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Cytology of Genetics Eukaryotes replication mitosis meiosis recombination chromosome variation number of sets number of chromosomes is a set chromosome modifications - Speciation due to chromosome modifications 1 Eukaryotes - cells with linear chromosomes in true nuclei bounded by nuclear envelopes and that undergo meiosis. DNA can be in three organelles in eukaryotic cells: nucleus - linear DNA, 96-98% of total DNA in the cell mitochondria - circular DNA, 1-2% of cellular DNA chloroplast - circular DNA, 1-2%of cellular DNA Nucleus 1. Nuclear membrane - has openings, ‘pores’, that allows passage of material such as nucleotides, enzymes into the nucleus and mRNA, tRNA, and rRNA out of the nucleus. 2 2. Nucleolus - dark staining body in the nucleus, region of high transcription of rRNA. 3. Chromatin - relaxed form of linear DNA found at interphase. Chromatin exists in two forms: euchromatin - stains lightly at interphase, regions of potential transcription. Heterochromatin - stains dark at interphase, regions of inactive or condensed DNA. 4. Chromosomes - condensed (tightly wound) DNA during mitosis and meiosis. The chromosome is actually a complex of DNA and proteins. There are two primary types of proteins: 1. basic histones 2. acidic 3 Histones - basic proteins rich in lysine and arginine. The positive charge allows the proteins to interact with the phosphate groups in the DNA. There are 5 types of histones: H1 - lysine rich H2A - slightly lysine rich H2B - slightly lysine rich H3 - arginine rich H4 - arginine rich The histone proteins H2A, H2B, H3, and H4 combine (2 copies of each) to form an octomer. DNA is coiled twice around an octomer in the nucleus. This gives the appearance of beads on a string. Each bead is called a nucleosome. 4 The nucleosomes facilitate condensing of the chromatin in interphase and to form chromosomes for mitosis and meiosis. Reason for condensing of the chromatin before cell division - to insure each daughter cell gets all the genetic information. First step in condensation is the connecting of the nucleosomes by histone H1. The next step is the coiling of the nucleosomes, further shortening the length of DNA. This coiled structure can exist during interphase. 5 The coiled DNA can be attached to proteins on the nuclear membrane during interphase forming loops. Prior to the start of either mitosis or meiosis the loops attach to a nonhistone (acidic) protein scaffolding. This further condenses the chromatin. The scaffolding can also have folds resulting in a densely packed DNA = chromosomes 6 Condensation of Chromatin to form Chromosomes DNA Diameter (nm) 2 Length 40 mm DNA + histones (nucleosomes) 10 - 11 8 mm Solenoid fibril 25 - 30 1 mm Looped domains 250 50 μm Radial loops + scaffold 850 4 μm 7 Chromosome shape 1. primary constriction - determines length of arms of the chromosome. It is also the site for attachment of the microtubules during mitosis or meiosis. Other names for the primary constriction are centromere or kinetochore. Primary constriction Based on the location of the primary constriction you can describe a chromosome as metacentric, acrocentric or telocentric. arms = 1 arm > other arm only one arm 8 2. Secondary constriction - on a few chromosomes there can be a second area that appears to be constricted. This is a region of late transcribing DNA that codes for rRNA genes. It is related to the nucleolus and is called the NOR or nucleolar organizing region. secondary constriction primary constriction 9 Cell Cycle stages G1 - growth stage for synthesis of products needed for DNA replication. S - DNA synthesis stage G2 - growth stage of products necessary for cell division. M - Mitosis or Meiosis Control of the cell cycle 10 Timing of the cell cycle is controlled by the presence and activity of specific genes and gene products. cdc genes cell division cycle genes MPF maturation promoting factors cdc2 protein + cyclin = MPF cyclin protein present during interphase that disappears at mitosis The presence of MPF triggers mitosis, the breakdown of the nuclear membrane, and cyclin degradation. The absence of cyclin allows mitosis to end. As mitosis ends cyclin and MPF are low 11 Summary of Control of the Cell Cycle Mitosis MPF cyclin As interphase progresses cyclin is synthesized and combines with cdc2 protein to produce inactive MPF. interphase cdc2 + cyclin = MPF protein inactive MPF is then modified to produce active MPF presence of active MPF induces mitosis mitosis (start) cyclin starts to be degraded loss of cyclin deactivates MPF mitosis (end) absence of cyclin allows mitosis to end 12 nuclear membrane starts to reform with the absence of MPF allowing the cell cycle to start again Mitosis - nuclear division with the resulting daughter cells having the same amount of DNA as the parent cell Four stages of mitosis 1. prophase 2. metaphase 3. anaphase 4. telophase 13 Meiosis - Two successive nuclear divisions that result in the reduction of the chromosome number to half that of the parental cell equational division N N 2N reductional division N N 2N somatic cell chromosome number N gametic cell chromosome number 14 Terminology for Meiosis Each chromosome in a cell has a duplicate based on gene location and order. Together these chromosomes are called homologous chromosomes What makes the chromosomes homologous is that they contain the same genes in the same linear sequence. The homologous chromosomes pair during meiosis. Chromosomes that do not pair at meiosis are called non-homologous chromosomes. 15 Meiosis can be divided into stages just like mitosis. Because of the events in the first division, prophase is divided into sub-stages. Reductional division 1. Prophase I a) leptotene b) zygotene c) pachytene d) diplotene e) diakinesis 2. Metaphase I 3. Anaphase I 4. Telophase I Interkinesis 16 Equational division 1. Prophase II 2. Metaphase II 3. Anaphase II 4. Telophase II 17 Prophase I a) Leptotene chromosomes begin to condense chromosome ends (telomeres) are attached to the nuclear membrane nucleolus is present sister chromatids are not visible. b) Zygotene chromosomes continue to condense pairing (synapsis) of the homologous chromosomes occurs. Paired chromosomes called a bivalent. synaptonemal complex starts to form nucleolus present 18 Prophase I c) Pachytene chromosomes continue to condense synaptonemal complex extends the length of the bivalent crossing-over between non-sister chromatids occurs sister chromatids begin to become visible nucleolus present 19 Prophase I d) Diplotene chromosomes continue to condense synaptonemal complex disappears centromeres become visible sites of crossing-over become visible. Called chiasmata - singular or chiasma - plural nucleolus starts to disappear 20 e) Diakinesis chromosomes continue to condense nucleolus disappears nuclear membrane disappears chiasma move to the ends of the bivalents Force of separation causes the tangled regions to move to the ends of the bivalent. classic ring bivalent 21 Metaphase I bivalents align on the metaphase plate. Anaphase I homologous chromosomes separate and move towards opposite sides of the cell. Telophase I homologous chromosomes reach their respective poles. Interkinesis the homologous chromosomes relax. This stage may or may not occur. 22 Equational division - similar to mitosis 1. Prophase II 2. Metaphase II 3. Anaphase II - separation and migration of sister chromatids 4. Telophase II The result of meiosis is the production of four cells with half (N) the number of chromosomes as the parental cell (2N) In the female only one of the four cells survives to be an egg cell (animals - ootid, plants - megaspore) 23 Post-meiotic mitotic divisions - additional cycles of mitosis that occur in the gametic cell after meiosis. In animals once meiosis has occurred additional cell divisions are not necessary to produce mature gametes. In plants additional mitotic divisions are necessary to produce mature gametes with the proper number of nuclei. males: require two additional mitotic divisions to produce 2 sperm nuclei and one generative nucleus. The generative nucleus directs the growth of the pollen tube. The two sperm nuclei are for the double fertilization of the egg and endosperm nuclei in the female. females: require three additional mitotic divisions to produce one egg nucleus, two polar nuclei, 2 synergid nuclei and 3 antipodal nuclei. 24 Eukaryotic Recombination 3 ways for recombination to occur: 1. Sorting of non-homologous chromosomes 2. Crossing-over between non-sister chromatids 3. Transposable elements 25 1. Sorting of non-homologous chromosomes The orientation of paternal and maternal chromosomes at metaphase I of meiosis is random. This results in the potential for different combinations of maternal and paternal chromosomes migrating to the centrioles. This results in the gametes having a unique assortment of genes from the mother and father This ‘mixing’ of genes by shuffling of nonhomologous chromosomes is the basis of Mendel’s law of independent assortment. 26 2. Crossing-over between non-sister chromatids - or recombination Prophase I: with synapsis of homologous chromosomes there is the potential for exchange of DNA between non-sister chromatids. For a difference to be observed the nonsister chromatids must vary slightly for the DNA sequence of a gene or genes. Crossing-over can occur between any of the non-sister chromatids. Crossing-over can occur at more than one site or between more than two non-sister chromatids. 27 - The Holliday model is the best for explaining how recombination occurs between non-sister chromatids. There are other models that have been proposed that address differences between what has been observed and what would be expected with the Holliday model. One such model is the Meselson-Radding model. 28 Transposable Elements First observed in maize 1938 - M. Rhodes 1950’s B. McClintock Observed that genes for certain mutations appeared to ‘move’. In fact the changes that were observed were due to pieces of DNA that could move and insert into specific DNA sequences. Insertion and disassociation caused altering of the DNA sequence of a gene resulting in the changing of expression of that gene. Transposable elements have now been identified in other eukaryotic systems (yeast, drosophila) and in prokaryotes (called transposons). 29 Characteristics of transposable elements A. Has regions of direct or inverted repeat DNA sequences. B. Region that may carry specific genes, possibly for movement of the element. Movement of transposable elements 30 1) insertion a) not necessary to have base complementarity with the gene where the transposable element will insert. The site of insertion may depend on a recognition sequence for the enzyme responsible for insertion. Insertion is initiated by a staggered cut in the target gene (endonuclease activity). b) After insertion the uneven ends are filled in resulting in direct repeat sequences flanking the transposable element. Migration of transposable elements 31 There are 3 different ways for transposable elements to move: 1) conservative transposition 2) replicative transposition 3) retro-transposition 32 Uses of transposable elements can be used to cause mutations problem - may be unstable autonomous element can move so would be unstable. non-autonomous element would not be capable of independent movement so would be stable unless another autonomous element was present in the genome. could be used to insert genes problem - would not know where the element would insert. 33 Chromosome variation Chromosome number among species Species 2N N human dog cat mouse fruit fly 46 78 38 40 8 23 39 19 20 4 barley corn tomato wheat potato 14 20 24 42 48 7 10 12 21 24 34 Chromosome number within specific tissues salivary glands in drosophila fruit fly have large polytene chromosomes that are the result of multiple chromosome divisions without cell division. Process is called endomitosis. The reason for the extra chromosome copies in the cell is to produce high levels of salivary proteins. Polytene chromosomes made it possible to see area of euchromatin and heterochromatin that corresponded to specific genes. Differential expression of genes could also be observed with these ‘large’ chromosomes. 35 Chromosomes with specific functions Sex chromosomes There are two types of chromosomes in a cell when considering sex determination: 1. sex chromosomes - carry genes that determine the sex of an individual. 2. autosomes - chromosomes that are not involved in sex determination. Example - humans 2N - 46 autosomes - 44 sex chromosomes - 2 36 In humans have X and Y chromosomes 2X = female = homogametic sex XY = male = heterogametic sex Because of this the female’s gametes will be 100% X while the male’s gametes are 50% X and 50% Y. So the male is the one who determines the sex of the progeny. in some species the male is homogametic and the female is heterogametic (turkeys, chickens) 37 What happens to the extra X chromosome in females? Good example of facultative heterochromatin in most cells one X is extremely condensed. This can be seen at interphase and is called a Barr body Females will have one Barr body Males will not have a Barr body In cats can tell that either X chromosome can condense. The way to do this is to have variation in a gene on the X chromosome. 38 Example - Coat color in tortoiseshell or calico cats. 1 X carries a gene for red coat color 1 X carries a gene for non-red color The color of a section of fur will depend on which X chromosome condensed during development. It also indicates that once an X is designated as a Barr body it remains a Barr body in that cell line. The result of random condensing of the X chromosomes is a female cat with a patchwork (red and non-red) coat. Why would you be surprised to see a male tortoiseshell or calico cat? 39 Chromosome modifications Euploidy - Changes in the number of sets of chromosomes. Aneuploidy - changes in the number of chromosomes in a set. Changes within a chromosome a) deletion b) duplication c) inversion d) translocation 40 Euploidy - change in the number of sets of chromosomes. If a species has more than two sets of chromosomes the organism is said to be a polyploid. 2 sets 3 sets 4 sets 6 sets diploid triploid tetraploid hexaploid To describe the number of chromosome sets in a species you use X to equal or represent one chromosome set. Humans - diploid 2N = 46 chromosomes N = 23 chromosomes 2X = 46 chromosomes X = 23 chromosomes - number of chromosomes in one set 41 Wheat - hexaploid 2N = 42 chromosomes N = 21 chromosomes 6X = 42 chromosomes X = 7 chromosomes 42 If all the sets are homologous, i.e. originating from the same species, then the species is called an autopolyploid. Examples of autopolyploids - potatoes, strawberries Advantages of an autopolyploid: - plant parts and fruits are larger - plant may be more vigorous Disadvantages of an autopolyploid: - problems in meiosis with chromosome pairing and even distribution of chromosomes. - low or no seed set Why is poor or no seed set not a problem with potatoes and strawberries? 43 Another way to have no seed set is to have an uneven number of chromosome sets in a polyploid. Example: banana, an autotriploid Possible origin: 1. multiple fertilization (not likely) 2. fertilization of an unreduced (2N) gamete 3. hybridization between a tetraploid and a diploid. 4X x 2X 3X 44 Problems in gamete formation and seed set are due to uneven chromosome pairing and division in the reduction division of meiosis. (i.e. how do you divided a trivalent equally?) Advantages of autotriploids plants - larger fruit no seeds oysters - no egg or sperm production 45 If the chromosome sets originated from more than one related species then the organism is said to be an allopolyploid. The chromosomes that are from related species but carry the same genes in relatively the same linear order but do not pair during meiosis are said to be homeologous chromosomes. Advantages of allopolyploids plants and seeds are larger more genetically diverse so able to adapt to more environments fertile because only bivalents are formed in meiosis because the homeologous chromosomes will not pair. Examples: wheat canola (rapeseed) 46 Example: wheat An allohexaploid with chromosome sets donated by 3 related species. All the donor species (AA, BB, DD) were diploids with a 2N number of 14. AA X BB AB double the number of chromosomes through endomitosis AABB now have allotetraploid (4X) AABB X DD ABD double the number of chromosomes by endomitosis AABBDD allohexaploid (6X) 2N = 42 N = 21 47 Chromosomes 1A, 1B, and 1D would be called homeologous chromosomes because they carry the genes for the same traits. Why are mules sterile? Example of an allodiploid Horse (HH) X Donkey (DD) Mule (HD) In meiosis would you have proper chromosome pairing and distribution? Chromosome changes 48 Changes in the number of chromosomes in a chromosome set will result in aneuploidy. With aneuploidy the addition or deletion of a chromosome may have a major or minor effect. Effect depends on the genes that are on the missing or additional chromosome and the tolerance of the species to aneuploidy. Mammals have a low tolerance while plants have a high tolerance, especially polyploids. Aneuploidy occurs due to non-disjunction of either the homologous chromosomes or the sister chromatids during meiosis. Non-disjunction during first or second division of meiosis 49 first division n+1 n+1 n+1 n-1 n-1 2n n-1 second division n+1 n n-1 n n 2n n Example of the addition of a chromosome 50 humans - 2N = 46 If one chromosome is added the condition is called trisomic with 2N+1 = 47 If a pair of homologous chromosomes are added the condition is called tetrasomic with 2N+2 = 48 If two non-homologous chromosomes are added the condition is called double trisomic with 2N+1+1 = 48 examples in humans trisomic for chromosome 21 - Down’s syndrome trisomic for a sex chromosome Klinefelter’s syndrome (XXY) Other examples of trisomy in plants include : 51 barley corn The presence of an extra chromosome can be useful in determining the chromosome location of a gene because the presence of the extra chromosome disrupts normal Mendelian segregation. Trisomics can also occur that are only partial chromosomes (acro-trisomic or telo-trisomic). The presence of an extra chromosome (trisomy) is observed more in diploid species than the deletion of a chromosome (monosomy) because diploids do not have the necessary gene redundancy to tolerate the loss of any genetic information. Example of the deletion of a chromosome 52 wheat - 2N = 42 If one chromosome is missing the plant is said to be monosomic with 2N-1 = 41. If a homologous pair of chromosomes is missing the plant is said to be nullisomic with 2N-2 = 40. If two non-homologous chromosomes are missing the plant is double monosomic with 2N-1-1 = 40. Example in humans Monosomic for a sex chromosome - Turner’s syndrome (X instead of XX) This form of aneuploidy in polyploid plants is useful for genetic analysis. Chromosome changes 53 changes within a chromosome a) deletions b) additions c) inversions d) translocations 54 Deletions Loss of a section of a chromosome. This loss could be from an end or from within the chromosome. Deletions can be caused by unequal crossing-over, by radiation or by movement of transposible elements. Can observe deletions in meiosis during prophase I if the individual is heterozygous for the deletion. 55 Duplications Increase in the number of copies of a gene or genes on a chromosome. Can be the result of uneven crossing-over. Example: Bar eye in drosophila. A physical change in the eye shape relates to the addition of a band or bands seen on polytene chromosomes. Polytene chromosome Eye shape phenotype 56 Inversions When the gene order in a section of a chromosome is reversed. Occurs when the chromosome is looped and breakage and reunion occurs but reunion is not to the original ends. 57 Two types of inversions can occur: a) paracentric inversion where the centromere is not involved in the inverted region. b) pericentric inversion where the centromere is involved in the inverted region. Problems can occur in meiosis if the individual is heterozygous (one normal one inverted chromosome) for the inversion. To get proper pairing of the chromosomes a loop structure must form. 58 The other requirement for a problem to occur is that a cross-over must occur within the inversion. If a cross-over occurs in the inverted region then duplications and deletions can occur resulting in non-viable gametes. The presence of the non-viable gametes makes the individual partially sterile. The type of duplication and deletion that may occur depends on whether it is a paracentric or pericentric inversion. Result of being heterozygous for a paracentric inversion. Anaphase I - dicentric bridge and acentric chromosome. End of Meiosis - two functional gametes and two non-functional gametes with deletions and possibly duplications. 59 Results of being heterozygous for a pericentric inversion. Anaphase I - no dicentric bridge, no acentric chromosome. End of meiosis - two functional gametes and two non-functional gametes with complementary duplications and deletions. 60 Translocations The transfer of a section of one chromosome to another chromosome. If an exchange occurs between two nonhomologous chromosomes you now have reciprocal translocations. 61 Only time that reciprocal translocations can be a problem is when an organism is heterozygous for the translocations. The individual will appear normal since there is no genetic information missing or duplicated. The problem occurs in meiosis when the homologous regions of the chromosomes attempt to pair in prophase I. Depending on how chromosomes separate at anaphase I, the resulting gametes could carry deletions and duplications of regions of the chromosomes involved in the translocations resulting in non-viable gametes. 62 When the chromosomes pair a cross-shaped structure can form. When they separate they can either separate in a adjacent formation or an alternate formation. Adjacent - open ring structure leads to nonviable gametes due to duplications and deletions. 63 Alternate - figure eight or twisted circle structure that leads to viable gametes because all the translocated chromosomes go to one pole and all the normal chromosomes go to the opposite pole. 64 Why do duplications and deletions result in non-viable gametes or if viable, result in a modified organism?, dose effect. Most systems depend on not only the presence of specific genes but also the proper level of expression of the gene. The absence or duplication of genes leads to abnormally low or high levels of expression of a gene product. Example in humans - translocation of chromosome 21 resulting in Down’s syndrome. 65 But, mate this individual with a person with normal chromosomes: 66 Speciation and Chromosome Modifications Inversions and Translocations can lead to speciation in two ways: 1. Creation of blocks of genes that are inherited as a unit due to suppression of recombination. - in a heterozygote for an inversion recombination will not be observed for genes within the inversion. 1. Increase in sterility of heterozygotes as the number of inversions and translocations increase. Example: Individual A x Individual B (3 inversions) (no inversions) F1 (heterozygous for 3 inversions) Level of fertility: .5 x .5 x .5 = .125 or 12.5% 67