria The average mitochondrion, whether in a mammalian or in a lower eucaryotic cell (e.g., yeast), has approximately the same dimensions as the bacterium Escherichia coli. It is most commonly observed as an oval particle, 1-2 um long and 0.5-1 um wide. Mitochondria, therefore, are sufficiently large to be seen in the light microscope, and the earliest descriptions of this organelle were based on observations of fixed and stained tissues. Mitochondria are the powerhouses of eucaryotic cells, producing most of the energy of the cell in the form of adenosine triphosphate (ATP). Mitochondria occupy a substantial portion of the cytoplasmic volume of eucaryotic cells, and they have been essential for the evolution of complex animals. Without mitochondria, present-day animal cells would depend on anaerobic glycolysis for all of their ATP. However, when glucose is converted to pyruvate by glycolysis, only a very small fraction of the total free energy potentially available from the glucose is released. In mitochondria, the catabolism of glucose and other fuel molecules is completed; the pyruvate is imported into the mitochondrion and is oxidized by molecular oxygen to CO2 and HjO. The energy released is harnessed so etTiciently that about 36 molecules of ATP are produced for each molecule of glucose oxidized. By contrast, only two molecules of ATP are produced by anaerobic glycolysis alone (Stryer, 1995). Mitochondria are usually depicted as elongated cylinders with a diameter of 0.5 to I .O |.im. Time-lapse microcinematography of living cells, however, shows that mitochondria are remarkably mobile and plastic organelles, constantly changing their shape and even fijsing with one another and then separating again. As they move about in the cytoplasm, they often appear to be associated with microtubules, which may determine the unique orientation and distribution of mitochondria in different types of cells. Thus, the mitochondria in some cells form long moving filaments or chains, while in others they remain fixed in one position where they provide ATP directly to a site of unusually high ATP consumption, such as being packed between adjacent myofibrils in a cardiac muscle cell or wrapped tightly around the fiagellum of a sperm. Although mitochondria are large enough to be seen in the light microscope and were first identified in the nineteenth century, real progress in the understanding of their flinction depended on procedures developed in 1948 for isolating intact mitochondria. For technical reasons, many biochemical studies have been carried out with mitochondria purified from liver; each liver cell contains 1000 to 2000 mitochondria, which in total may occupy a fifth of the cell volume. Each mitochondrion is bounded by two highly specialized membranes that play a crucial part in its activities. Together, they create two separate mitochondrial compartments: the internal matrix space and a narrow intermembrane space. The outer membrane contains many copies of a transport protein called porin, which forms large aqueous channels through the lipid bilayer. This membrane thus resembles a sieve that is permeable to all molecules of 5000 daltons or less, including small proteins. Such molecules 6 can enter the intermembrane space, but most of them cannot pass the relatively impermeable inner membrane. Other proteins in this membrane include enzymes involved in mitochondrial lipid synthesis and enzymes that convert lipid substrates into forms that are subsequently metabolized in the matrix. The intermembrane space contains several enzymes that use the ATP passing out of the matrix to phosphorylate other nucleotides. The major working part of the mitochondrion is the matrix space and the inner membrane that surrounds it. The inner membrane is highly specialized. It contains a high proportion of the "double" phospholipid cardiolipin, which contains four fatty acids and may help make the membrane especially impermeable to ions. It also contains a variety of transport proteins that make it selectively permeable to those small molecules that are metabolized or required by the many mitochondrial enzymes concentrated in the matrix space. The matrix enzymes include those that metabolize pyruvate and fatty acids to produce acetyl CoA and those that oxidize acetyl CoA in the citric acid cycle. The principal end products of this oxidation are CO2, which is released from the cell as waste, and NADH, which is the main source of electrons for transfer among the respiratory chain components. The enzymes of the respiratory chain are embedded in the inner mitochondrial membrane, and they are essential to the process of oxidative phosphorylation, which generates most ATP in animal cells. The matrix contains a highly concentrated mixture of hundreds of enzymes, including those required for the oxidation of pyruvate and fatty acids and for the citric acid cycle. The matrix also contains several identical copies of the mtDNA genome, special mitochondrial ribosomes, tRNAs and various enzymes required for expression of the mitochondrial genes. 7 The inner membrane is folded into numerous cristae, which greatly increases its total surface area. It contains proteins with three types of functions: (1) those that carry out the oxidation reactions of respiratory chain, (2) an enzyme complex called ATP synthase that makes ATP in the matrix, and (3) specific transport proteins that regulate the passage of metabolites into and out of the matrix. Because an electrochemical gradient that drives the ATP synthase is established across this membrane by the respiratory chain, it is important that the membrane be impermeable to most small ions (Alberts et al., 1994). Mammalian mtDNA Eucaryotic organisms possess several identical copies of mtDNA in a single mitochondrion (Robin and Wong, 1988). The genes that mtDNA encodes have been highly conserved, but the structure and size vary dramatically (Morin and Cech, 1988). Most eucaryotes contain a circular double-stranded mtDNA genome (Gray, 1982), but ciliated protozoa Tetrahymena and Paramecium have linear mtDNA of about 50 kb (Suyama and Miura, 1968; Goddard and Cummings, 1975), and hydra mtDNA consists of two linear plastids of 7.5 kb each (Warrior and Gall, 1986). Complete mitochondrial nucleotide sequences have been determined for human (Anderson a/., 1981), mouse (Bibb e/fl/., 1981), bovine (Anderson e/a/., \9Z2), Xenopus laevis (Roe et ai, 1985), Drosophilayakuha (Clary and Wolstenholme, 1985), Ascaris suiim (Wolstenholme et al., 1987), sea urchin (Jacobs et al., 1988; Cantatore et al., 1989), rat s (Gadaleta et al., 1989), chicken {Desjiardins and Morals, 1990), fin whale (Arnason et al., 1991), and the blue moilusk My/ilii.s eJiili.s (Hoffman el ai, 1992). The mitochondrial genome is much larger in yeast. The exact size varies quite widely among different strains of Sacchromyces cerevisiae, it is roughly about 84 kb long. There are about 22 mitochondria per yeast cell, which corresponds to 4 genomes per organelle. In growing cells, the proportion of mtDNA can be as high as 18% of total cellular DNA. Plants show an extremely wide range of variation in mtDNA size, with a minimum size of 100 kb. The function of the additional DNA, in comparison with mammalian mtDNA, has not been defined decisively, but it seems likely that much of it is noncoding. The size of the genome makes it difficult to isolate intact mtDNA, but restriction mapping in several plants suggests that each plant mtDNA has a single sequence, and it is organized as a circle. Within this circle, there are short homologous sequences. Recombination between these elements generates smaller, subgenomic circular molecules that coexist with the complete 'master' genome, explaining the apparent complexity of plant mtDNAs (Lewin, 1994). Mammalian mtDNA is approximately 16.5 kb in duplex circles. The mammalian mtDNA genome is very compact in contrast to that of fungi and plants. The organization and expression of the human mitochondrial genome reflects a strictly economical use of genetic material (Barrell et al., 1980a; Anderson et a/., 1981). The genome has tRNA genes interspersed amongst the rRNA and protein coding sequences and is very compact with relatively few intergenic nucleotides and no detectable introns (Ojala et al., 1980; Battey and Clayton, 1980; Anderson t?/a/., 1981). Because the two strands ofnitDNA have different base compositions and can be separated by their buoyant density in CsCI gradients on the basis of their density, one strand of mtDNA is named as the heavy strand (H-strand) and the other complementary strand is termed as the hght strand (L-strand),. The ratios of guanine (G) and thymine (T) in H-strand to those of L-strand, are 2.27 for G and 1.25 for T in human mtDNA, whereas the corresponding ratios are 1.92 and 1.20 in mouse mtDNA, respectively (Anderson et al, 1981; Bibb et al., 1981). A distinguishing feature of mtDNA is the formation of a transient triplex helical structure in the region of the replication origin where a short nascent strand (H-strand) of DNA is maintained at the origin of replication (Clayton, 1984). This region is located between the proline transfer RNA (tRNA) and phenylalanine tRNA and is about 900 nucleotides in length. Because the nascent strand DNA separates and displaces the parental double strands of DNA to form a loop within this region, the region is commonly called the D-loop (displacement loop) region. The D-loop region contains the origin of replication for H-strand mtDNA and the promoters for both L-strand and H-strand transcription, which are located at the 5' end of the D-loop upstream from the origin of H-strand replication (Clayton, 1982; 1984; Channel a/., 1987; Nelson, 1987). Over 90% of the mtDNA genome is the tRNA, rRNA, and protein coding region. Mammalian mtDNA codes for a small (12S) and a large (16S) ribosomal RNA (rRNA) subunit, 22 tRNAs and 13 polypeptides involved in oxidative phosphorylation (Anderson et ai, 1982). These polypeptides are cytochrome c oxidase subunits L and III; NADH 10 dehydrongenase subunits 1, 2, 3, 4, 5, 6, and 4L; ATPase subunits 6 and 8; and cytochrome b. The tRNAs are spaced throughout the genome and are located between protein coding regions (Barrell cV al., 1980; Anderson d a/., 1981). The significance of the alternation of tRNA genes with rRNA and protein coding genes is that the tRNA indicate sites of RNA cleavage. By cleaving the primary transcript on either side of each tRNA gene, all of the genes except ATPase 6 and cytochrome c oxidase subunit III give rise to monocistronic products. The rRNA molecules seem to be synthesized in greater amounts than are the mRNAs. This difference could be caused by premature termination of some proportion of the transcripts at some point after the two rRNA genes have been transcribed. Another character that distinguishes mtDNA from chromosomal DNA is that the mitochondrial genome reportedly has a high frequency of ribonucleotides incorporated. The ribosubstitution occurs more frequently near the origin of H-strand replication and L-strand replication, in the cytochrome c oxidase III, 12S rRNA and 16S rRNA, the tRNA''*""" and the tRNA^"' genes than in the other regions. Other regions throughout the mtDNA, however, also are ribosubsitituted at a low frequency (Brennicke and Clayton, 1981). Mitochondrial DNAs are closed circular molecules, but a large portion of mammalian mtDNA inside the cells exists as a circular dimer or concatemers, just like links in a chain (Clayton and Vinograd, 1967). Two mtDNA strands usually are twisted around with about 100 negative supercoils (Clayton, 1982). The exposure of mouse cells to nalidixic acid, which selectively inhibits bacterial type II DNA topoisomerases, caused the loss of mtDNA. 11 Therefore, the type II DNA topoisoinerase is essential for the maintenance of functional mtDNA (Lawrence t'/a/., 1990). The copy number of mtDNA per mitochondrion or per cell varies among species and tissues and seems to be regulated by the need of energy within specific tissues. Bogenhagen and Clayton (1974) found that mouse L cells contain about 1100 copies per cell and that Hela cells have approximately four times as many molecules of mtDNA as normal epithelial cells. More recently, Robin and Wong (1988) found that the number of mtDNA molecules per mammalian ceil varies from 220 to 1720 copies/cell in rabbit lung macrophages. They also found, however, that the number of copies of mtDNA molecules per mitochondrion seems to remain constant at around 2.6 copies/mitochondrion within a species (Robin and Wong, 1988), This finding is contradicted by the study of Vetri et al. (1990) who found that the number of copies per mitochondrion varies from 1 in mouse heart to 4.3 in mouse brain. Williams (1986) found that the amount of mtDNA per gram of tissue varies directly with the oxidative capacity of the tissue. Upon repeated contraction of muscle for 21 days, cytochrome b mRNA becomes elevated coordinately v/ith mtDNA copy number (Williams et al., 1986), suggesting that gene dosage may be important in the regulation of mtDNA expression. Replication of mtDNA Mitochondria contain multiple copies of a closed circular DNA genome that is replicated autonomously within the organelle matrix. It has been known for some time that 12 the control of replication for mtDNA is more relaxed than is that for chromosomal DNA. There is an apparent lack of restriction on mtDNA replication with regard to cell cycle phase (Clayton, 1982). The major form of mtDNA observed in vertebrate animals is those with D-loop. It is formed through maintaining a short piece of nascent DNA strand at the leading-strand origin of replication. The D-loop then consists of a three-stranded structure with a short nascent Hstrand whose 5' end is located at the origin of H-strand replication (On). These short D-loop strands seem to be metabolically unstable in that their turnover rate is much higher than that of genomic replication. A replicative intermediate is termed as a molecule in which DNA synthesis has proceeded past the D-Ioop region around the molecule; this process occurs in a unidirectional manner with an ever-increasing displacement of the parental H-strand. When the new Hstrand has elongated to two-thirds of its total length, the origin of L-strand replication ( O l) is exposed on the displaced parental H-strand and then the initiation of daughter L-strand synthesis is started. Thus, mammalian mtDNA have two separate and distinct origins of replication. The asynchrony of mtDNA replication on H-strands and L-strands is caused by the dominant effect of H-strand origin of replication and spatial distance between the two origins of replication. During replication, dissimilar progeny DNA circles are produced and segregated; one progeny DNA strand requires gap filling and subsequent introduction of superhelical turns (Clayton, 1982), 13 A commitment to mtDNA replication begins by initiation of H-strand synthesis that results in strand elongation over the entire length of the genome. Initiation of L-strand synthesis only occurs after Oi, is exposed as a single stranded template, but replication has never been found to begin at this origin. Thus, the H-strand (leading strand) origin is dominant with respect to dictation of a replication event, whereas the L-strand (lagging strand) is in effect, a secondary, albeit required, replication origin element. Interestingly, Oi, seems to have been deleted (or was never present) in the chicken mtDNA (Desjardins and Morais 1990), though the organization of the five tRNA genes has been conserved in this region. It is not clear whether H-strand synthesis is continued by extending the triplex D-loop structure or by proceeding from the origin of H-strand replication as a single event. However, replication priming and transcription begin at exactly the same site. Therefore, the replication priming for O h is an event dependent upon transcription promoter fimction (Clayton, 1991a). Another interesting feature of the origin of H-strand is that the transitions from RNA synthesis to DNA replication occur at the region with three short conserved sequence elements, which are termed conserved sequence blocks (CSBs) I, II, III. These sequences had been identified previously by Walberg and Clayton (1981) based on the analysis of available mtDNA sequence information. These blocks seem to be highly conservative within the region of mtDNA that is otherwise highly divergent. On the basis of this observation, it was speculated that the CBSs would have some regulatory role in mtDNA replication or expression. A search for an enzymatic activity that might have the ability to recognize 14 features of these CSBs in order to process nucleic acid from this origin region proved successtlil The enzyme is a ribonucleoprotein termed RNase MRP (for mitochondrial RNA processing), and it has been characterized from both human and mouse (Clayton 1991). The origin of L-strand replication is very short in size, approximately 30 nucleotides, and it is within a cluster of five tRNA genes (Chang and Clayton, 1985). It is a small noncoding region tlanked by coding sequences, and it has the potential to form a stem-loop structure that is highly conserved among sequenced vertebrate mtDNAs, except that of chicken. The stem-loop structure is the only conserved feature. Therefore, the exact nature of the primary sequence and its relationship to the population of surrounding tRNA genes may be different. Although the identification of O l was unambiguous (Martens and Clayton, 1979), our current understanding of how Oi. functions requires the development of an in vitro replication system capable of accurate initiation of L-strand synthesis. The use of an in vitro run-off replication assay (Wong and Clayton, 1985a,b) demonstrated that human mitochondria contains a mtDNA primase with the apparent ability to recognize Oi. and to initiate RNA priming and DNA synthesis. The essential points are that daughter L-strand synthesis is primed by RNA, which is complementary to a T-rich loop structure in O l and that a transition from RNA synthesis to DNA synthesis occurs near the base of the stem. The site of transition from RNA synthesis to DNA synthesis is actually part of a tRNA gene and is entirely conserved in the human and bovine mtDNA,. Hixson et fl/.(1986b) demonstrated that both the conserved stem-loop and divergent 5'-flanking sequences are necessary and sufficient to 15 support initiation of DNA replication and that the short pentanucieotide sequence at the base of the Oi, stem is required to maintain a functional region. DNA polymerase gamma (mtDNA polymerase) is necessary for initiation and chain elongation of mtDNA. There are several reports of an associated 3' to 5' exonuclease activity being an intrinsic part of the polymerase (Kunkel and Soni 1988; Insdorf and Bogenhagen 1989b; Kaguni and Olson 1989; Kunkel and Mosbangh 1989). The exonuclease activity may confer a proofreading capacity consistent with studies on the error rate of DNA. Polymerase gamma, unlike DNA polymerase alpha that acts on chromosomal DNA, acts more efficiently on single-stranded templates (Kaguni et al., 1988). Polymerase gamma has been found to replicate DNA with a remarkably high degree of fidelity. This high fidelity is a surprising discovery because it had been considered that the high error rate of polymerase gamma was responsible for the high rates of ribosubstitution and rapid evolution of mtDNA. The processivity of polymerase gamma is very low. Polymerase gamma polymerizes approximately 270 nucleotides per minute per strand. Compared with 60,000 nucleotides per minute for DNA polymerase III holoenzyme of E. coli, the processivity of polymerase gamma is about 200 times lower than that of E. coli DNA polymerase III (Clayton, 1982). Polymerase gamma purified from Xenopus laevis (Insdorf and Bogenhagen, 1989a) possesses a 140 kdal subunit. Single-strand DNA binding proteins (SSB) also play an important role in the replication of mtDNA. Two single-stranded DNA binding proteins already have been isolated from Xenopus laevis (Bogenhagen et a/., 1990). Another SSB has been isolated and characterized from rat liver mitochondria (Hoke et al., 1990). 16 A mtDNA primase has been isolated and characterized by Wong and Clayton (1985a). This complex makes an RNA primer of 9 to 25 nucleotides that is attached covalently to the nascent DNA strand. The primase complex has an associated structural RNA, which is encoded by a chromosomal gene that is necessary for primase function (Wong and Clayton, 1985a; 1986). It has been known for some time that mammalian mitochondria do not have efficient system for removal of pyrimidine dimers, nor do they exhibit anything in the way of a normal rate of recombination activity (Clayton, 1982). However, certain types of repair enzymes have been identified in mitochondrial fractions (Tomkinson et ciL, 1990). Transcription and RNA Processing in Mitochondria The D-loop region of vertebrate mtDNA has evolved as the control site for both transcription and replication. Each strand of the circular mtDNA genome is transcribed from a single major promoter in the D-loop region, Approximately 150 DNA base pairs separate the transcription start sites in mammals (Chang and Clayton 1984). The H-strand promoter (HSP) and L-strand promoter (LSP) were determined by mutational analyses. The two promoters on mouse and human mtDNA do not overlap and thus function as independent entities (Chang and Clayton 1984). The processes of transcription for both strands of mtDNA are initiated in the D-loop region. The promoters for L- and H-strand transcripts have been located near the origin of H-strand replication (Clayton, 1984). These promoters contained at least two types of functional domains, a short sequence at the transcription start site that was absolutely necessary for transcription initiation and an upstream region that, when present. 17 resulted in an augmented etficiency of initiation (Hixson and Clayton, 1985; Topper and Clayton, 1989). Linker analysis provided the evidence that the HSP or LSP each is composed of approximately 50 base pair sequences that are separated into two functionally distinct elements (Topper and Clayton, 1989). Mitochondrial RNA (mtRNA) polymerase has been purified successftilly from rat, X. laevis, and yeast. The enzyme from rats showed a strong template preference for the supercoiled recombinant plasmid that consists of pBR322 vector and D-loop region of rat mtDNA carrying the promoter origin of replication for H-strand (Yaginuma ct a/., 1982), Mitochondrial RNA polymerase required a dissociable transcription factor for specific transcription of.V. /acvis and S. ccrevista mtDNA (Bogenhagen and Insdorf, 1988; Ticho and Getz, 1988; Schinlel el al., 1988). The mtRNA polymerase and the transcription factor together are necessary and sufficient for active transcription from promoters in the D-loop region. The first trans-acting transcription factor in a mitochondrial system was isolated from human mitochondria (Fisher and Clayton 1985). This mitochondrial transcription factor 1 (mtTFl) binds specifically to upstream sequences of the transcription start sites (Fisher et a/., 1987). Promoter-specific transcription of human mtDNA required the presence of mtTFl in addition to a mtRNA polymerase fraction. Mitochondrial RNA polymerase alone is not effective for transcription. The mtTFl interacts with the mtDNA template by direct binding mechanism. The mtTFl binding sites that have been assigned are located upstream of both the HSP and LSP from position -12 to -39 of the respective transcription start sites, but the 18 affinity ofmtTFl for each of the promoters is different (Fisher ei al., 1987; Fisher and Clayton, 1988). It is interesting that the relative binding atTinity is greater for the LSP than for HSP. L-strand promoter is a stronger promoter than is HSP both in vivo and in vitro. Mitochondrial TFl has been purified to homogeneity from human cells (Fisher and Clayton, 1988; Fisher ct a/., 1991). By virtue of its targeting to mtDNA template immediately upstream of the transcription start site, it is logical to assume that mtTFl activates transcription as a result of DNA binding. It has been revealed that mtTFl has the capacity to unwind and bind DNA, thereby suggesting the manner in which at least part of the transcription initiation process takes place. During transcription, the L- and H-strands of the mtDNA are transcribed from the separate L- and H-strand promoters in the D-loop region. Two polycistronic RNAs are synthesized from the two respective promoters. Each polycistronic RNA contains all the genetic messages from the H-strand or L-strand of the mitochondrial genome (Chang et ai, 1987). Heavy strand transcription promoter regions of human mtDNA have been defined by Montoya et al. (1982; 1983). The first promoter region is located 19 nucleotide upstream of the 5' end of tRNA"""'. The second promoter region is located close to the 5' end of the 12S rRNA coding region. Transcription from the first promoter region is believed to produce only 12S rRNA, 16S rRNA, tRNA'^", and tRNA^"', The transcript ends at the 3' end of the 16S rRNA. The second promoter region near the 12S rRNA is believed to transcribe past the 3' end of the 16S rRNA (Ojala et al, 1981). Initiation at the first promoter is believed to occur at a higher frequency than at the second promoter. Complete transcripts from the second 19 promoter of H-strand will start at 5' end of 12S rRNA and include all the structural genes and tRNAs downstream of the H-strand origin of transcription (Attardi et a/., 1983; Motoya et al., 1982; Motoya c'/cv/., 1983). The initiation site for L-strand transcription is near nucleotide 407 in human mtDNA (Chang and Clayton, 1985; Hixson and Clayton, 1985). The L-strand initiation site has been determined to be composed of nucleotides ATACCGCCAAAAGAT in human mtDNA. Mutation analysis revealed that a mutation of one of the underlined nucleotides decreased the efficiency of the promoter function (Hi.xson and Clayton, 1985). The replacement of the C with other nucleotides to the right of the G in this sequence destroys the promoter function. The second A after the underlined sequence has been determined as the initiation site of Lstrand synthesis. Light strand transcripts are in low abundance and seem to have a rapid turnover rate (Cantatore and Attardi, 1980). Once initiated, transcription of mtDNA proceeds around the entire genome, yielding a polycistronic RNA molecule from each strand (Clayton, 1984). Then, the primary transcript is processed by a series of endonuclolytic cleavages. An RNase P-type endonuclease that has been purified from human mitochondria may be responsible for endonucleolytic cleavage of the polycistronic RNA transcripts (Doerson el al., 1985). Because tRNAs are located between rRNA genes and most of protein genes, excising the tRNAs from the primary transcript will generate monocistronic RNAs (Battey and Clayton, 1980). This model provides the reasonable explanation for transcript processing in which tRNA excision is the major processing event to free RNA molecules for fijrther modification. However, the activity 20 of RNase P has not been demonstrated to cleave miRNA (Clayton, 1991a). Conversely Karvvan et al (1991) showed that RNase P may be involved in multisile RNA processing. Therefore, the role of the RNase P in mtRNA processing is not clear yet. The 3' ends of mitochondrial mRNA precursors are polyadenylated, but the 5' end of niRNA is not modified (Claylon, 1984). Polyadenylation is an important modification because many of the protem coding genes contain incomplete termination codons and rely on polyadenylation to create a functional stop codon (Anderson ei a/., 1981). When these genes are transcribed as part of a large RNA precursor and adjacent gene transcripts are separated by endonucleolytic processing, polyadenylation of the immature mRNAs creates in-phase U.A.A codons (Barrell et al., 1980a; Anderson tv (//., 1981; Ojala cv «/., 1981). The processing of these mtRNAs must operate with the precision similar to that observed for RNA splicing. Even a small error in the site of polyadenylation would result in translation of a poly(A) tail and thus produce polylysine-terminated proteins. However, the mitochondrial and the nuclear polyadenylation processes are significantly different, because the AAUAAA sequence found upstream of poly(A) addition sites in nuclear RNAs (Proudfoot and Brovvnlee, 1976) is absent from most of the mitochondrial mRNAs (Anderson t'/a/., 1981; Ojala 1981). A polymerase for the polyadenylation of 3' mitochondrial mRNAs in rats has been identified (Jacob, 1974). It has been reported that the nutritional status of the rats could indirectly regulate the activity of the poly(A) polymerase. Mitochondrial poly(A) polymerase activity significantly decreased after rats were starved for 24 to 48 hours. However, the normal activity was restore within 1 to 2 hours by administering an amino acid mixture orally to rats (Jacob et al., 1976). 21 The concentrations of different mitochondrial niRNAs remain quite stable. However, the mature rRNA and mRNA of mitochondria are metabolically unstable and the ratio of two specific types of niRNAs may vary as much as 10 fold (Gelfand and Attardi, 1981; Bhat et a!., 1985). Because the transcription from HSP or LSP is polycistronic, these mRNA population differences must be caused by the stability of different transcripts. It has been suggested that different lengths of poIy(A) additions to transcripts 3' end could lead to greater stability of transcripts (Avadhani, 1979). Protein Tnuishition in Mitochondria Mammalian mitochondrial genetic code is different from the "universal" genetic code in that UGA codes for tryptophan rather than termination, AUA for methionine and initiation rather than isoleucine, and AGA and AGG for termination rather than for arginine (Barrell et al., 1979; Barrell e! ai, 1980 a.b; Anderson et a!., 1981). This code is composed of four termination codons and 22 codon "families", each of which is read by a single mitochondrial tRNA. For families containing two codons, the decoding mechanism relies on G-U basepairing by the wobble position nucleotide in the tRNA. However, those tRNAs that read four-codon families all have a U in the wobble position, and hence evidently decode by using U-N or "2-out-of-3" base pairing (Barrell ct al., 1980b). Mammalian mitochondrial tRNAs lack several "invariant" features found in all previously studied nonmitochondrial tRNAs (Anderson et al., 1981), and likewise the 12S and 16S rRNAs exhibit only low homology to nonmitochondrial rRNAs (Epron et a/., 1980). 22 DNA and RNA sequence analyses indicate that animal mitochondrial mRNAs contain tew or no nucleotides as 5' or 3' untranslated leaders (Attardi, 1985; Ojala d al., 1980), Hence, this system will not use a Shine Dalgarno sequence type of interaction to specify the start AUG (Shine and Dalgarno, 1974). Furthermore, the lack of a 5' leader or cap structure indicates that the mitochondrial ribosome will not use a cap binding and scanning mechanism for initiation such as that found in the eukaryotic cytoplasmic system (Kozac, 1983). The small subunit of the mitochondrial ribosome seems to have the ability to bind mRNA tightly in a sequence-independent manner (Liao et al, 1989). Structures of rRNAs of mitochondrial have been proposed (Dams et a/., 1988; Gutell and Fox, 1988; Neefs cl al., 1990). During translation, the ribosome contacts the mRNA over about 80 nucleotides, with the small subunit having a binding site for guanyl nucleotides (Denslow el al., 1991) that is not present in either prokaryotic or eukaryotic cytoplasmic ribosomes. This interaction requires an RNA having a minimal length and seems to be independent of the secondary structure of the RNA (Liao et al., 1990a). It has been suggested that the first step in the initiation cycle in this organelle may be the binding of the small subunit of ribosome to the mRNA. Mitochondrial initiation factor 2 (IF-2mt) was the first animal mitochondrial initiation factor detected and partially purified (Liao et al., 1990b). This factor promotes the binding of fmet-tRNA to the mitochondrial ribosome. The binding reaction requires the presence of a message such as poly (A, U, G) and GTP. The IF-2mt is more similar to the prokaryotic initiation factor than to the eukaryotic cytoplasmic initiation factor (Liao et al., 1991). Eberly et al. (1985) found that bovine mitochondrial ribosomes are active only with homologous 23 elongation factor G (EF-G), but elongation factor Tu (EF-Tu) can be replaced by the EF-Tu from many other biological sources, including K. coli. Bacillus stihlHis, and Eitglena chloroplasts. It has been suggested that the translation of mitochondrial mRNA may be regulated. Chomyn and Attardi (1987) showed that translational etTiciency varies over an eight-fold range and suggested that the variation in translation rates may be responsible for different syntheses of mRNAs. However, the synthesis of mitochondrial proteins does not correlate with the amount of mitochondrial niRNAs, suggesting that translation may also be regulated (Polosa and Attardi, 1991). Import of Mitochondrial Proteins Most mitochondrial proteins are encoded in the nucleus, synthesized in the cytoplasm, and imported into the mitochondria (Schatz and Butow, 1983). Pathways for the import of a number of mitochondrial proteins have been elucidated by two basic approaches: pulselabeling studies with intact cells in the presence and absence of inhibitors, followed by subcellular fractionation and the incubation of isolated mitochondria or mitochondrial fractions with radiolabeled protein precursors synthesized in vitro. The following key steps have been identified: 1) synthesis of a precursor in the cytoplasm, 2) recognition of the mitochondrial surface by the precursor, 3) transfer of the precursor into or across one or both mitochondrial membranes, 4) conversion of the precursor to the mature form by proteolytic 24 cleavage, conformational change, or other modification, and 5) assembly of the polypeptide into an active protein complex. The import of proteins into mitochondria is a complex, multi-step process. In recent years, a number of components of the mitochondrial import machinery have been identified. Most proteins destined to be transported from the cytoplasm into the matrix are synthesized with N-terminal amphiphilic targeting sequences that direct the proteins into mitochondria. The precursor protein then binds to the mitochondrial surface and is transported across the outer and the inner membrane at sites where the two membranes are closely opposed. Passage across the two membranes requires that the protein be loosely folded. Once the protein has reached the matrix, its targeting sequence is removed and the mature polypeptide folds into native conformation (Attardi and Schatz, 1988; Hartl and Neupert, 1990). Each of these steps is catalyzed by specific proteins. Premature folding in the cytoplasm is prevented by cytosolic 'chaperones', which selectively bind and stabilize partially folded proteins; specific binding to the mitochondrial surface is mediated by mitochondrial surface receptors. Transport across the mitochondrial membrane is catalyzed by proteins forming 'proteintransport pores' across these membranes. The removal of the targeting sequence is effected by a specific protease. The folding of protein transported is controlled by matrix-located chaperones in the matrix. The mitochondrial members of this protein family collectively represent the mitochondrial protein import machinery. The biogenesis of mitochondria requires the coordinated action of both nuclear and mitochondrial genomes (Grivell, 1989). Proper function of mitochondrial processes depends 25 on accurate import and suborganellar sorting of many preproteins synthesized on cytosolic ribosomes. Protein import into mitochondria is a complex process that requires two separate machinery in the outer and inner membranes, each consisting of a large number of components (Pfanner and Neupert, 1990; Click and Schatz, 1991; Pfanner cV «/., 1992; Emtage and Jensen, 1993; Segui-Real cV a/., 1993b). The translocation machinery of the mitochondrial outer membrane (MOM) is comprised of at least six components organized in a complex (Kiebler ct al., 1990; Moczko el a/., 1992). MOM 19 and MOM72 of Neiirospora crassa mitochondria have been reported to be involved in the initial step of recognition and binding of preproteins to the mitochondrial surface (Sollner et ai, 1989; 1990). Recently MOM22 has been shown to function in the passage of preproteins from this receptor binding stage to a site where proteins are fully inserted into the outer membrane (Kiebler et al., 1993). At least part of this site, the so-called 'general insertion pore' (Pfaller et al., 1988), consists of MOM38 and M0M7 (Sollner et al., 1992). Genetics of Mammalian mtDNA Eukaryotic cells contain a class of cytoplasmic DNA molecules, which are found only within the mitochondria where they replicate and are transcribed. This mtDNA constitutes the mitochondrial genome and carries genetic information essential to mitochondrial function. There is evidence that the mitochondrial genome of a fungus or amphibian individual is derived solely from the maternal parent (Reich el al., 1966; Bernardi el al., 1968; Dawid et 26 a/., 1972). In a well-known experiment, Hutchinson ct al. (1974) provided evidence that the mammalian mitochondrial genome exclusively is inherited maternally. The study of the inheritance of mtDNA was performed by isolating pure mtDNA from hybrids between two closely related mammalian species. The two parental mtDNAs are readily distinguishable by restriction enzyme cleavage analysis; analysis of the mtDNA from the hybrid animals yielded a fragment pattern characteristic of only the maternal animal. None of the DNA fragments specific for the paternal animal was observed (Hutchinson et al., 1974). In another study, use of litters of rats with two types of mtDNA for interbreeding showed that the mtDNA type of progeny was the same as that of dams. This maternal inheritance of mtDNA could be observed in all organs tested and thus suggests that during development paternal mtDNA is not localized to a certain specific organ where it proliferates (Hayashi et ol., 1978), These authors concluded that maternal inheritance of mtDNA is strict. By examining the reciprocal crosses of two widely divergent species of mice, Gyllensten et al. (1985) reached the same conclusion. The study spanned eight generations of backcrossing and showed that mtDNA inheritance was at least 99.9 percent maternal. For over 20 years, it has been assumed, on the basis of low-resolution experiments, namely, restriction fragment length polymorphism (RFLP), that mtDNA has an exclusively maternal mode of inheritance in animals. However, Gyllensten et al (1991) demonstrated the existence of the paternal inheritance of mtDNA by the backcrossing of mice for 26 generations and by performing a more sensitive test, that is polymerase chain reaction (PGR), to amplify paternally inherited mtDNA selectively. The proportion of paternal mtDNA that 27 can enter the egg cytoplasm and persist has been calculated about 10"' per generation. By using the PCR, paternally inherited mtDNA molecules have been detected in mice at a frequency of lO'"*, which is relative to the maternal contribution. Biparental inheritance of mtDNA has been detected in mussels (Hoeh i't a/., 1991) and yeast (Bernadi et a/., 1968). There are no exact reasons for the seemingly strict maternal inheritance of mtDNA in mammals, because the middle piece of the mammalian spermatozoa that contains the mitochondria usually enters the egg during fertilization (Austin, 1961). In the mouse, mitochondria from the sperm have been observed to scatter in the cytoplasm of the egg (Gresson, 1940). In light of these observations, it is possible that the maternal inheritance may result from a quantitative preponderance of maternal (egg) mitochondria in the zygote. It is amusing to consider some numerological consequences of the mechanism. It has been estimated that a bull sperm contains 72 mitochondria (Bahr and Engler, 1970). Mammalian eggs are known to be rich in mitochondria (Austin, 1961; Gresson, 1940), and, if the concentration of mtDNA is similar to that found in the cytoplasm of echinoderm eggs (Piko et a!., 1967; Piko et al, 1968), an entire egg would contain about lO*" molecules of mtDNA. These figures would imply that if paternal and maternal mitochondria replicate at equal rates, then only about lO"* of the mtDNA in a mammal would be derived paternally. This fraction would be below the limit of detection by RFLP analyses. Furthermore, the sperm mitochondria could be partitioned into cells during the formation of the blastocyst and before the onset of mitochondrial replication. As a consequence, many clones of cells in the developing embryo may be completely free of paternal mtDNA, even if paternal mitochondria 2S are viable. It therefore seems unnecessary' to postulate any replicational defect in the paternal mitochondria to account for the observed maternal inheritance of mtDNA. The possibility still exists that paternal mitochondrial genomes are present in very small numbers in mammalian tissues and that sufficiently sensitive experiments (such as PCR) could detect their presence (Gyllensten f/«/., 1991). Mutation and Sequence Variations of mtDNA The mtDNA of many organisms has been isolated and studied, and the complete mtDNA sequences of certain organisms have been obtained (Anderson et a/., 1981; Anderson el a/., 1982; Bibb t'/a/., 1981; Roe cv a/., 1985; Clary and Wolstenholme, 1985; Wolstenholme et a/., 1987; Jacobs et ai, 1988; Cantatore et at., 1989; Gadaleta e! a/., 1989; Desjardins and Morais, 1990; Arnason t'/a/., 1991; Hoffman f/«/., 1992). The mammalian lines that ultimately gave rise to humans and cows diverged from each other about 80 million years ago (Wilson el a/., 1977). Because the rate of DNA sequence divergence is relatively fast for mammalian mitochondrial genomes (Upholt and Dawid, 1977; Brown el a/., 1979), those portions of the mtDNA sequence that have been conserved are likely to have been subject to function-related constraints during mammalian evolution. Functionally important higher order structures in those macromolecules coded for by mammalian mtDNA are also likely to have been conserved; so, comparative analysis of mtDNA sequences can be used to derive secondary or tertiary structure models for mitochondrial rRNAs and tRNAs. 29 Brown et al. (1979) demonstrated that the rate of evolution of the mitochondrial genome exceeds that of the single-copy fraction of the nuclear genome by a factor of 5 to 10, and the high rate may be due, in part, to an elevated rate of mutation in mtDNA. The nucleotide substitution rate is estimated at 0.02 substitutions per base pair per million years. The nucleotide variation in mtDNA within an animal species was first described by Brown and Vinograd (1974), who used RFLP techniques to study polymorphism in human mtDNA. Since then, the rapid intraspecies variation in mitochondrial genotypes has been noted for a number of mammalian species (Potter cr al., 1975; Brown el al., 1979; Brown, 1974; Upholt and David, 1977; Kroon et al., 1977; Buzzo et al., 1978; Hanswirth et al., 1980). In the majority of these studies, variation was detected by restriction site polymorphism and was explicable by simple gains or losses in sites. Nucleotide sequence data for several EcoR I derived polymorphisms in rat mtDNA confirmed this view (Buzzo et al., 1978; Hayashi et al., 1978). In one case, RFLPs suggested that insertional events occurred in the D-loop region (Upholt and Dawid, 1977). The variations of nucleotide sequences in the D-loop region of mtDNA have been found in humans (Cann e?/a/., 1987), primates (Brown tV o/., 1982), mice (Ferris £?/«/., 1983), and cattle (Hanswirth and Laipis, 1982; Olive et al., 1983; Watanabe et al., 1985; Koehler, 1989). Lindberg (1989) determined sequences of D-loop regions of mtDNA from cows from 38 maternal lineages of Holstein cattle and found 48 sites of nucleotide substitutions, one deletion site, and two variable-length poly G/C regions. The most frequent variation was the transition event, followed by transversion, and then deletion or insertion. It is accepted 31) generally that the D-loop region is the least conserved ponion of the mammalian mitochondrial genome. Modification of nucleic acid sequences generates variations in nuclear and nitDNA. The most common nuclear and mtDNA modifications are transitions (purine to purine substitution or pyrimidine to pyrimidine substitution). A mechanism for the spontaneous occurrence of transitions was suggested by Watson and Crick (1953) in their classic paper on the DNA double helix. They noted that some of the hydrogen atoms on each of the four bases can change their location to produce a tautomer. An amino group (-NH2) can tautomerize to an imino form (=NH). Likewise, a keto group (-C=0) can tautomerize to an enol form (=COH). The fraction of each base in the form of these imino and enol tautomers is about lO"*. These transient tautomers can form non-standard base pairs that fit into a double helix. For example, the imino tautomer of adenine can pair with cytosine. This A-C pairing would allow C to become incorporated into a growing DNA strand where T was expected, so that it would lead to a mutation if left uncorrected. Following replication, one of the daughter DNA molecules will contain a G-C base pair in place of the normal A-T base pair. Mutations also can be caused by either the modification of the incorporated bases or the incorporation of base analogs into the DNA strand during polymerization. Chemicals that can react with DNA could alter bases directly. For example, nitrous acid will oxidize amino groups of bases to keto groups. The significant conversions are A to hypoxanthine, C to U, and methylcytosine to T. Each of the converted bases forms hydrogen bonds in such a way as to cause transitions, and the G-C to A-T type has been reported most frequently. However, 31 even transversions are observed occasionaliy. Free radicals of oxygen, produced by radiation or as byproducts of aerobic metabolism, have been suggested to cause base modifications and to drive the high rate of mutation known to occur in mitochondria (Brown, 1981). Alkylating agents or base analogs can directly change the hydrogen bond property and cause mutation of DNA The localization of length differences to a homopolymer region suggests that a replication slippage mechanism may have generated some variation. DNA polymerases may use a mispaired primer-template in which one strand has slipped relative to its complement to generate insertions or deletions. Models using mispairing of homopolymers and larger repeats during replication have been used to explain length variation in a number of prokaryotic, eukaryotic, nuclear, and mitochondrial genes ( Efstratiadis et ai, 1980; Farabaugh et al., 1978; Brown and DesPosiers, 1983). Mitochondrial DNA Heteroplasniy in Animals Heteroplasmy means that more than one type of mtDNA exists within a single mitochondrion, a single cell, or an individual animal. There are two types of heteroplasmy. The term length heteroplasmy or size variation is used when different sizes of mtDNA exist within an individual animal. Site variants occur when mtDNA differs in nucleotide sequence (the genome is the same size) as defined by RFLP or direct nucleotide sequencing. 32 Heteroplasmy has been found in insects (Harrison el al., 1985), fish (Bermingham et a/., 1986), scallops (Snyder cv tj/., 1987), lizards (Densmore f/«/., 1985), humans with diseases caused by mutations in mtDNA (Holt et al., 1988), and cattle (Hausvvirth and Laipis, 1982b; Hauswirth el al., 1984; Ashley el a/., 1989). Heteroplasmy of size variation is more common in lower animals that in mammals (Bermingham et a!., 1986; Harrison et ai, 1985; Buroker et o/., 1990; Densmore et a/., 1985). Heteroplasmy usually can be explained by differences in the copy number of tandemly repeated sequences among otherwise identical molecules (Snyder t'/a/., 1987; Harrison c/a/., 1985; Desmore t'/a/., 1985). Buroker c?/. (1990) cloned and sequenced the D-loop region of two extreme length variants and found that extensive length polymorphism and heteroplasmy of the D-loop region are observed in mtDNA of the white sturgeon. The nucleotide sequence of this region, for both a short and long form, shows that the differences are the result of variable numbers of a perfect 82 base pair (bp) direct repeat. These data allow Buroker and colleagues (1990) to propose that the length heteroplasmy may be the result of frequent competitive misalignment in the repeat region prior to replication. An alternative mechanism proposed by Rand and Harrison (1989) suggests that frequent intermolecular recombination may cause heteroplasmy in crickets. The frequency with which heteroplasmy has been observed in animals suggests that it occurs rather frequently; yet, recombination in mtDNA has not been shown in mammals (Hayashi et a!., 1985). The evolution of heteroplasmy in maternal lineages has been examined in cattle (Hauswirth and Laipis, 1982; Ashley a/., 1989; Koehler t?/«/., 1991), DfosophUa 33 mauziiiana (Solignac et uL, 1984; Soliunac tv a!., 1987), and Giylliisfirnuis (Rand and Harrison, 1986). By following the transmission of a heteroplasmic intDNA through four generations of Holstein cows, Ashley a al. (1989) documented that substantial shifts in the frequency of heteroplasmy occurred between single mammalian generations. The neutral mitochondrial genotypes segregated in different directions in otTspring of the same female and a return to homoplasmy occurred in only two or three generations. Koehler et al. (1991) found the bovine mitochondrial genome can be replaced completely by a nucleotide sequence variant within a single generation, and no replication advantage or other selective bias could explain the rapid fixation of the mutation at the specific site. Solignac et al. (1987) studied the mitochondrial genotype of all F1 female offspring (426 individuals) of a single Di osophila maiiritiana female that was heteroplasmic for two types of mtDNA, a short genome characterized by a duplication of 470 bp sequence and a long genome characterized by a triplication of the same sequence. They found that all descendants were heteroplasmic. An increase of the percentage of long DNA in offspring was observed as the female became older. The variance of the genotypes increased as rounds of stem cell division progressed. In crickets, a heteroplasmic mother also transmitted heteroplasmy to her off^spring, suggesting that the segregation of mtDNA variation in cricket germ cell lineages does not occur rapidly (Harrison et al, 1985). Heteroplasmy of mammalian mtDNA usually involves point mutations and limited length variations affecting essentially noncoding regions (Hauswirth et ai, 1984; Ashley et al., 1989; Koehler et al, 1991). One case of heteroplasmy involving a large change in mtDNA 34 size has been reported to occur in mammals. Boursot ct al. (1987) found a mitochondrial mutant with a very large deletion in a coding region in European mice. The deletion is 5 kb long and encompasses six tRNA coding regions and seven protein genes. The mice were heteroplasmic: they contained a mixture of normal mtDNA and the deletion mutant. Although the mutant mtDNA is functionally defective, it represents 78% - 79% of the mtDNA molecules in both animals. Size heteroplasmy also was demonstrated in humans by Holt et al. (1988). The normal size of mtDNA co-existed with mtDNA containing deletions up to 7 kb in length in a patient with mitochondrial disease. Theoretically, site heteroplasmy should be common considering the high frequency of mutation in mtDNA. When a mutation occurs, different types of mtDNA should co-exist at some point of time, but site heteroplasmy rarely has been detected in normal mammals (Ashley et al., 1989; Koehler et al., 1991). Nevertheless, site heteroplasmy is very common in human mitochondrial disease with point mutations in mtDNA (Kobayashi et ai, 1994; Hayashi et ai, 1994; Pastores et al., 1994; Holt et al., 1990). Holt et al. (1990) reported that blood and skeletal muscle from patients with retinitis pigmentosa, demention, seizures, ataxia, proximal neurogenic muscle weakness, and sensory neuropathy contained two populations of mtDNA, one of which had a restriction site for /Ira I. Sequence analysis showed that the heteroplasmy was the result of a point mutation of nucleotide 8993, resulting in an amino acid change from a highly conserved leucine to an arginine in subunit 6 of mitochondrial H"-ATPase. The fraction of mutant mtDNA in patients is correlated positively with clinical severity. The heteroplasmy is inherited from their mothers. 35 Homoplasmy, in which only one type of mtDNA exists, usually is considered as a consequence of maternal inheritance of mammalian mtDNA. Therefore, the frequency of heteroplasmy within individuals will depend on the rates of mutation and rate of transmission or segregation. A high mutation rate coupled with a rapid segregation could lead to rapid evolution of mtDNA and the absence of individual heteroplasmy (Koehler el a/., 1991). Under these conditions, the heteroplasmic state, which must intervene if maternal lineages are to be differentiated, exists only briefly (Rand and Harrison, 1986). Phenotypic Effects of mtDNA Variations Variation of mtDNA sequence can change the quantity or quality of products encoded by mtDNA genes and then alter the phenotype. The polypeptides encoded by mtDNA are involved exclusively in mitochondrial membrane electron transport and oxidative phosphorylation to yield the common energy currency of living cells—ATP. Therefore, any change in the quantity and quality of the mtDNA gene protein can exert a direct influence on oxidative phosphorylation and energy production inside mitochondria. Bunn et ol. (1974) demonstrated that yeast strains with mutations in mitochondrial genes that encode components of electron transport have altered mitochondrial respiratory characteristics. Studies carried out with genetically divergent animals have confirmed genetic influences on mitochondrial enzyme activities and oxidative phosphorylation. For example, mitochondria with different mtDNA genotypes have different rates of oxidative phosphorylation in DrosophUa (McDaniel and Grimwood, 1971), rabbits (Dzapo et ai. 36 1973), sheep (Wolanis el al., 1980), chickens (Dziewiecki and Kolataj, 1980), mice (Brown et ciL, 1987, Lindberg ei ci/., 1989), and swine (Dzapo and Wassmuth, 1983). It has been reported that a specific change in mtDNA conferred resistance of chloramphenicol in a mammalian cell line (Blanc cl a/., 1981). Recently, a variety of human diseases that involve the brain, heart, skeletal muscle, kidney, and endocrine glands have been attributed to mutations in mtDNA (Holt ef al., 1988; Holtt'/n/., 1990; Kobayashi c/a/., 1994; Hayashi tV a/., 1994; Pastores £.'/a/., 1994). The first pathologic mtDNA mutations identified were associated with rare syndromes such as Leber's optic neuropathy (LHON), myoclonic epilepsy and ragged-red fiber disease, and the Kearns-Sayre Syndrome (Wallace, 1993) However, the characteristics of late onset tissue- specific diseases and progression of these diseases suggest a variety of more common degenerative disorders also might be caused by mitochondrial mutations. The tissue-specific diseases also could be explained by a combination of the tissue-specific accumulation of somatic mtDNA mutations with age and the variation between tissues in the expression of nuclear genes that encode mitochondrial function (Wallace, 1993). Different tissues and organs rely on mitochondrial energy to various extents, with the central nervous system, heart, skeletal muscles, kidney, and endocrine glands being particularly oxidative and therefore especially dependent on energy produced by the mitochondria. As the proportion of mutant mtDNAs increases among maternal relatives, mitochondrial energy output declines until the ATP-generating capacity of the cell falls below 37 the energy threshold necessary for normal tissue function. Then, disease ensues (Wallace, 1992). Thus, it is clear that mutation of mtDNA. can have profound etTects on phenotype. In mammals, the only known extranuclear genetic material is mtDNA. It has been documented that cytoplasmic genetic (extranuclear) effects also contribute to differences in milk production efficiency among dairy cattle. It has been shown that 2% of variation in milk production and 3.5% of variation in milk fat percentages were explained by cytoplasmic effects (Bell c/ o/., 1985). Schutz el at. (1992; 1993; 1994) found that the within-herd variation in milk production traits and health condition were associated with mtDNA genotypes. Huizinga et al (1986) documented that 10% of the variation in milk economic returns were the result of cytoplasmic components. Faust et al. (1989) found large cytoplasmic effects on number of days from calving to first service, conception rate, and number of services per conception. Therefore, evidently mtDNA genotype of cows can influence animal production traits significantly. Mitochondrial DNA Genetics in Cattle Bovine mtDNA is 16,336 nucleotides in length (Anderson et a!., 1982). The bovine mtDNA sequence has high homology to that of human mtDNA (Anderson et al., 1981), and the genes are organized in virtually identical fashion. The protein coding genes of bovine mtDNA show 63 to 79% homology to their human counterparts, and the nucleotide differences occur in the third positions of the codons. The bovine and human mitochondrial tRNA coding regions exhibit more interspecies variation than do their cytoplasmic :i8 counterparts, whereas DNA sequence in the bovine D-ioop region is only slightly homologous to the corresponding region in the human mtDNA. This region is also quite variable in length and accounts tor the bulk of the size ditTerence between human and bovine mtDNAs. Few studies have been carried out to evaluate the variation of bovine mtDNA sequences. Hauswirth and Laipis (1982) first demonstrated that two mitochondrial genotypes coexisted within one Holstein cow maternal lineage. The variation was detected by the appearance of an extra Hae III recognition site in one genotype, and this variation was caused by a point mutation within an open reading frame at the third position of a glycine codon. The mutation did not change the amino acid sequence. They concluded that the shift of mtDNA genotype took place in no more than 4 years. By sequencing part of the D-loop region of 14 maternally related Holstein cows, Olivo et at. (1983) found that four different D-loop sequences existed in the mtDNA of cows from the same maternal lineage. They concluded that the genotypic shifts in the bovine nUDNA D-loop were relatively rapid. The first case of intra-animal heterogeneous sequences of bovine mtDNA was reported to be located within an evolutionary conserved C homopolymer sequence near the 5' end of the D-loop region (Hauswirth et o/., 1984). Laipis et al. (1988) reported that the relative ratio of the two heteroplasmic molecules varies 3-fold among sibling dairy cows, and they postulated that the unequal partitioning of bovine mitochondrial genotypes among siblings was the basis for rapid DNA sequence variation. Ashley ct al (1989) also provided evidence of rapid segregation of heteroplasmic bovine mitochondria by following the transmission of a heteroplasmic mtDNA mutation through four generations of Holstein cows, 39 whereas Koehler el al. (1991) observed a complete change of bovine mtDNA genotype within one generation. There is enough evidence that a new mtDNA genotype could arise and replace old ones in a relatively short period of time. Lindberg (1989) has cloned and sequenced mtDNA D-loop region of Holstein cows from 38 maternal lineages. Forty-eight sites of nucleotide substitutions, one deletion site and two variable length poly G/C regions have been detected within the 910-bp D-loop region when compared with the published sequence (Anderson et a/., 1982). The nucleotide diversity has been calculated to be 3.8x10'' from nucleotide substitution data in the D-loop region. It is interesting that many nucleotide substitutions found in cows were thought to be located within the CSB in humans. There were no sequence variations in the central GC-rich region from 16,142 to 16,202. Phylogenetic trees based on nucleotide substitution data were constructed and showed two major divisions of mtDNA genotypes present in Holstein cattle. Johnston et al. (1991) investigated the sequences of a 2735-base pair region of mtDNA that includes both of the mitochondrial rRNA genes (12S and 16S rRNA) and three mitochondrial tRNA genes (tRNA* '"', tRNA' '""', and tRNA'"""') from 29 maternal lineages of Holstein cattle. Ten transitions and one variable length homopolymer region were observed. All the sequence variants were located in the rRNA genes. The possible effects of those sequence variants on the predicted secondary structure of the rRNAs and the nucleotide sequence identity with other rRNAs have been examined. It has been postulated that two variants would increase the potential number of base pairs in the rRNA, whereas one sequence variant is in a base-paired structure which would not affect the secondary structure. One 40 sequence variant was thougiit to increase the nucleotide sequence identity with other known rRNAs. The variable-length homopolymer with either six or seven A residues was located within 16S rRNA gene. No heteroplasmic state was detected at this site. References Alberts, B., Bray, D., Lewis, J., RafF, M., Roberts, K., and Watson, J. D. (1994). Molecular Biology of the Cell, pp 653-683. Garland Publishing, New York and London. Albring, M., Griffith, J., and Attardi, G. (1977), Association of a protein structure of probable membrane derivation with HeLa cell mitochondrial DNA near its origin of replication. Proc. Natl. Acad. Sci. USA. 74: 1348-1352. Anderson, S., Bankier, A. T., Barrel, B. G., DeBruijn, M. H. L., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. 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Nucleic Acids Res. 10: 75317542. 56 SEQUENCE HETEROPLASMY OF D-LOOP AND rRNA CODING REGIONS IN MITOCHONDRIAL DNA FROM HOLSTEIN COWS OF INDEPENDENT MATERNAL LINEAGES A paper to be submitted to The Proceedings of the National Academy of Science USA Jianming Wu, Renotta K. Smith, A. E. Freeman, Gary L. Lindberg, Ben T. McDaniel, and Donald C. Beitz Abstract Mitochondrial DNA (mtDNA) D-loop and rRNA coding regions of two Holstein cows from each of 18 maternal lineages were cloned and sequenced to determine the presence of hypervariable sites and the existence of heteroplasmic mtDNA. Seventeen sites of nucleotide sequence variations were observed within D-loop and rRNA coding regions. The hypervariable sites in the D-loop and rRNA coding regions were located at nt 169, 216, and 1594 and between nt 352 to 364. Heteroplasmic mtDNA was detected within D-loop and rRNA coding regions of mtDNA within independent maternal lineages. The presence of heteroplasmic mtDNA in cattle of the same maternal lineages suggests that mtDNA of one genotype could have been maintained for at least 11 generations in their respective maternal lineages. Because heteropiasmy was observed frequently and seemingly is persistent, selected amplification of specific mtDNA may not be a part of mtDNA inheritance. The mechanism of mtDNA deletions and insertions is proposed. 57 Mutations by transversions, insertions, and deletions invariably occurred at the end or within nucleotide homopolymers in the D-loop and rRNA coding regions. Introduction Bovine mitochondrial DNA (mtDNA) consists of 16338 base pairs and codes for two rRNAs, 22 tRNAs, and 13 polypeptides. Mitochondria carry multiple copies of double-stranded circular DNA that is replicated and e.xpressed within the organelle, and it is believed that mammalian mtDNA is inherited maternally (1,2). The complete sequence of mtDNA has been determined for cattle, humans, mice, and toads (3-7); however, apart from the mode of maternal inheritance, segregation and transmission of mtDNA are not well understood. Variation in nucleotide sequences of mtDNA can affect mitochondrial function. Use of genetically divergent animals has confirmed genetic influences on mitochondrial enzyme activities and oxidative phosphorylation. For example, different rates of oxygen consumption have been observed for mitochondria of different genotypes of Drosophila, rabbits, sheep, chicken, mice, and swine (8-13). Recent data indicate that 5% of within-herd variation in total amount of milk fat and milk fat percentage of dairy cattle is associated with maternal lineages as defined by mtDNA genotype (14). Moreover, maternally inherited diseases such as mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episode, Kearns-Sayre syndrome, Leber's hereditary optic myopathy, and other mitochondrial myopathies are caused by mtDNA deletions and point mutations (15, 16). Estimates based upon restriction fragment length polymorphisms (RFLP) in mtDNA of humans and other mammals indicated that mtDNA mutates at a rate of perhaps 10 times that of nuclear 58 DNA (17). This rate would be an underestimate if mtDNA polymorphisms are manifestations only of heritable mutations. Theoretically, any animal that has a mtDNA mutation must be heteroplasmic or possess more than one type of mtDNA at some point in time. The heteroplasmic state is defined as a mixture of mtDNA sequence variants that exist within a single mitochondrion, a single cell, or a single animal. For mutant mtDNA to be predominant in future generations, different types of mutant mtDNA must separate from the majority of parental mtDNA molecules to become the sole or dominant mitochondrial genomes in germ cells of successive generations. Despite the great volume of mammalian mtDNA data, most of the known examples of large scale, within-species polymorphisms and heteroplasmy of mtDNA occurs in insects, fishes, scallops, toads, lizards, and humans (18-23). The mechanism of transmission of heteroplasmic mtDNA and the mechanism of segregation of these genomes are not known. Ashley et al. (24) reported the rapid segregation of heteroplasmic bovine mitochondria by following the transmission of a single heteroplasmic mtDNA mutation by using RPLP analysis through four generations of Holstein cows and suggested that a return to homoplasmy may occur within only two or three generations. By using RFLP analysis, the replacement of bovine mtDNA by a sequence variation can occur within one generation (25). It is considered that the evolution of heteroplasmic mtDNA is rapid, based on data obtained fi-om RFLP analysis. For over 20 years, it has been assumed that mtDNA has an exclusively maternal mode of inheritance in animals. However, evidence of paternal inheritance of mammalian mtDNA has begun to emerge (26). 59 Our hypothesis is tiiat inheritance of mtDNA may be the result of random passing from ancestor to progeny instead of the result of selection. Therefore, the study was designed to determine the nucleotide sequence variation sites in D-loop and rRNA coding regions, to analyze the phenomenon of heteroplasmy (different types of mtDNA exist within an individual animal), and to investigate the characteristics of bovine mtDNA inheritance. We report here for the first time that multiple heteroplasmic mtDNA states within the Dloop and rRNA coding regions of mtDNA existed extensively in Holstein cows. Heteroplasmy was found in independent maternal lineages by determining the mtDNA sequences of different clones {rom the same cows. One mtDNA genotype observed in some Holstein cows could have been maintained for more than 11 generations in the maternal lineages. Hypervariable sites in the D-Ioop and rRNA coding regions are located at nt 169, 216, and 1594 and between nt 352 and 364. Deletions, insertions, and transversions were found within or at the end of nucleotide homopolymers. Different types of mtDNA from the same cow were found to differ by as many as three sites within the D-loop and rRNA coding regions. Therefore, it is concluded that the mtDNA population within an individual animal is a mixture of different mitochondrial genotypes of mtDNA and that mutations could accumulate on the same mtDNA molecule at many different sites. Furthermore, because heteroplasmy was observed frequently and seemingly is persistent, selected amplification of specific mtDNA may not be a part of mtDNA inheritance. 61) Materials and Methods Thirty-six Hoistein cows were from 18 maternal lineages (two cows from each maternal lineage), which were from two research herds owned by the North Carolina Department of Agriculture (NCDA) and one herd by North Carolina State University (NCSU). One NCDA herd was established in about 1981 from cows purchased in the northeastern region of the United States, and the other NCDA herd was started in 1950 with cows from an older herd plus a few from another herd that has been dispersed since then. The NCSU herd was established in 1922 from cattle purchased in the state of Pennsylvania. Cows were traced by female lineages to common female ancestors in the Hoistein Herd Book. Four hundred milliliters of whole blood were taken from each cow via jugular venipuncture, and the red blood cells were lysed hypotonically with an equal volume of 10 mM KHCOj, 150 mM NH4CI, and 1 mM EDTA. The treated blood was centrifriged at 1400 xg, and the resulting leukocyle pellets were transported on dry ice within 24 hours from North Carolina to Iowa State University. Mitochondrial DNA was isolated from the leukocytes as described by Brown et al. (27). The 4.3 kilobase (kb) fragment obtained from P.stl-Sacl enzymatic digestion of mtDNA was cloned into the multiple cloning site of the pUCl 19 phagemid vector, and E. coli strain TGI was transformed. Out of many recombinant clones obtained from each cow, two clones were chosen randomly for flirther analysis. Single-stranded DNA (ssDNA) template was prepared by infecting E. coli containing recombinant phagemid with M13K07 helper phage and subsequent recovery of phage particles by precipitation with 20% polyethylene glycol. Pure ssDNA was obtained by phase extraction of 61 phage particles with organic solvents followed by ethanol precipitation. Nucleotide sequencing was performed by the dideoxy chain termination method with [a-"'S] dATP incorporation (USB Sequenase Version 2.0 sequence kit, USB, Cleveland, OH). Twelve oligonucleotide primers were used for the DNA sequencing reactions. The oligonucleotide primers and their locations on bovine mtDNA are listed in Table 1. DNA fragments were separated by electrophoresis on a 6% polyacrylamide gel, transferred to blotting paper, dried under vacuum, and exposed to Kodak XOmat AR film for approximately 24 hours at room temperature. For studies of mutagenesis in bacteria, recombinant clones were inoculated into 10 ml of LB medium (28) containing 50 |.ig/m! ampicillin and grown to an OD goo of 1.0 (passage 1). A 0.1 Table 1. 5' nt' Oligonucleotide primers for D-loop and rRNA coding region sequence analysis. D-loop region 5' nt' rRNA coding region 496 5' GGGGTGTAGATGCTTGC 3' 694 5' ACTCCTGTTAGCTTGGG3' 355 5' GGGGCCTGCGTTTAT 3' 809 5' CTAGTAGTACTCTGGCG 3' 155 5' CTGGTGCTCAAGATG 3' 1120 5' TCTTCCCATTTCATAGG 3' 16305 5' GACCGTTTTAGATGAGA 3' 1328 5' ACTTGTCTCCTCTCAGT 3' 16151 5' TCTAATGGTAAGGAAT 3' 1536 5' TTAGATTTCTATTCTCC 3' 1760 5' CTTACAAATCTTCTCAC 3' 2755 5' CCTATTGTCGATATGG 3' "The 5' nucleotide indicates the startin" nucleotide of a primer, which is numbered as by Anderson etai (4). 62 ml aliquot was inoculated into 10 ml of fresh LB medium (ampicillin 50 |.ig/ml) and grown to an ODcoo of 1.0 (passage 2); subsequent further "passages" were generated by inoculating 0.1 ml into 10 ml of fresh LB medium. After the eighth passage, ssDNA was prepared, and the nucleic acid sequence was determined as described previously to determine the stability of recombinant clones. Results Seventeen nucleotide sequence variants among 18 maternal lineages were observed within the D-loop and rRNA coding regions of mtDNA (Table 2). Four types of mutations (insertions, deletions, transitions, and transversions) were found in D-loop and rRNA coding regions. Among 17 nucleotide variations, transitions (purine to purine or pyrimidine to pyrimidine) were the most frequent events; 11 instances of substitutions were transitions. These transition substitutions all occurred in the D-loop region. Two deletion and three insertion sites were observed in the D-loop and rRNA coding regions. Two transversions (purine to pyrimidine or pyrimidine to purine) were observed. Among all the variations, transition from adenine to guanine at nt 169, cytosine insertion at nt 216, transversion from cytosine to guanine at nt 364, length variation of cytosine homopolymer between nt 352 and 364, and deletion of adenine at nt 1594 were the most common mutation events and sites. Mitochondria! DNA heteroplasmy could be defined by at least three criteria: 1) mtDNA differences among females within a maternal lineage, 2) mtDNA differences among different tissues within an individual, and 3) mtDNA differences within a tissue of an individual. In this study, 63 Table 2. Summary of nucleotide variations in the D-loop and rRNA coding regions of bovine mtDNA. Sites" Substitution events'' Numbers of lineages with the substitution Numbers of cows across lineages 364' C->G transversion 11 17 1677 T->A transversion 1 1 216' C insertion 17 33 C deletion or insertion 12 18 352-364' 1062 A insertion 2 3 1594' A deletion 13 20 169' A-^G transition 12 16 364 C-»T transition 1 1 16022 G—>A transition 2 3 16050 C-^T transition 2 4 16057 G-»A transition 1 1 16135 T->C transition J 4 16141 T-^C transition 1 1 16196 G—>A transition 2 2 16200 G^A transition 2 2 16231 C->T transition 1 1 16247 C->T transition 3 4 " Nucleotide numbering according to Anderson et al.{A). '' Nucleotide on left side of arrow is replaced by that on right. " Hypervariable sites. 64 heteroplasmic cows refer to cows that contain different types of mtDNA in the same tissue. Multiple clones of mtDNA from individual animals revealed mtDNA heteroplasmy within animals at eight sites (Table 3). The most frequent heteroplasmic site was polycytosine between nt 352 and 364; sixty-seven percent of cows surveyed were heteroplasmic at this region. The percentage of cows that were heteroplasmic at other sites ranged from 6% to 50% (Table 3). Although the nucleotide substitution at nt 169 occurred at high frequently (44%), no evidence ofheteroplasmy was found at this site. Table 3. Sites within mtDNA where heteroplasmy within cows has been observed. Sites and events Number of cows with the substitution' Heteroplasmic cow with the nucleotide substitution 17 Percentage of heteroplasmic cows'* 2 12 2 6 18 12 67 1062 A insertion 3 1 33 1594 A deletion 20 5 25 16050 C—>T transition 4 1 25 16135 T->T transition 4 1 25 16200 G—>A transition 2 1 50 364 C-^G transversion 216 C insertion 352-364 C deletion or insertion Thirty-six cows in 18 maternal lineages were examined. ''Percentage of the heteroplasmic cows is defined as number of heteroplasmic cows over the total number of cows with the substitution. 65 The most trequent transversion was cytosine to guanine at nt 364 at the end of a homopolymer of cytosines in the D-loop region. Mitochondrial DNA of 17 cows from 11 maternal lineages possessed this kind of substitution. Another transversion was located at nt 1677, which was also at the end of a homopolymer of adenine within the 16S rRNA coding region. Table 4 shows the common features of the transversions at nt 364 and 1677, the insertions at nt 216 and 1062, the deletion at nt 1594, and the variation of the length of polycytosines between nt 352 and 364. These events occurred either at the end of homopolymers or within the A and C homopolymers. Table 4. Neighboring homopolymer sequences of transversion, deletion, and insertion mutation sites Mutation sites (Region) Event Neighboring sequence"" (D-loop) C^G transversion G-'^"GCCCCCCCCCCCCe®' (16S rRNA) T—>A transversion a'"° acaaaatgaatttta"^^^ (D-loop) C insertion A''"TATATCCCCCCTTCA^'^ 1062 (12S rRNA) A insertion C'"''"TA A A A AGGAAAAAAAGTA'"^*^ 1594 (16S rRNA) A deletion A'^^AAGAAAAAAACTAAAG'®^ (D-loop) C insertion or deletion g''"gccccccccccccc'^ 364 1677 216 352-364 " Bold face and underlined letters indicate the nucleotide transversion site or insertion and deletion regions. 66 The nature and transmission of mtDNA were studied by the analysis of heteroplasmic mtDNA derived from animals within the same maternal lineages. Two cattle in maternal lineage 10126 were sisters, but they possessed mtDNA with differing nucleotide substitutions (Table 5). Heteroplasmic cow 4167 had two types of mtDNA: one with substitutions at nt 216, 364, and 1594; another with substitutions at nt 216 and 364. The other cow in the same maternal lineage had one type of mtDNA with substitutions at nt 169, 1594, and 16200. This observation means that the dam of two cows might have had a mixture of three types of mtDNA, unless numerous substitutions occurred in daughter cows. Both cows in maternal lineage 79171 were heteroplasmic (Table 6), and had three different types of mtDNA that differed at two sites. Cow 1968 had one type of mtDNA with substitutions Table 5. Mitochondrial DNA heteroplasmy within a maternal lineage and within individual animals. Cow number^ Clone number 4167 46.4 216 364 46.7 216 364 4239 Substitution sites'*' 1594 _ 47.4 169 1594 16200 47.7 169 1594 16200 " Heteroplasmic cow 4167 and cow 4239 are sisters in the maternal lineage (lineage 10126). '' Substitution sites are the specific nucleotides that were different from the published sequence by Anderson el al. (4). Different substitution sites among clones were at nt 216, 364, 169, 1594, and 16200, 67 Table 6. Mitochondrial DNA heteroplasmy within a maternal lineage and within individual animals. Cow number' Clone number 1968 40.2 169 216 40.6 169 216 1594 41.3 169 216 1594 41.6 169 216 1594 2138 Substitution sites''" 16050 16050 16050 Heteroplasmic cow 1968 and 2138 were two and three generation descendants of the same dam, respectively (lineage 79171). Substitution sites are the specific nucleotides that were different from the published sequence by Anderson et al. (4). " Different substitution sites among clones were at nt 1594 and 16050. at nt 169, 216, and 16050. Cov^' 2318 in the same maternal lineage had another type of mtDNA with substitutions at nt 169, 216, and 1594. Both cows also had one common type of mtDNA in which all the substitutions (at nt 169, 216, 1594, and 16050) found were exactly the same, but the two different types of mtDNA in two different cows of the same lineage were not only different from the common type of mtDNA but also differed from each other at two sites (nt 1594 and 16050). Table 7 shows that the heteroplasmic cow 1907 in maternal lineage 70080 had two different types of mtDNA. One type of mtDNA had substitutions at nt 216 and 1594, and the other had substitutions at nt 216 and 364. Two types of mtDNA differed at two sites (at nt 364 68 Table 7. Mitochondrial DNA heteroplasmy within a maternal lineage and within individual animals. Cow number'' Clone number 1907 49.7 216 49.9 216 50.4 216 1594 50.8 216 1594 2064 Substitution sites'^ '' 1594 364 Cow 2064 and heteroplasinic cow 1907 were four- and five-generation descendants of the same dam, respectively (lineage 70080). Substitution sites are the specific nucleotides that were different from the published sequence by Anderson et al. (4). Different substitution sites among clones were at nt 364 and 1594. and 1594). Cow 2064 in the same lineage only had one type of mtDNA with substitutions at nt 216 and 1594. Both cows still had one common type of mtDNA. In maternal lineage 89109 (Table 8), the heteroplasmic cow 2094 had one type of mtDNA with substitutions at nt 364 and 16247, and another with substitutions at nt 16247, 1062, and 1594. Two different types of mtDNA differed at three sites (nt 364, 1062, and 1594) within the same cow. The other cow in the same maternal lineage had one type of mtDNA with substitutions at nt 1062, 1594, and 16247. Two cows in the same lineage still had one common type of mtDNA after seven generations, The observations suggest that one type of mtDNA might not change sequence within the D-loop and rRNA coding regions after seven generation and that the different substitutions could occur on the other type of mtDNA repeatedly or simultaneously. 69 Table 8. Mitochondrial DNA iieteroplasmy within a maternal lineage and within individual animals. Cow number'' Clone number 2118 43.43 1062 1594 16247 43.44 1062 1594 16247 2094 59.21 59.22 Substitution sites'' " 364 16247 1062 1594 16247 ' Cow 2118 and heteroplasmic cow 2094 were seven- and four-generation descendants of the same dam, respectively (lineage 89109), '' Substitution sites are the specific nucleotides that were different from the published sequence by Anderson ct a/. (4). Different substitution sites among clones were at nt 364, 1062, and 1594. Table 9 shows that both cows in lineage 59002 were heteroplasmic; they had one type of mtDNA with substitutions at nt 16022 and 16050 and another type with substitutions at nt 216, 16022, and 16050. The mtDNA of both cows was not different, and two cows were seven- and 11-generation descendants of the same dam, respectively. Even though there were other substitution sites on mtDNA molecules, exactly the same two types of mtDNA still existed in cows after seven and 11 generations, which suggests that the heteroplasmic mtDNA were inherited from a common ancestor of the two cows. Alternatively, the same mutations could have arisen at different points in the lineage. It is unlikely, however, that the same nucleotide substitutions could occur in the same way 11 generations later. In the maternal lineage 59500 (Table 10), two cows were nine- and 11-generation descendants of the same dam and still had a common type of mtDNA with substitutions at nt 70 Table 9. Mitochondrial DNA heteroplasmy within a maternal lineage and within individual animals. Cow number' Clone number Substitution sites'' '^ 2888 11.13 16022 16050 16022 16050 16022 16050 16022 16050 11.18 3240 216 12.14 12.19 216 Heteroplasmic cows 2888 and 3240 were seven- and 11-generation descendants of the same dam, respectively (lineage 59002). Substitution sites are the specific nucleotides that were different from the published sequence by Anderson et a/. (4), Different substitution sites among clones were at nt 216. Table 10. Mitochondrial DNA heteroplasmy within a maternal lineage and within individual animals. Cow number' Clone number 1972 29.50 216 29.58 216 55.6 216 1594 55.10 216 1594 3451 Substitution sites'"'"" 1594 16135 16135 16200 16135 Heteroplasmic cows 1972 and 3451 were nine- and 11-generation descendants of the same dam, respectively (lineage 59500). '' Substitution sites are the specific nucleotides that were different from the published sequence by Anderson ei ciL (4). "Different substitution sites among clones were at nt 1594, 16135, 16200. 71 216, 1594, and 16135. Cow 1972 had another type of mtDNA with substitutions at nt 216 and 16135. Cow 3451 had the other copy of mtDNA with substitutions at nt 216, 1594, and 16200. There were three different sites between two different types of mtDNA from two cows in the same maternal lineage (nt 1594, 16135, and 16200). Table 11 shows that cow 4324 in maternal lineage 10177 was heteroplasmic; it had one type of mtDNA with substitutions at nt 216 and 364 and another type with substitution at nt 216, 364, and 1594. The other cow in the same lineage had only one type of mtDNA with substitutions at nt 216 and 364. Two cows were 10- and 11-generation descendants of the same dam; and they still had one type of mtDNA with substitutions at nt 216 and 364 in common. Table 11. Mitochondrial DNA heteroplasmy within a maternal lineage and within individual animals. Cow number' Clone number 4324 53.6 216 364 53.9 216 364 54.6 216 364 54.9 216 364 4307 Substitution sites'' '" 1594 ' Cow 4307 and heteroplasmic cow 4324 were 10- and 11-generation descendants of the same dam, respectively (lineage 10177). '' Substitution sites are the specific nucleotides that were different from the published sequence by Anderson et al. (4). Different substitution sites among clones were at nt 1594. 72 Within all the maternal lineages in which the heteroplasmic cows have been found, except for maternal lineage 10126, cows in the same maternal lineage had one common type of mtDNA even after three, five, seven, nine, 10, and 11 generations. To confirm that the indication of heteroplasmy is not artifactual, 10 recombinant clones were passaged in LB medium for eight generations. No changes were found in nucleotide sequences of the D-loop and rRNA coding regions of mtDNA in these recombinant clones. Discussion In mammals, mitochondrial heredity is predominantly maternal, and heteroplasmy can be generated only by mutations affecting the female mtDNA. After the occurrence of a mutation in a mtDNA molecule, a heteroplasmic state must exist for at least some time if the mutant mtDNA molecule is maintained through subsequent generations. Very often, RFLP analysis fails to detect this intra-individual heterogeneity. The heteroplasmic state depends on mutation frequency and whether the mutation remains in the mtDNA as opposed to the mutant type not remaining in the organism. Because it has been detected rarely, the heteroplasmic state has been thought to be fugacious. The inheritance of heteroplasmy has been studied for Drosophila and crickets (29, 30). For crickets, no substantial variation in the level of heteroplasmy was noted in 10 single-generation offspring from heteroplasmic individuals. In a more extensive study of Drosophila, the distribution of mtDNA genotypes among individuals was consistent with an unbiased stochastic partitioning of heteroplasmic genotypes at each maternal generation (31). Despite the high mtDNA mutation rate. 73 which implies a need for heteroplasmy during the transition from one genotype to another, very few examples of such a state have been reported in higher animals. Hauswirth ct a/. (32) first reported heterogeneous length of a polycytosine run between nt 352 and 364 within the nitDNA D-loop sequence in bovine tissue. It has been found that 67% of cows surveyed in this study were heteroplasmic at the polyc>losine region between nt 352 and 364 of the mtDNA D-Ioop. Therefore, this polycytosine region of mtDNA variation between nt 352 and 364 is not a useflil genetic marker. It has been reported that a substantial shift in the percentage of heteroplasmy at nt 364 could occur within a single generation, and it has been suggested that a return to homoplasmy may occur in only two or three generations (24). Koehler et al. (25) reported replacement of bovine mtDNA by a sequence variant at the same site (nt 364) within one generation, but they only used the RFLP analysis to detect homoplasmic states. In the latter instance, these authors failed to find heteroplasmic cattle as intermediates in the genetic fixation process. A significant amplification both in number of mitochondrial genotypes and in mtDNA content occurs during follicular development of a specific oocyte. For example, Piko and Matsumoto (33) calculated that the mature mouse oocyte contains 100-1000 times more mtDNA than is found in somatic cells. Hauswirth and Laipis (34) also found 100-fold more in mtDNA in mature bovine oocytes than in undeveloped ones. Also, a limited number of genotypes of mtDNA may be used as templates for DNA amplification during oocyte development, and certain types of mtDNA may be amplified more than others. But, the heteroplasmic state still should exist persistently in the fijily developed embryo, even though the profile of mtDNA genotypes is 74 changed. It is a reasonable assumption that the mtDNA with the C to G transversion at nt 364 already has existed in the cattle gemi line for a long time because the majority (11 out of 18) maternal lineages surveyed contained mtDNA with this variation. Therefore, it is possible that the either one of two types of mtDNA with variations at nt 364 could be amplified to a predominant level so that the other type of mtDNA could not be detected easily by insensitive methods like RFLP analysis. This observation might explain the phenomenon of replacement of one type of mtDNA genotype with another within one generation, which was reported by Koehler el al. (25). Persistent heteroplasmy also has been found in humans where mtDNA deletions or point mutations are associated with maternal inherited disorders (35-38). It has been argued that selection may exist for one of the two variants because, in most instances, the severity of disease positively or negatively correlates with the degree of heteroplasmy. Thus, one sequence variant is likely to result in nonfijnctional or partly functional mitochondria, providing selection for the fijlly fijnctional alternative sequence. If more clones from each individual animal were chosen and longer mtDNA regions were sequenced, it seems reasonable that more heteroplasmic sites and animals could have been found. Therefore, the absolute percentage of heteroplasmic dairy cattle could be greater than what we observed. Furthermore, the absolute homoplasmic state may exist rarely in dairy cattle. In one instance, two clones from the same cow differed at three sites (cow 2094 in maternal lineage 89109). To accumulate three substitutions on a specific mtDNA molecule in successive generations, heteroplasmic states of certain types of mtDNA might be maintained for a long period of time. No mechanism seems to exist to eliminate the once dominant mtDNA genotypes 75 completely from mtDNA populations because cows in the same maternal lineage had one common type of mtDNA even after three, five, seven, nine, 10, and 11 generations. Therefore, the mtDNA population in a specific animal would be a mixture of different genotypes if more genetic markers are used because random passing of mtDNA from mother to progeny seems to be the primary mechanism of mtDNA inheritance. This explanation also could explain why persistent heteroplasmy exists in a human pedigree that manifests mtDNA disorders. We suggest that heteroplasmy exists extensively in individual dairy cattle, but the percentage of a specific type of mtDNA genome in the total population of mtDNA may be so variable that it is difficult to detect all genotypes or that the detection of different individual animals with different mtDNA genotypes in successive generations could be missed easily. This difficulty will result in an underestimation of heteroplasmy and an overestimation of mtDNA segregation rates. Moreover, hypervariable sites within D-loop and rRNA coding regions are located at nt 169, 216, 364, and 1594 and between nt 352 and 364. If substitutions at these sites are easily reversible in the maternal lineage, another question that should be answered is whether all these hypervariable sites are suitable as genetic markers in studying the genetics of mtDNA alone. Up to now, all the published papers related to the mtDNA segregation transmission of heteroplasmic cows have not used the nt 364 cytosine-to-guanine transversion as a genetic marker. Therefore, it is questionable that a single hypervariable marker could be used to estimate the segregation rate of different types of mtDNA, even though almost all the heteroplasmic cows we surveyed had one identical copy of mtDNA to that of the other cows in the same maternal lineages even after 11 generations, and the mtDNA seemed fairly stable at these hypervariable sites. We strongly suggest, therefore, that 76 multiple genetic markers should be used to calculate the rate of segregation and transmission of mtDNA. All transversions, insertions, and deletions that we found in D-loop and rRNA coding regions occurred in the homopolymer regions of mtDNA (Table 4). One instance of transition from cytosine to thymine also occurred at nt 364. The reference sequence (4) contains six sequential cytosines around nt 216, 12 cytosines around nt 364, five adenines around nt 1062, seven adenines around nt 1594, and four adenines around nt 1677. This finding suggests that the origin of sequence variation could have a common mechanism. The occurrence of small deletion or insertion mutations usually correlates with the presence of repeated bases in the DNA sequence. Streisringer et a/. (39) suggested that runs of repeated nucleotides can promote mutations by allowing relatively stable heteroduplexes to form when complementary DNA strands are misaligned. In this instance, such misalignment on the variable regions of D-loop and rRNA coding regions could occur via DNA strand slippage during the replication of the run of different nucleotides. Relatively stable duplexes resulting from such events would be generated if slippage occurred only within the run of homopolymeric nucleotides or if the misalignment extended into the nearby runs of other nucleotides. Following a subsequent cycle of DNA replication, a mutated mtDNA molecule with insertion, deletion, or transversion would be generated. Actually, Brown and DesRosier (40) found the variation of thymine runs in Rattus norvegiciis. In this study, the most common sites of deletions, insertions, and transversions in D-loop and rRNA coding regions are exclusively either within or at the end of homopolymer regions. Therefore, our data provide 77 additional evidence that replication slippage could be the mechanism to cause some of bovine mtDNA mutations. This research was supponed in part by BARD Research Project No. US-1519-88 of the United States - Israel Binational Agricultural Research and Development Fund of the United States Department of Agriculture and by 21st Century Genetics Cooperation. Journal Paper No. J-16072 of the Iowa Agriculture and Home Economics Experiment Station, Ames, lA, Project No. 3020. References 1. Hutchison, C. A., Newbold, J. E., Potter, S. S. & Edgell M. H. (1974) Nature (London) 251, 536-538. 2. Gray, M, W. (1989) Annu. Rev. Cell Biol. 5, 25-50. 3. Anderson, S., Bankier, A. T., Barrel, B. G., DeBruijn, M. H. L., Couison, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger F., Schreier, P. H., Smith, A. J. H., Staden, R. & Young, I. G. { 1 9 S \ ) N a t u r e ( L o n d o n ) 290, 457-465. 4. Anderson, S., DeBruijn, H. L., Couison, A. R., Drouin, J., Eperon, I. C., Sanger F. & Young I. G. (1982)./. Mol. Biol 156, 683-717. 5. Bibb, M. J., Van Etten, R. A., Wright, C. T., Walberg, M. 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M., George, Jr. M. & Wilson, A. C. (1979) Proc. Nail. Acad Sci. USA 76, 1967-1971. 18. Harrison, R. G., Rand, D. M. & Weeler, W. C. (1985) Science 228, 1446-1448. 19. Bermingham, E., Land, T. E. & Avise, J. C. (1986) J. Hered. 77, 249-252. 20. Snyder, M, Fraser, A. R., Lakoche, J., Gartner-Kepkay, K. E. & Zouros, E. (1987) Froc. Nail. Acad. Sci. [ISA 84, 7595-7599. 21. Monnerot, M., Mounolou, J. C. & Solignac, M. (1984) Bio/. Cell 52, 213-218. 22. Densmore, L. D., Wright, J. W. & Brown, W. M (1985) Genetic.s 110, 689-707. 23. Holts, I. J., Harding, A. E. & Morgan-Hughs, J. A. ( 1 9 8 S ) N a t u r e ( L o n d o n ) 331, 717719. 24. Ashley, M. V., Laipis, P. J. & Hauswirth, W. W. (1989) Nucleic Acid.s Res. 17, 73257331. 25. Koehler, C. M., Lindberg, G. L., Brown, D. R., Beitz, D. C., Freeman, A. E., Mayfield, J. E., & Myers, A. M. (1991) Genetic.n 129, 247-255. 26. Gyllensten, U., Warton, D., Josefsson, A., & Wilson, A. C. (1991) A'i7///re (London) 352, 255-257. 27. Brown, D. R., Koehler, C. M., Lindberg, G. L., Freeman, A. E., Mayfield, J. E., Myers, A. M., Schutz, M. M. & Beitz, D. C. (1989) J. Anini. Sci. 67, 1926-1932. 7') 28. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular (lo/iing: A laboratory niamial (New York: Cold Spring Harbor Laboratory Press), p. A. 1. 29. Solignac, M, Monnerot, M. & iVIounolou, J. C. (1983) Proc. Nail. Acad Sci. USA 80, 6942-6946. 30. Harrison, R. G., Rand, D. M. & Wheeler, W. C. { \ 9 S 1 ) M o l . B i o l . K v o l . 4, 144-158. 3 1. Solignac, M., Genermot, J., Monnerot, M & Mounolou, J. C. (1984)/V/o/. Gen. Genet. 197, 183-188. 32. Hausvvirth, VV. W., Van de Walie, M. J., Laipis, P. J. & Olive, P. D. (1984) Cell 31, 1001-1007. 33. Piko, L. & Matsumoto, L. (1976) Dev. Bid. 49, 1-10. 34. Hausvvirth, VV. VV. & Laipis, P. J. (1982) Proc. Nail. Acad. Sci. USA 79, 4686-4690. 35. Holt, L J., Harding, A. E. & Morgan-Hughes, J. A. (1988) Nature (Lottdon) 331, 717719. 36. Holt, L J., Harding, A. E., Petty, R. K. H. & Morgan-Hughes, J. A. (1990)/I/??. J. Hum. Genet. 46, 428- 433. 37. Holt, 38. Schoffner, J. M., Lott, M. T., Lezza, A. M. S., Seibel, P., Ballinger S. W. & Wallace, D. C. (1990) Cell 61, 931-937. 39. Strisinger, G., Okada, Y., Emrich, J., Newton, J., Tsugita, A., Terzaghi, E. & Inouye, M. (1966). Cold Sprii!}^ Harbor Symp. Oiiant. Biol. 31, 77-84. 40. Brown, G. G. & DesRosier, L. J. (1983). Nucleic Acid.s Res. 11, 6699-6708. J., Miller, D. H. & Harding, A. E. (1989) J. Med. Genet. 26, 739-743. 8(» SEQUENCE VARIATIONS IN tRNA AND PROTEIN CODING GENES OF BOVINE mtDNA A paper to be submitted to Genetics Jianming Wu, A. Eugene Freeman, Gary L. Lindberg, and Donald C. Beitz Abstract The tRNA and protein coding regions of bovine mtDNA, which includes genes of COIII, tRNA'^'^ ND3, tRNA '^s, ND4L, ND4, tRNA'"", tRNA'^^ and tRNA'"' and portion of ND5 and ATPase subunit 6 from 13 cows were cloned and sequenced. Four different nucleotide substitutions were detected in these regions. The first substitution was a transition from G to A at nt 12023 within the T-loop of the tRNA''^ coding region. Only one cow had a substitution at this site. The second substitution occurred at nt 8916 within the ATPase 6 gene; two cows had a C to T transition at this site. The third substitution was located at nt 10349 of ND4L gene where G was substituted by A. Two cows had this type of transition. The last substitution was a transversion at nt 9682 where G was substituted by C within the COIII gene. All 13 cows showed exact the same variation at nt 9682. The frequency of nucleotide substitutions in tRNA and protein coding region was much less than that in D-loop and rRNA coding regions that was reported previously. The transition substitution was the major type of mutation events in these tRNA and protein coding regions. The transitions occurred at the third nucleotide of the codon or in the T-loop region of 81 tRNA and thus will not change the amino acid encoded by the codon or the overall structure of tRNA. The transversion at nt 9682 from G to C is an exception because the conservative amino acid is alanine in other animal species. Therefore, the "true" sequence of bovine mtDNA in cattle population could be C at nt 9682, and the originally published sequence could have contained a rare substitution in that cow; alternatively, there could have been a sequence divergence between mtDNA of cows in this study and the originally published sequence. No deletions or insertions were observed in this study. No significant difference of fijnction would be expected from all different gene types of mtDNA within the tRNA and protein coding regions. Introduction The mammalian cell contains several thousand copies of a species-specific, circular mitochondrial genome that is replicated autonomously, transcribed within the organelle, and inherited maternally (Hutchison ct a/., 1974; Gray, 1989). Mitochondria maintain a complete protein-synthesizing system that is physically and genetically distinct fi'om the remainder of the c>1oplasmic system. Several components of the system, including rRNA, tRNA and mRNA, are encoded by mitochondrial DNA (mtDNA), whereas others are nuclear gene products and must be imported into the organelle. The majority of proteins present in mitochondria are encoded by nuclear genes, but a small number of proteins are encoded by mtDNA and translated on mitochondrial ribosomes (Chomyn el al., 1985; 1986). The mtDNA codes for seven subunits (NDl, 2, 3, 4L, 4, 5, and 6) of the respiratory complex I (NADH : ubiquinone oxido reductase), three subunits (COI, II, and III) of complex IV (cytochrome c oxidase), two subunits (ATPase 6 82 and 8) of complex V (ATP synthase), and one subunit (cyt b ) of complex III (ubiquinol: cytochrome c oxido reductase). In addition, the nitDNA encodes large and small rRNAs and a set of 22 tRNAs. Those mitochondrial translation products that have been identified are components of the inner mitochondrial membrane that functions in electron transport and oxidative phosphorylation. Bovine mtDNA is heterogeneous in the population of dairy cattle, with many differences being point mutations within the D-loop (Hauswirth and Laipis, 1982; Laipis et a/., 1982; Olivo et al., 1983; Watanabe cva/., 1985; Koehler, 1989; Lindberg, 1989; Smith 1993) and rRNA coding regions (Johnston, 1992; Wu t'/a/., 1995). This conclusion is derived from RFLP analysis and from sequencing of mtDNA with comparison to the published bovine mtDNA sequence (Anderson et al., 1982). This information allowed assignment of point mutations to the D-loop and rRNA coding regions. Mutations in the D-loop may affect the abundance of mtDNA gene transcripts subserving the ATP synthetic capacity or the size of the mtDNA population in the cell because the origins of replication for the H-strand and L-strand of mtDNA and the transcription start site for the H-strand are located in the D-loop region (Chang and Clayton, 1984). Mitochondrial fijnction can be affected also by small changes in the protein coding sequences of mtDNA. For example, yeast strains with mutations in mitochondrial genes that encode the components of electron transport have altered mitochondrial respiratory characteristics (Bunn et al., 1974). Studies carried out with genetically divergent animals have confirmed genetic influences on mitochondrial enzyme activities and oxidative phosphorylation. For example, the mitochondria with different mtDNA genotypes have different rates of oxidative 83 phosphorylation in Drosophila (McDaniel and Grimwood, 1971), rabbits (Dzapo el a/., 1973), sheep (Wolanis et al., 1980), chickens (Dzievviecki and Kolataj, 1980), mice (Brown et al., 1987; Lindberg et al., 1989), and swine (Dzapo and Wassmuth, 1983). It has been reported that a specific change in mtDNA conferred resistance to chloramphenicol in a mammalian cell line (Blanc et al., 1981). Recently, a variety of human diseases that involve the brain, heart, skeletal muscle, kidney, and endocrine glands have been attributed to mutations in mtDNA (Holt et al., 1988; Holt et al., 1990; Kobayashi et al., 1994; Hayashi et al., 1994; Pastores et al., 1994). The first pathologic mtDNA mutations identified were associated with rare human diseases such as Leber's optic neuropathy, myoclonic epilepsy and ragged-red fiber disease, and the KearnsSayre syndrome (Wallace, 1993). Therefore, nucleotide sequence differences in tRNA and protein coding genes of cattle could exert some effects on gene products and thus on production, reproduction, and health traits of animals. Hiendleder er a/. (1991) found three polymorphisms within the genes for NADH dehydrogenase subunit 5 and cytochrome b in sheep. Watanabe ei al. (1985) found seven polymorphic restriction sites in protein and tRNA coding regions of mtDNA from nine native Philippine cattle, but the effects of these polymorphisms are not clear. As for dairy cattle, the extent and the types of substitutions for tRNA and protein coding regions of mtDNA have not been documented at the nucleotide sequence level. Therefore, the purpose of this study was to investigate the extent and type of sequence variation of bovine mtDNA including certain tRNA and protein coding regions and to predict the effects of these nucleotide substitutions on gene fijnction and expression of mtDNA of dairy cows. 84 Materials and Methods Cows used in this experiment were from the Iowa State University breeding research herd that was established in 1968 from foundation cows that were purchased from 38 sources. Most cows were registered with the Holstein Association, and pedigree information was traceable to 1885. All cattle were traced to their earliest origins in the Holstein Herd book through maternal lineage pedigrees (Chenery, 1885). Approximately 400 ml of blood per animal was collected from selected cattle by jugular venipuncture and mixed with 4 ml of 0.5 M EDTA to prevent coagulation. Erythrocytes were lysed hypotonically with an equal volume of 10 mM KHCOj, 150 mM NH4CI, and 1 mM EDTA solution, and Ieukoc\tes were sedimented by centrifugation (Brown et ai, 1989). The leukocytes or white blood ceils were resuspended and lysed by the addition of Triton X-100. Nuclei were pelleted by centriftigation, and the remaining nucleic acids in the supernatant were purified by extraction once with chloroform ; isoamyl alcohol (24 ; 1; v : v) and twice with phenol : chloroform : isoamyl alcohol (25 : 24 : 1; v : v : v). Total nucleic acids were precipitated in two volumes of ethanol (Linderberg, 1989). Mitochondrial DNA was cut by Psil and EcoRl into five fragments (1.1 Kb, 3.3 Kb, 3.6 Kb, 3.9 Kb, and 4.3 Kb), and the 3.6 Kb fragment was isolated by preparative agarose gel electrophoresis. The 3.6 Kb fragment then was cloned into a phagemid vector (pBS SK+), which was used to transform XLl blue E. coli cells (Sambrook et al., 1989). Mitochondrial DNA was cut by restriction enzyme EcoRl into three fragments, and the digested mtDNA was separated by electrophoresis in 1% agarose gel overnight at 5 V/cm. The gel 85 slice containing the 4.8 Kb fragment was excised from the agarose gel, and mtDNA was recovered from the gel slice by using the QIAGEN DNA gel extraction protocol (QIAGEN Inc., Chatsworth, CA). The 4.8 Kb fragment then was cloned into the EcoRl cloning site within the cWoramphenicol-resistance gene of vector pACYC184 (ATCC 37033). E. coli DH5a was transformed, and transformants were selected on LB media containing 10 ug/ml chloramphenicol (Sambrook el ai, 1989). A 2.2 Kb mtDNA fragment was recovered from the recombinant plasmid by digestion with /'.v/1 and BaniHl. The 2.2 Kb fragment was purified from agarose gel by QIAGEN DNA extraction Kit (QIAGEN Inc., Chatsworth, CA) and subcloned into the multiple cloning site of pUC119 in E. coli strain TGI. The recombinant clones were screened first by white^lue selection on LB media that contained X-gal. Positive clones were confirmed by restriction enzyme digestion, and positive clones were analyzed fijrther by production of singlestranded DNA (ssDNA) and by DNA sequencing (Sambrook el al., 1989). Single-stranded DNA template was prepared by growth and coinfection of E. coli cells contairting recombinant phagemid with helper phage M13K07 according to the supplier's specification (Promega, Madison, \V1). The recombinant phage in the supernatant was precipitated by 20% polyethylene glycol. The ssDNA was purified from the phage pellet by two phenol extractions, two chloroform extractions, and precipitation with two volumes of ethanol. Singlestranded DNA template was annealed to the primers in the dideoxy chain-termination sequencing reaction by using [a-'^S] dATP (USB Sequenase Version 2.0 sequence kit, USB, Cleveland, OH). Fourteen oligonucleotide primers (primers 1 to 14) were used to obtain the DNA sequence of the first group of four cows, and eight primers (primers 7 to 14) were used to obtain DNA sequence of 86 the other nine cows. The sequences and locations of the primers on bovine mtDNA are listed in Table 1. Figure 1 shows the bovine mtDNA genetic map and the regions that have been sequenced in this experiment. Sequencing reactions were applied to 6% polyacrylamide gels of SEQUAGEL"" (National Diagnostics, Atlanta, GA). The gels were run for 4 to 6 hours at 1000 Table 1. H-strand sequencing primers used for sequencing the tRNA and protein coding region of bovine mtDNA. Primer Nucleotide number at 5' end" Sequence of primer 1 8996 5' ATGATAAGCATGAGTTTGGT 3' 2 9399 5' TAGAAACTCCGGAAGCCAAT 3' 3 9795 5' AGCTGATTGGAAGTCAGCTG 3' 4 10198 5' CATTTATTTTATTTTAAACT 3' 5 10399 5' GTAAAATGTGAGTTGAGGAT 3' 6 10599 5' CAAATTATATTATTTTTTGA3' 7 10798 5' GTTAGGTTTTCTTTTGATAG 3' 8 10996 5' GCCAGCTAGTGTATAGAATA 3' 9 11196 5' GCTTCTACGTGAGCTTTAGG 3' 10 11400 5' ACAGAGGAGTATGCGATGAG 3' 11 11609 5' GTAGTCATCAGGTGGCTATT 3' 12 11809 5' CGAGATATTATTAATGTGGT 3' 13 12008 5' GCATAGAATTAGCAGTTCTT 3' 14 12208 5' GTGGGTAGTTGGAAGGTTT 3' ^Nucleotide numbering according to Anderson et al. (1982). 87 D-loop V. Bovine mtDNA Figure 1. 00 Genetic map of bovine mitochondrial DNA. tRNA genes are represented by the single-letter amino acid code; NDl, ND2, ND3, ND4, ND4L, ND5, and ND6: subunits of NADH dehydrogenase; CO I, COII, and COIII: subunits of cytochrome c oxidase; ATPase 6 and ATPase 8: subunits of H^-ATPase. The DNA sequence betv/een nt 8783 to 12412 (numbering according to Anderson el ai, 1982) were sequenced in this study. 88 volts. Gels were fixed in water: acetic acid : methanol (18 : 1 : 1; v : v : v) for 15 minutes, dried, and exposed to X-ray film (Kodak X Omat AR) for 24 hours at room temperature. Ail sequences were determined once from reactions that were run in one direction. Results The cloned 3.6 Kb fragment includes cytochrome c oxidase subunit III (COIII), tRNA*^"''', NADH dehydrogenase subunit 3 (ND3), tRNA ''^ ND4L, ND4, tRNA' '", tRNA''" and tRNA'"" and part of ND5 and ATPase subunit 6 (Figure 1). Four nucleotide substitutions were found within this 3.6 Kb mtDNA fragment. Figure 2 shows a G-to-A transition at nt 12023 within the tRNA'^" gene as compared with the reference sequence (L-strand sequence numbering according to Anderson ef al., 1982), which was present in one out of four cows surveyed. Figure 3 shows the C-to-T transition at nt 8916 within ATPase subunit 6 and the C-to-T transition at nt 8916 was found in two of 13 cows. All 13 cows varied from the reference sequence by a C transition at nt 9682 within the COIII gene (Figure 4). Two of 13 cows had a G-to-A transition at nt 10349 within the ND4L gene (Figure 5) Overall, four substitutions were found in the 3 .6 Kb region of mtDNA from this study. Three of them were within the protein coding region, and one was within the tRNA^" gene. Three substitutions were transitions, and one was a transversion. No insertions and deletions were found in tRNA and protein coding genes in this study. 89 12023A Figure 2. Variation at nt 12023 within tRNA'^'"^ gene of mtDNA from dairy cattle. The sequence of L-strand is read from top to bottom with one set of G, A, T, and C lanes representing mtDNA sequence of one cow. G at nt 12023 is substituted by A within the tRNA'^"' gene of mtDNA from a cow indicated on the left side of the figure. 90 G A T C Figure 3. G A T C Variation at nt 8916 within ATPase subunit 6 gene of mtDNA from dairy cattle. The sequence of L-strand is read from top to bottom with one set of G, A, T, and C lanes representing mtDNA sequence of one cow. C at nt 8916 was replaced by T on the mtDNA from a cow indicated on the left side of the figure. 91 Figure 4. Variation at nt 9682 within COIII gene of mtDNA from dairy cattle. The sequence of L-strand is read from top to bottom with one set of G, A, T, and C lanes representing mtDNA sequence of one cow. Ail 13 cows surveyed had C instead of G at nt 9682, which is at the second base of amino acid codon where glycine was replaced by alanine in 13 cows. The figure shows that two cows had C at nt 9682, which is different from the published sequence by Anderson d al. (1982). 'J 2 G A T C G A T C G A T C 10349 A Figure 5. Variation at nt 10349 within ND4L gene of mtDNA from dairy cattle. The sequence of L-strand is read from top to bottom with one set of G, A, T, and C lanes representing mtDNA sequence of one cow. The G at nt 10349 is substituted by A within the ND4L gene of mtDNA from two cows indicated on the left side of the figure. The sequence of mtDNA from one cow, indicated on the right side of the figure, is the same as the reference sequence (Anderson et ai, 1982). 93 Discussion There are two types of tRNA^'"'' on the mtDNA genome. One type of tRNA^" contains a 5'-UGA-3' anticodon and recognizes the 5'-UCN-3' codon on mRNA (N represents A, U, C, or G). The other contains a 5'-GCU-3' anticodon recognizing the 5'-AGY-3' codon (Y represents U or C). The transition of tiie G to A occurred in the tRNA^" with the 5'-AGY-3' anticodon and was at the T-loop of the tRNA secondary structure. When comparing the sequence of the tRNA''" of bovine mtDNA with the corresponding one in humans, mice, and toads (Anderson et ai, 1981; Bibb ctaL, 1981; Roe e/o/., 1985), the corresponding site is occupied by an A in human and mouse and a C in toad (Table 2). Therefore, the G-to-A substitution in cattle conferred an evolutionary conservative base at this site. This comparison suggests that no effect would be Table 2. Comparison of T-loop sequences of tRNA^" gene of cattle and other vertebrate mitochondrial genomes. Source^ T-loop sequence of mitochondrial tRNA^'""' gene"" Human TCTAACAA Cattle TCTAATAG Mouse TTTAATAA Toad TTCAATTC " Based on the published sequences by Anderson etal. (1981), Anderson etal. (1982), Bibb etal. (1981), and Roe e/a/. (1985). '' The bold and underlined nucleotide stands for the comparative location where the nucleotide substitution was found in dairy cattle. 94 caused by this transition in cattle. Furthermore, no secondary structure changes could be expected because this nucleotide substitution was in the T-loop. Therefore, no immediate phenotypic effect could be expected trom this G-to-A substitution. The C-to-T substitution at nt 8916 of ATPase subunit 6 gene caused no change in the amino acid sequence. Isoleucine is coded by ATC, and C is at third position of the isoleucine codon in the reference sequence (Anderson ct a/., 1982). After the C is substituted by T, the resulting ATT still encodes for isoleucine. The isoleucine is within the conserved amino acid sequence of the carboxyl end of ATPase 6. This specific isoleucine is encoded by ATC in humans, toads, and cattle but by ATT in mice (Table 3). For many organisms, the pattern of codon usage in a gene may reflect the availability of the tRNAs that are used to decode the message for different Table 3. Conserved amino acid sequence at C-terminus of ATPase 6 among diflFerent animal species and codon for isoleucine within the region. Source' Amino acid sequence'' Codon mutation site'' Human AVAUQAYVFTLLVS ATC Cattle AVAMIQAYVFTLLVS ATC Mouse AVAUQAYVFTLLVS ATT Toad AVAMIQAYVFTLLVS ATC ' Based on the published sequences by Anderson ef al. (1981), Anderson et al. (1982), Bibb etal. (1981), and Roe el al. (1985). '' The bold and underlined amino acid stands for the comparative location where the nucleotide substitution has been found in dairy cattle. ' The bold and italicized letter represents where the nucleotide substitution that occurred in cattle is located. 95 genes; hence, the relative concentrations of the different tRNA species for the same amino acid could determine the basal level of expression of any given gene. Indeed, codon usage patterns for the same amino acid have been found to correlate with gene product abundance in bacteria (Grantham et a!., 1980). It is unlikely, however, that the expression of ATPase 6 relative to other mitochondrial genes is governed by codon usage and specifically of this isoleucine codon change, because in mammalian mitochondria only a single tRNA species is used to decode each codon family (Barrell et a!., 1980). Therefore, no change in the function and expression of the ATPase subunit 6 could be expected from this base substitution. In the mitochondrial system, methionine is encoded by two codons; one is ATG, the other is ATA, and there is no codon preference in the recognition and translation of mRNA by tRNA and other translational components. In this study, two cows had the substitution in the methionine codon at nt 10349 in the ND4L gene where G is replaced by A. The substitution of G by A at nt 10349 did not change the amino acid sequence, because the G is at the third base of the codon. Table 4 shows pan of the amino acid sequence in ND4L among different animal species and their respective codons at this site. For many animal species, the third nucleotide of the codon is A {e.g., humans, mice, and toads), but it is a G in Holstein cattle. The majority of the Holstein cattle (11 out of 13) retained a G at position 10349, which is the same sequence as published (Anderson et al., 1982). Only two cows had the substitution of A for G at this codon. The amino acid at this site could be highly divergent as long as they are nonpolar amino acids such as methionine in humans and cattle, valine in mice, and leucine in toads (Table 4). Therefore, the fijnction or the translation efficiency of the ND4L mRNA probably will be unaffected. This specific nucleotide 96 Table 4. The amino acid sequence within ND4L among different animal species and codon for the specific amino acid within the region. Source' Amino acid sequence*" Codon mutation site" Human LLCLEGMMLSLF AJA Cattle LLCLEGMMLSLF ATC; Mouse LLCLEGMVLSLF GTA Toad LLCLEGMLLMSM CIA Based on the published sequences by Anderson et at. (1981), Anderson e/ al. (1982), Bibb et al. (1981), and Roe et al. (1985). '' The bold and underlined amino acid stands for the comparative location where the nucleotide substitution has been found in dairy cattle. The bold and italicized letter represents where the nucleotide substitution (nt 10349) occurred in dairy cattle is located. is very conservative according to the sequence data from humans, mice, and toads. The mtDNA with substitution of G by A seems to be an old genotype in the view of evolution because the humans, mice, and toads all have an A at this specific site (Table 4). The mtDNA of all cattle in this study had C instead of G at nt 9682, Table 5 shows the conservative amino acid sequence of different animal species and their codons used for this site within the COIII gene. Except for cattle, the second nucleotide of the codon is C in humans, mice, and toads. In the published sequence (Anderson et a/., 1982), however, bovine mtDNA at nt 9682 (the second nucleotide of the codon) is G instead of C. This change from G to C is a transversion. According to data from 36 cows in another herd, transversions occurred invariably at the end or within homopolymer regions of mtDNA in the D-loop region and the rRNA coding regions (Wu 97 Table 5. The amino acid sequence within COIII among different species of animals and the codon for the specific amino acid within the region. Source"" Amino acid sequence'' Codon at mutation site'' Amino acid Human FGFEAAAWYWH GCC Ala Cattle FGFEAGAWWH GGT Gly Cattle'' FGFEAAAWYWH GCT Ala Mouse FGFEAAAWYWH GCA Ala Toad FGFEAA-WYWH GCA Ala Based on the published sequences by Anderson cf al. (1981), Anderson et al. (1982), Bibb et al. (1981), and Roe et al. (1985). The bold and underlined amino acid stands for the comparative location where the nucleotide substitution has been found in dairy cattle. " The bold and italicized letter represents where the nucleotide substitution (nt 9682) occurred in dairy cattle is located. Cattle in this study. e t a l , 1995). It is uncommon for a transversion to occur at the second nucleotide of the codon in the reading frame of a polypeptide and at the same time change the amino acid encoded (Anderson etai, 1981; 1982). Furthermore, the specific amino acid is occupied by alanine in humans, mice, and toads but glycine for cattle (Table 5). It seems that glycine in cattle is an unusual amino acid change on the basis of the amino acid sequences of other animal species (Table 5). The substitution of C for G in all the cows will revert the codon from one for glycine to one for alanine, and alanine is highly conserved among other animal species. Therefore, it is reasonable to consider that the "true" nucleotide occupied at this specific site should be a C instead of a G so that the ys amino acid can remain as alanine, vviiich is conserved highly among other animal species. Moreover, it is possible that Anderson c/ al. (1982) could have sequenced a cow with a rare mutation at this site; alternatively, there could have been a sequence divergence between mtDNA of the cows in this study and the originally published sequence. Several phagemid vectors (pGEM, pBluscript, and pUCl 19) were used for the cloning of the 3.6 Kb /^.v/I-Aa;RI fragment of bovine mtDNA, but cloning with these vectors was not very successful. Therefore, a lower copy number plasmid, pACYC 184, was used instead, and the 4.8 Kb /x'oRl fragment of mtDNA was cloned successfully into pACYC184. In this study, all the recombinant clones obtained with vector pACYC184 contained only one orientation of the £coRI insert. The opposite orientation might have resulted in toxic products killing the bacterial host or, alternatively, it might interfere with plasmid replication. Bacteria also could delete or rearrange the inserted harmflil foreign DNA. Others have reported similar problems with cloning of specific regions of mtDNA, especially protein coding regions. Drouin (1980) reported that several regions of human mtDNA were refractory to cloning in E. coli. Three of 23 Mhol fragments of human mtDNA were strikingly underrepresented in a collection of human mtDNA-pBR322 recombinant clones. One of these three fi'agments was detected only once among 705 recombinant clones. In another study. Tapper al al. (1983) reported that a 2.8 Kb 'fagl fragment could not be isolated as a stable insert in either of two Ml 3 vectors. Futterer el al. (1988) also reported that a region of the cauliflower mosaic virus genome was found to direct the expression of a nucleic acid-binding protein in E. coli. 99 but the protein was toxic and led to the destabilization of plasmids containing that region (Futterer eta!.^ 1988). Any substitution affecting tRNA secondary structure or anticodon flinction could affect protein translation of mtDNA genes that, in turn, will affect the chance of the organism for survival. Therefore, the neutral nucleotide substitution would occur normally at the loop regions of tRNA. Because of anticodon wobble during translation, most base changes that occur at the third position of a codon will not change the amino acid that it encodes. Therefore, more substitutions could be expected in protein coding regions than within those for tRNAs. In the conserved amino acid sequence of mitochondrially synthesized proteins, any critical amino acid change will affect the fijnction or activity of the enz\'me, but, in those nonconserved amino acid sequences, an amino acid change may have only a small effect on flinction. Two of the nucleotide substitutions occurred in conserved amino acid sequence regions; an amino acid change, however, did not occur because the nucleotide change occurred at the wobble position of the codon. In summary, four different nucleotide substitutions were found in the 3.6 Kb Rv/I-iTcoRI mtDNA fragment that includes COIII. tRNA°'- , ND3, tRNA'^-", ND4L, ND4, tRNA^'", tRNA^'" tRNA"'\ part of ND5, and part of ATPase subunit 6. One substitution was located in tRNA^" and the other three were located at ATPase 6, COIII, and ND4L, respectively. It is concluded that the frequency of nucleotide substitutions in tRNA and protein coding regions was much less than that observed in the D-loop region and rRNA coding regions (Lindberg, 1989; Wu el a/., 1995). The transition substitution was the major type of mutation in the tRNA and protein coding regions. Usually transitions occurred at the third nucleotide of the codon of mRNA, and therefore a change 100 in the amino acid did not occur. Tiie transversion at nt 96S2 from G to C was considered to be rare because the "true" sequence of bovine mtDNA in the germ line should be C when based on data from other animal species. No significant physiological differences among animals could be e.xpected from the different genotypes of mtDNA found in this study, and no insertions or deletions were observed in tRNA and protein coding regions of bovine mtDNA. References Anderson, S., Bankier, A. T., Barrel, B. G., DeBruijn, M. H. L., Coulson, A. R., Drouin, J., Eperon, I. C , Nierlich, D. P , Roe, B. A., Sanger F., Schreier, P. H., Smith, A. J. H., Staden, R., and Young I. G. (1981) Sequence and organization of the human mitochondrial genome. Nature 290: 457-465. Anderson, S., DeBruijn, H. L., Coulson, A. R., Drouin, J., Eperon, I. C,, Sanger F., and Young I. G. (1982). Complete sequence of bovine mitochondrial DNA. J. Mol. Biol. 156: 683-717. Barren, B. G., Anderson, S., Bankier, A. T., De Bruijn M. H. L., Chen, E,, Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger F,, Schreier, P. H., Smith, A, J. H., Staden, R., and Young 1. G. (1980). Different pattern of codon recognition by mammalian mitochondrial tRNAs. Proc. Natl. Acad. Sci. USA. 77: 3164-3166. Bibb, M. J., Van Etten, R. A., Wright, C. T., VValberg, M W., and Clayton D. A. (1981). Sequence and gene organization of mouse mitochondrial DNA. Cell 26:167-180. Blanc, H., Wright, C. T., Bibb, M. J., Wallace, D. C., and Clayton, D. A. (1981). Mitochondrial DNA of chloramphenicol-resistant mouse cells contains a single nucleotide change in the region encoding 3' end of large ribosomal RNA. Proc. Natl. Acad. Sci. USA. 78: 3789-3793. Brown, D. R., DeNise, S. K., and McDaniel, M. F. (1987). Phenotypic variation in respiratory metabolism and complementation of murine hepatic mitochondria. Theor. Appl. Genet. 75: 189-193. Brown, D. R., Koehler, C. iVI., Lindberg, G. L., Freeman, A. E., Mayfield, J. E., Myers, A. M., Schutz, M., and Beitz, D. C. (1989). Molecular analysis of cytoplasmic genetic variation in cows. J. Anim. Sci. 67: 1926-1932. 101 Bunn, C. L., Wallace, D. C., and Eisenstadt, J. M. (1974). Cytoplasmic inheritance of chloramphenicol resistance in mouse tissue culture cells. Proc. Natl. Acad. Sci. USA 71: 1681-1685. Chang. D. D., and Clayton, D. A. (1984). Precise identification of individual promoters for each strand of human mitochondrial DNA. Cell 36: 635-643. Chenery, W. W. (1885). Holstein cattle. Holstein Herd Book. 1:9-43. Chomyn, A., Cleeter, M. W. J., Ragan, C. I., Riley, M, Doolittle, R. F., and Attardi, G. (1986), URF6, last unidentified reading frame of human mtDNA, codes for an NADH dehydrogenase subunit. Science 234: 614-618. Chomyn, A., Mariottini, P., Cleeter, M. W. J., Ragan, C, I., Matsuno-Yagi, A., Hatefi, Y., Doolittle, R. P., and Attardi, G. (1985). Six unidentified reading frames of human mitochondrial DNA encode components of the respiratory chain NADH dehydrogenase. Nature 314: 592-597. Drouin, J. (1980). Cloning of human mitochondrial DNA in Escherichia coh. J. Mol. Biol. 140: 15-34. Dzapo, v., Reuter, H., and Wassmuth, R. (1973). Heterosis and mitochondrial complementation. Z. Tierzuchtg. Zuchtgbiol. 90: 169-180. Dzapp, v., and Wassmuth, R. (1983). Mitochondrial metabolism and heterotic effects in pigs. Results of a reciprocal crossbreeding experiment. 11. Activity of oxygen uptake of cells and oxidative phosphorylation in heart, liver and scrotal mitochondria, Z. Tierzuchtg. Zuchtgbiol. 100: 280-295. Dziewiecki, C. and Kolataj, A. (1980). Atmung von lebermitochondrien in anwesenheit von verschiedener substraten bei reirassinger hiihnern und ihren beiderseitigen kreuzungen. Z. Tierzuchtg. Zuchtgbiol. 97: 50-57. Futterer, J., Gordon, K., Pfeiffer, P., and Hohn, T. (1988). The instability of a recombinant plasmid, caused by a prokaryotic-like promoter within the eukaryotic insert, can be alleviated by expression of antisense RNA. Gene 67: 141-145. Grantham, R., Cautier, C., and Gouy, M. (1980). Codon frequencies in 119 individual genes confirm consistent choices of degenerate bases according to genome type. Nucl. Acids Res. 8: 1893-1912. Gray, ivl. W. (1989). Origin and evolution of mitochondrial DNA. Annu. Rev. Cell Biol. 5: 25-50. 102 Hausvvirth, W. VV., and Laipis, P. J. (1982). Mitochondrial DNA polymorphism in a maternal lineage of Holstein cows. Proc. Natl. Acad. Sci. USA. 79: 4686-4690. Hayashi, J.-l., Ohta, S., Kagavva, Y., Takai, D., Miyabayashi, S., Tada, K., Fukushima, H., Inui, K., Okada, S., Goto, Y., and Nonaka, 1. (1994). Functional and morphological abnormalities of mitochondria in human cell containing mitochondrial DNA with pathogenic point mutations in tRNA genes. J. Biol. Chem. 269: 19060-19066. Hiendleder, S., Hecht, \V , and Wassmuth, R. (1991). Restriction enzyme analysis of cytoplasmic genetic variation in sheep. J. Anim. Breeding Gen. 108: 290-298. Holt, I. J., Harding, A. E., and Morgan-Hughes, J. A. (1988). Deletions of muscle mitochondrial DNA in patient with mitochondrial myopathies. Nature 331: 717-719. Holt, I. J., Harding, A E., Petty, R. K. H., and Morgan-Hughes, J. A. (1990). A new mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am. J. Human Genet. 46: 428-433. Hutchison, C. A., Newbold, J. E., Potter, S. S., and Edgell M. H. (1974). Maternal inheritance of mammalian mitochondrial DNA. Nature 251: 536-538. Johnston, S. D. (1991). Sequence heterogeneity of bovine mitochondrial ribosomal RNA genes. M.S. Thesis, Iowa State University. Ames, lA. Kobayashi, Y., Sharpe, H., and Brown, N. (1994). Single-cell analysis of intercellular heteroplasmy of mtDNA in Leber hereditary optic neuropathy. Am. J. Human Genet. 55: 206-209. Koehler, C. M. (1989). Molecular characterization of bovine mitochondrial DNA. M.S. Thesis, Iowa State University. Ames, lA. Laipis, P. J., Van de Walle, M. J., and Hauswirth, VV. W. (1988). Unequal partitioning of bovine mitochondrial genotypes among siblings. Proc. Natl. Acad. Sci. USA. 85: 81078110. Lindberg, G. L. (1989). Sequence heterogeneity of bovine mitochondrial DNA. Ph.D. dissertation, Iowa State University, Ames, lA. Lindberg, G. L., Shank B. B., Rothschild M. P., Mayfield, J. E., Freeman, A. E., Koehler, C. M., and Beitz, D. C. (1989). Characteristics of mammary mitochondrial in lines of mice genetically divergent from milk production. J. Dairy Sci. 72: 1175-1191. McDaniel, R. G. and Grimwood, B. G. (1971). Hybrid vigor in drosophila: Respiration and mitochondrial energy conservation. Comp. Biochem. Physiol. 38B: 309-314. 103 Olivo, P. 0., Van cie Walle, M. J., Laipis, P. J., and Hauswirth, W. VV. (1983). Nucleotide sequence evidence for rapid genotypic shifts in the bovine mitochondrial DNA D-loop. Nature 306: 400-402. Pastores, G. M., Santorelli, F. M, Shanske, S., Gelb, B. D., Fyfe, B., Wolfe, D., and Willner, J. P. (1994). Leigh syndrome and hypertrophic cardiomyopathy in an infant with a mitochondrial DNA point mutation (T8993G). Am. J. Med. Genet. 50: 265-271. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press. Tapper, D. P., Van Etten, R. A., and Clayton, D. A. (1983). Isolation of mammalian mitochondrial DNA and RNA and cloning of the mitochondrial genome. Meth. Enzymol. 97: 426-434. Wallace, D. C. (1993). Mitochondrial diseases: Genotype versus phenotype. Tr. Genet. 9: 128-133. Watanabe, T., Hayashi, Y., Semba, R., Ogasawara, N. (1985). Bovine mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites. Biochem. Genet. 23: 947-957. Wu, J., Smith, R. K., Freeman, A, E., Lindberg, G. L., McDaniel, B. T., and Beitz, D. C. (1995). Sequence heteroplasmy of D-loop and rRNA coding regions in mitochondrial DNA from Holstein cows of independent maternal lineages. FASEB J. 9: A1041. 104 GENERAL SUMMARY Mammalian mitochondria carry multiple copies of double-stranded circular DNA (mtDNA) that is replicated and expressed within the organelles, and it is believed to be inherited maternally. The complete sequence of mtDNA has been determined in dairy cattle, humans, mice, and other species, and sequence variations have been detected within most of these species. Beyond its maternal inheritance and its rapid evolution, the segregation and transmission of different types of mtDNA in mammals are not clear. Furthermore, the extent and types of sequence variations in tRNA and protein coding regions of mtDNA are not documented in dairy cattle. The study of the genetics of bovine mtDNA and the effect and types of nucleotide variations in tRNA and protein coding regions will allow us to assess accurately the presence and evolution of different types of mtDNA in animal germ lines, the effects of different types of mtDNA on animal production traits, and the genetic basis of human mitochondrial diseases. Thirty-six Holstein cows from 18 maternal lineages (two cows from each maternal lineage) were used for the study of heteroplasmy and sequence variations in the D-loop and rRNA coding regions. By cloning and sequencing the mtDNA of cows from different maternal lineages, multiple heteroplasmic mtDNA states within the D-loop and rRNA coding regions of mtDNA were found to exist extensively in Holstein cows. By analyzing heteroplasmic cows in the same maternal lineages, the nature and transmission of mtDNA were investigated. The mtDNA population in a specific animal was found to be a mixture of 105 different genotypes. Because heteroplasmy was observed frequently and seemingly is persistent, selected amplification of specific mtDNA may not be a part of mtDNA inheritance. The random passage of different types of mtDNA from mother to progeny could account for the presence of multiple mtDNA heteroplasmy. At same time, four hypervariable sites were located at nt 169, 216, and 1594 and between nt 352 to 364, and over half of the cows studied possessed nucleotide substitutions at these sites. All transversions, insertions and deletions in the D-loop region and rRNA coding regions occurred at the end or within the homopolymer regions of mtDNA The data suggest that replication slippage could be a common cause of the transversion, insertion, and deletion. In another study, a 3.6 Kb fragment of mtDNA from 13 Holstein cows was cloned and sequenced. This fragment contained the tRNA and protein coding regions which includes genes of com, tRNA^'^ ND3, tRNA'"^, ND4L, ND4, tRNA^'^", tRNA^", and tRNA"'' and portions of ND5 and ATPase subunit 6. Four different nucleotide substitutions were detected in these regions. One substitution was a transition from G to A at nt 12023 within the T-loop of the tRNA^" coding region. Only one out of four cows had a substitution at this site. The second substitution occurred at nt 8916 within the ATPase 6 gene; two out of 13 cows had a C-to-T transition at this site. The third substitution was located at nt 10349 of the ND4L gene where G was substituted by A; two out of 13 cows had this type of transition. The last substitution was a transversion at nt 9682 where G was substituted by C within the COIII gene. All 13 cows showed exactly the same variation at this site. The frequency of nucleotide substitutions in tRNA and protein coding regions was found to be much less than that in the D-loop and rRNA coding regions. Transitions were the 106 major t^'pe of mutations in tRNA and protein coding regions. All transitions occurred at the third nucleotide of the codon or the T-loop region of tRNA, and thereby did not change the amino acid encoded by the codon or the general structure of the tRNA. The transversion at nt 9682 from G to C was considered to be an exception because the conservative amino acid is alanine as based on the data from other animal species. Therefore, the "true" sequence of bovine mtDNA in the germ line could be cytosine at nt 9682, and the original published sequence might be a rare substitution in that cow; alternatively, there could have been a sequence divergence between mtDNA of the cows in this study and the originally published sequence. No deletions or insertions were observed in tRNA and protein coding regions. No significant physiological differences could be expected from all the different genotypes of mtDNA. Some regions of mtDNA were refractory to cloning and the problem was solved by using a low copy number plasmid and bidirectionally cloning techniques. 107 ACKNOWLEDGMENTS I am greatly indebted to Dr. Don Beitz who gave me the chance to reunite with my wife and to study in the Nutritional Physiology Group. I really appreciate his help and guidance, and I am blessed to have him as my major professor. Dr. Gary Lindberg, my co-major professor, and Dr. Gene Freeman, my POS committee member, who have advised, counseled, supported, and encouraged me in doing the research with the bovine mtDN.^ I thank them very much for their efforts and kindness. I also want to express my thanks to Dr. Chris Minion and Dr. Alan Myers who served as my POS committee members; I have learned a lot from them. Thanks also go to Renotta Smith, Andrea Falk, Jenny Puis, and Betsy Swanson who worked with me. Thank them for their technical assistance. Finally, I would like to thank the whole Nutritional Physiology Group, which is a fun group of people to work with. I have enjoyed staying with them and learned a lot from them. My dear wife, Ling, deserves a separate paragraph of my thanks. I thank her very much for her bed-time suggestions, arguments, and encouragement. Without her support and encouragement, I don't know where I would be now.