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Genome Organization Genome Complete set of instructions for making an organism • master blueprints for all enzymes, cellular structures & activities an organism‘s complete set of DNA The total genetic information carried by a single set of chromosomes in a haploid nucleus Located in every nucleus of trillions of cells Consists of tightly coiled threads of DNA organized into chromosomes Typical viral genome DNA or RNA 4-200 genes Viral genomes Viral genomes: ssRNA, dsRNA, ssDNA, dsDNA, linear or circular Viruses with RNA genomes: • Almost all plant viruses and some bacterial and animal viruses • Genomes are rather small (a few thousand nucleotides) Viruses with DNA genomes (e.g. lambda = 48,502 bp): • Often a circular genome. Replicative form of viral genomes • all ssRNA viruses produce dsRNA molecules • many linear DNA molecules become circular Molecular weight and contour length: • duplex length per nucleotide = 3.4 Å • Mol. Weight per base pair = ~ 660 Procaryotic genomes Generally 1 circular chromosome (dsDNA) Usually without introns Relatively high gene density (~2500 genes per mm of E. coli DNA) Contour length of E.coli genome: 1.7 mm Often indigenous plasmids are present Typical Procaryotic genome one circular doublestranded DNA chromosome 500-12,000 genes often plasmid(s) Bacterial genomes: E. coli 4288 protein coding genes: • Average ORF 317 amino acids • Very compact: average distance between genes 118bp Numerous paralogous gene families: 38 – 45% of genes arisen through duplication Homologues: • H. influenzae (1130 of 1703) • Synechocystis (675 of 3168) • M. jannaschii (231 of 1738) • S. cerevisiae (254 of 5885) Easy problem Bacterial Gene-finding Dense Genomes Short intergenic regions Uninterrupted ORFs Conserved signals Abundant comparative information Plasmids Extra chromosomal circular DNAs -lactamase ori Found in bacteria, yeast and other fungi foreign gene Size varies form ~ 3,000 bp to 100,000 bp. Replicate autonomously (origin of replication) May contain resistance genes May be transferred from one bacterium to another May be transferred across kingdoms Multipcopy plasmids (~ up to 400 plasmids/per cell) Low copy plasmids (1 –2 copies per cell) Plasmids may be incompatible with each other Are used as vectors that could carry a foreign gene of interest (e.g. insulin) Agrobacterium tumefaciens Characteristics • Plant parasite that causes Crown Gall Disease • Encodes a large (~250kbp) plasmid called Tumorinducing (Ti) plasmid Portion of the Ti plasmid is transferred between bacterial cells and plant cells T-DNA (Tumor DNA) Agrobacterium tumefaciens T-DNA integrates stably into plant genome Single stranded T-DNA fragment is converted to dsDNA fragment by plant cell Then integrated into plant genome 2 x 23bp direct repeats play an important role in the excision and integration process Agrobacterium tumefaciens Tumor formation = hyperplasia Hormone imbalance Caused by A. tumefaciens • Lives in intercellular spaces of the plant • Plasmid contains genes responsible for the disease Part of plasmid is inserted into plant DNA Wound = entry point 10-14 days later, tumor forms Agrobacterium tumefaciens What is naturally encoded in T-DNA? • Enzymes for auxin and cytokinin synthesis Causing hormone imbalance tumor formation/undifferentiated callus Mutants in enzymes have been characterized • Opine synthesis genes (e.g. octopine or nopaline) Carbon and nitrogen source for A. tumefaciens growth Insertion genes • Virulence (vir) genes • Allow excision and integration into plant genome Ti plasmid of A. tumefaciens 1. Auxin, cytokinin, opine synthetic genes transferred to plant 2. Plant makes all 3 compounds 3. Auxins and cytokines cause gall formation 4. Opines provide unique carbon/nitrogen source only A. tumefaciens can use! Eucaryotic genomes Located on several chromosomes Relatively low gene density (50 genes per mm of DNA in humans) Contour length of DNA Carry organellar genome as well Typical eukaryotic genome 4-224, linear chromosomes 5,000 - 125,000 genes Fungal genomes: S. cerevisiae First completely sequenced eukaryote genome Very compact genome: • Short intergenic regions • Scarcity of introns • Lack of repetitive sequences Strong evidence of duplication: • Chromosome segments • Single genes Redundancy: non-essential genes provide selective advantage Human Genomes Human 50,000 genes X 2 kbp=100 Mbp Introns=300 Mbp? Regulatory regions=300 Mbp? •Only 5-10% of human genome codes for genes - function of other DNA (mostly repetitive sequences) unknown but it might serve structural or regulatory roles Plant genomes It contains three genomes The size of genomes is given in base pairs (bp) The size of genomes is species dependent The difference in the size of genome is mainly due to a different number of identical sequence of various size arranged in sequence The gene for ribosomal RNAs occur as repetitive sequence and together with the genes for some transfer RNAs in several thousand of copies Structural genes are present in only a few copies, sometimes just single copy. Structural genes encoding for structurally and functionally related proteins often form a gene family Genetic information is divided in the chromosome The DNA in the genome is replicated during the interphase of mitosis Size of the genome in plants and in human Genome Arabidopsis thaliana Zea mays Vicia faba Human Nucleus 70 Millions 3900 Millions 14500 Millions 2800 Millions Plastid 0.156 Millions 0.136 Millions 0.120 Millions Mitochondrion 0.370 Millions .570 Millions .290 Millions .017 Millions Plant genomes: Arabidopsis thaliana A dicotyledonous plant A weed growing at the roadside of central Europe It has only 2 x 5 chromosomes It is just 70 Mbp It has a life cycle of only 6 weeks A model plant for the investigation of plant function Contains 25,498 structural genes from 11,000 families The structural genes are present in only few copies sometimes just one protein Structural genes encoding for structurally and functionally related proteins often form a gene family Plant genomes: Arabidopsis thaliana Cross-phylum matches: • Vertebrates 12% • Bacteria / Archaea 10% • Fungi 8% 60% have no match in non-plant databases Evolution involved whole genome duplication followed by subsequent gene loss and extensive local gene duplications Global Increase in Genome Size Polyploidization (whole genome duplication): Allopolyploidy: combination of genetically distinct chromosome sets. (Wheat…) Autopolyploidy: multiplication of one basic set of chromosomes. (Goldfish, rose…) Regional duplication Repetitive Structure of Eukaryotic Genome Eukaryotic genomes contain various degrees of repetitive structure: satellites, micro/minisatellites, retrotransposons, retrovirus, etc. Repetitive sequence size correlates with genome size: Heterochromatin (*109bp) Gorrila gorilla Symphalangus syndactylus Pan troglodites Homo sapiens Hylobates muelleri Genome size (*109bp) Mechanisms for Regional Increase in Genome Size Duplicative transposition Unequal crossing-over Replication slippage Gene amplification (rolling circle replication) Gene Duplication duplication of a part of the gene: domain/internal sequence duplication enhance function, novel function by new combination duplication of a complete gene (gene family) invariant duplication: dose repetitions, variant duplication: new functions. duplication of a cluster of genes Internal Gene Duplication 5’ 1 2 3 4 5 3’ 6 Ancestral trypsinogen gene Deletion 6’ 1 5’ 3’ Thr Ala Ala Gly 4 fold duplication + addition of spacer sequence 6’ 1 5’ Internal duplications + addition of intron sequence 5’ 1 1 2 3 4 5 6 3’ Spacer: Gly 7 … 37 38 Antifreeze glycoprotein gene 39 40 41 6’ 3’ Complete Gene Duplication Invariant duplication: RNA specifying genes: Number of tRNA and rRNA correlates with genome size. Variant duplication: X-linked autosomal Trichromatic Human female Trichromatic Human male Human male (color blind) New world monkey female or or Dichromatic Trichromatic New world monkey female New world monkey male Dichromatic or Dichromatic Gene Loss Duplicated genes unprocessed pseudogenes. Single-copy genes devoid of selection pressure unitary pseudogenes. Loss of L-gulono--lactone oxidase in humans, guinea pigs, etc. comparing to other vertebrates: the enzyme at the terminal step of synthesizing L-ascorbic acid (vitamin C). Genome organization Protein Coding Gene A segment of DNA which encodes protein synthesis DNA sequence encoding protein Gene classification coding genes Chromosome (simplified) intergenic non-coding region genes Messenger RNA Structural RNA Proteins transfer RNA Structural proteins Enzymes ribosomal RNA other RNA Coding region Nucleotides (open reading frame) encoding the amino acid sequence of a protein The molecular definition of gene includes more than just the coding region Noncoding regions Regulatory regions • RNA polymerase binding site • Transcription factor binding sites Introns Polyadenylation [poly(A)] sites Eukaryotic genes Most have introns Produce monocistronic mRNA: only one encoded protein Large Appearance of genomes One to many chromosomes Repeat sequences common in some genomes e.g. 35% of human are transposable elements 10% Alu, 14.6% LINE1 sequences Gene structure varies – no. and length of introns What does 50 kb of sequence look like? repeat Pseudogene Intron-exon components of a gene Human – very few genes - repeats Yeast – many genes (~25) – few repeats Maize – mostly repeats What do the genes encode? Microbes highly specialized Basic functions + Yeast – simplest eukaryote Fly – complex development Genes for basic cellular functions such as translation, transcription, replication and repair share similarity among all organisms Worm – programmed development Arabidopsis – plant life cycle Gene families expand to meet biological needs. Repetitive DNA Moderately repeated DNA • Tandemly repeated rRNA, tRNA and histone genes (gene products needed in high amounts) • Large duplicated gene families • Mobile DNA Simple-sequence DNA • Tandemly repeated short sequences • Found in centromeres and telomeres (and others) • Used in DNA fingerprinting to identify individuals Types of DNA repeats Tandem repeats (e.g. satellite DNA) 5’-CATGTGCTGAAGGCTATGTGCTGCGACG- 3’ 3’-GTACACGACTTCCGATACACGACGCTGC- 5’ Inverted repeats (e.g. in transposons) 5’-CATGTGCTGAAGGCTCAGCACATCGACG- 3’ 3’-GTACACGACTTCCGAGTCGTGTAGCTGC- 5’ • Form stem-loop structures Palindroms = adjacent inverted repeats (e.g. restriction sites) • Form hairpin structures Loop Stem Hairpin Repetitive sequences Satellite DNA Chromosomal DNA Repeats in the mouse genome Caesium chloride density gradient Type No. of Repeats Size Percent of genome Highly repetitive Moderately repetitive > 1 Mill < 10 bp 10 % > 1000 ~ 150 - ~300 bp 20 % Mobile DNA Move within genomes Most of moderately repeated DNA sequences found throughout higher eukaryotic genomes • L1 LINE is ~5% of human DNA (~50,000 copies) • Alu is ~5% of human DNA (>500,000 copies) Some encode enzymes that catalyze movement Transposition Movement of mobile DNA Involves copying of mobile DNA element and insertion into new site in genome Why? Molecular parasite: “selfish DNA” Probably have significant effect on evolution by facilitating gene duplication, which provides the fuel for evolution, and exon shuffling RNA or DNA intermediate Transposon moves using DNA intermediate Retrotransposon moves using RNA intermediate Types of mobile DNA elements LTR (long terminal repeat) Flank viral retrotransposons and retroviruses Contain regulatory sequences Transcription start site and poly (A) site LINES and SINES Non-viral retro-transposons • RNA intermediate • Lack LTR LINES (long interspersed elements) • ~6000 to 7000 base pairs • L1 LINE (~5% of human DNA) • Encode enzymes that catalyze movement SINES (short interspersed elements) • ~300 base pairs • Alu (~5% of human DNA) Mitochondrial genome (mtDNA) Number of mitochondria in plants can be between 502000 One mitochondria consists of 1 – 100 genomes (multiple identical circular chromosomes. They are one large and several smaller Size ~15 Kb in animals Size ~ 200 kb to 2,500 kb in plants Mt DNA is replicated before or during mitosis Transcription of mtDNA yielded an mRNA which did not contain the correct information for the protein to be synthesized. RNA editing is existed in plant mitochondria Over 95% of mitochondrial proteins are encoded in the nuclear genome. Often A+T rich genomes Chloroplast genome (ctDNA) Multiple circular molecules, similar to procaryotic cyanobacteria, although much smaller (0.001-0.1%of the size of nuclear genomes) Cells contain many copies of plastids and each plastid contains many genome copies Size ranges from 120 kb to 160 kb Plastid genome has changed very little during evolution. Though two plants are very distantly related, their genomes are rather similar in gene composition and arrangement Some of plastid genomes contain introns Many chloroplast proteins are encoded in the nucleus (separate signal sequence) The family of plastids Buchannan et al. Fig. 1.44 Endosymbiosis Well accepted that chloroplasts and mitochondria were once free living bacteria Their metabolism is bacterial (e.g. photosynthesis) Retain some DNA (circular chromosome) • Protein synthesis sensitive to chloramphenicol • Cytosolic P synthesis sensitive to cycloheximide Most genes transferred from symbiont to nucleus • Requires protein tageting DNA for chloroplast proteins can be in the nucleus or chloroplast genome Buchannan et al. Fig. 4.4 Import of proteins into chloroplasts Buchannan et al. Fig. 4.6 Biochemistry inside plastids Photosynthesis – reduction of C, N, and S Amino acids, essential amino acid synthesis restricted to plastids • Phenylpropanoid amino acids and secondary compounds start in the plastids (shikimic acid pathway) • Site of action of several herbicides, including glyphosate • Branched-chain amino acids • Sulfur amino acids Fatty acids – all fatty acids in plants made in plastids “Cellular” Genomes Viruses Procaryotes Eucaryotes Nucleus Capsid Plasmids Viral genome Bacterial chromosome Chromosomes (Nuclear genome) Mitochondrial genome Chloroplast genome Genome: all of an organism’s genes plus intergenic DNA Intergenic DNA = DNA between genes Methods of regulation Gene expression • Normally slow relative to metabolic control that will be discussed most of the time in this course • Allows metabolism to be changed in response to environmental factors • Transcriptional control most common Sometimes variation in transcription rate not reflected in enzyme amount • Translational control also found No change in mRNA levels but changes in protein amounts Gene structure relevant to metabolic regulation Promoters Exploring metabolism by genetic methods Antisense – what happens when the amount of an enzyme is reduced • not clear how antisense works Knockouts • Often more clear-cut since all of the enzyme is gone • Use of t-DNA, Salk lines Overexpression • Use an unregulated version of the protein or express on a strong promoter • Sometimes leads to cosuppression RNA interference • 21 to 26 mers seem very effective in regulating translation