2. Genome  Complete set of instructions for making an organism master blueprints for all enzymes, cellular structures & activitiesmaster 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

3. 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

4. Bacterial genomes: E. coli 4288 protein coding genes: 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 Numerous paralogous gene families: 38 – 45% of genes arisen through duplication Homologues: Homologues: H. influenzae (1130 of 1703) Synechocystis (675 of 3168) M. jannaschii (231 of 1738) S. cerevisiae (254 of 5885)

5. Procaryotic genomes Generally 1 circular chromosome (dsDNA) Generally 1 circular chromosome (dsDNA) Usually without introns Usually without introns Relatively high gene density (~2500 genes per mm of E. coli DNA) Relatively high gene density (~2500 genes per mm of E. coli DNA) Contour length of E.coli genome: 1.7 mm Contour length of E.coli genome: 1.7 mm Often indigenous plasmids are present Often indigenous plasmids are present

6. Easy problem Bacterial Gene-finding Dense Genomes Dense Genomes Short intergenic regions Short intergenic regions Uninterrupted ORFs Uninterrupted ORFs Conserved signals Conserved signals Abundant comparative information Abundant comparative information Complete Genomes Complete Genomes

7. Genomes Gene Content E. coli 4000 genes X 1 kbp/gene=4 Mbp Genome=4 Mbp !

8. Plasmids Extra chromosomal circular DNAs Found in bacteria, yeast and other fungi Found in bacteria, yeast and other fungi Size varies form ~ 3,000 bp to 100,000 bp. Size varies form ~ 3,000 bp to 100,000 bp. Replicate autonomously (origin of replication) Replicate autonomously (origin of replication) May contain resistance genes May contain resistance genes May be transferred from one bacterium to another May be transferred from one bacterium to another May be transferred across kingdoms May be transferred across kingdoms Multipcopy plasmids (~ up to 400 plasmids/per cell) Multipcopy plasmids (~ up to 400 plasmids/per cell) Low copy plasmids (1 –2 copies per cell) Low copy plasmids (1 –2 copies per cell) Plasmids may be incompatible with each other Plasmids may be incompatible with each other Are used as vectors that could carry a foreign gene of interest (e.g. insulin) Are used as vectors that could carry a foreign gene of interest (e.g. insulin)  -lactamase ori foreign gene

9. Agrobacterium tumefaciens Characteristics Characteristics Plant parasite that causes Crown Gall DiseasePlant parasite that causes Crown Gall Disease Encodes a large (~250kbp) plasmid called Tumor- inducing (Ti) plasmidEncodes a large (~250kbp) plasmid called Tumor- inducing (Ti) plasmid Portion of the Ti plasmid is transferred between bacterial cells and plant cells  T-DNA (Tumor DNA ) Portion of the Ti plasmid is transferred between bacterial cells and plant cells  T-DNA (Tumor DNA )

10. Agrobacterium tumefaciens T-DNA integrates stably into plant genome T-DNA integrates stably into plant genome Single stranded T-DNA fragment is converted to dsDNA fragment by plant cell Single stranded T-DNA fragment is converted to dsDNA fragment by plant cell Then integrated into plant genome Then integrated into plant genome 2 x 23bp direct repeats play an important role in the excision and integration process 2 x 23bp direct repeats play an important role in the excision and integration process

11. Agrobacterium tumefaciens Tumor formation = hyperplasia Tumor formation = hyperplasia Hormone imbalance Hormone imbalance Caused by A. tumefaciens Caused by A. tumefaciens Lives in intercellular spaces of the plantLives in intercellular spaces of the plant Plasmid contains genes responsible for the diseasePlasmid contains genes responsible for the disease Part of plasmid is inserted into plant DNA Part of plasmid is inserted into plant DNA Wound = entry point  10-14 days later, tumor forms Wound = entry point  10-14 days later, tumor forms

12. Agrobacterium tumefaciens What is naturally encoded in T-DNA? What is naturally encoded in T-DNA? Enzymes for auxin and cytokinin synthesisEnzymes for auxin and cytokinin synthesis Causing hormone imbalance  tumor formation/undifferentiated callus Causing hormone imbalance  tumor formation/undifferentiated callus Mutants in enzymes have been characterized Mutants in enzymes have been characterized Opine synthesis genes (e.g. octopine or nopaline)Opine synthesis genes (e.g. octopine or nopaline) Carbon and nitrogen source for A. tumefaciens growth Carbon and nitrogen source for A. tumefaciens growth Insertion genes Insertion genes Virulence (vir) genesVirulence (vir) genes Allow excision and integration into plant genomeAllow excision and integration into plant genome

13. Ti plasmid of A. tumefaciens

15. 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!

16. Fungal genomes: S. cerevisiae First completely sequenced eukaryote genome First completely sequenced eukaryote genome Very compact genome: Very compact genome: Short intergenic regions Scarcity of introns Lack of repetitive sequences Strong evidence of duplication: Strong evidence of duplication: Chromosome segments Single genes Redundancy: non-essential genes provide selective advantage Redundancy: non-essential genes provide selective advantage

17. Eucaryotic genomes Located on several chromosomes Located on several chromosomes Relatively low gene density (50 genes per mm of DNA in humans) Relatively low gene density (50 genes per mm of DNA in humans) Contour length of DNA Contour length of DNA Carry organellar genome as well Carry organellar genome as well

18. 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 2300 Mbp=???

19. Plant genomes It contains three genomes It contains three genomes The size of genomes is given in base pairs (bp) The size of genomes is given in base pairs (bp) The size of genomes is species dependent 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 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 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 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 Genetic information is divided in the chromosome The DNA in the genome is replicated during the interphase of mitosis The DNA in the genome is replicated during the interphase of mitosis

20. 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

21. Plant genomes: Arabidopsis thaliana A weed growing at the roadside of central Europe A weed growing at the roadside of central Europe It has only 2 x 5 chromosomes It has only 2 x 5 chromosomes It is just 70 Mbp It is just 70 Mbp It has a life cycle of only 6 weeks It has a life cycle of only 6 weeks A model plant for the investigation of plant function A model plant for the investigation of plant function Contains 25,498 structural genes from 11,000 families Contains 25,498 structural genes from 11,000 families The structural genes are present in only few copies sometimes just one protein 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 Structural genes encoding for structurally and functionally related proteins often form a gene family

22. Plant genomes: Arabidopsis thaliana Cross-phylum matches: Cross-phylum matches: Vertebrates 12% Bacteria / Archaea 10% Fungi 8% 60% have no match in non-plant databases 60% have no match in non-plant databases Evolution involved whole genome duplication followed by subsequent gene loss and extensive local gene duplications Evolution involved whole genome duplication followed by subsequent gene loss and extensive local gene duplications

23. Complex Genome DNA ~10% highly repetitive (300 Mbp) ~10% highly repetitive (300 Mbp) NOT GENESNOT GENES ~25% moderate repetitive (750 Mbp) ~25% moderate repetitive (750 Mbp) Some genesSome genes ~25% exons and introns (800 Mbp) ~25% exons and introns (800 Mbp) 40%=? 40%=? Regulatory regionsRegulatory regions Intergenic regionsIntergenic regions

24. Genome organization

25. “Nonfunctional” DNA Higher eukaryotes have a lot of noncoding DNA Higher eukaryotes have a lot of noncoding DNA Some has no known structural or regulatory function (no genes) Some has no known structural or regulatory function (no genes) 80 kb

26. Duplicated genes Encode closely related (homologous) proteins Encode closely related (homologous) proteins Clustered together in genome Clustered together in genome Formed by duplication of an ancestral gene followed by mutation Formed by duplication of an ancestral gene followed by mutation Five functional genes and two pseudogenes

27. Pseudogenes Nonfunctional copies of genes Nonfunctional copies of genes Formed by duplication of ancestral gene, or reverse transcription (and integration) Formed by duplication of ancestral gene, or reverse transcription (and integration) Not expressed due to mutations that produce a stop codon (nonsense or frameshift) or prevent mRNA processing, or due to lack of regulatory sequences Not expressed due to mutations that produce a stop codon (nonsense or frameshift) or prevent mRNA processing, or due to lack of regulatory sequences

28. Repetitive DNA Moderately repeated DNA Moderately repeated DNA Tandemly repeated rRNA, tRNA and histone genes (gene products needed in high amounts)Tandemly repeated rRNA, tRNA and histone genes (gene products needed in high amounts) Large duplicated gene familiesLarge duplicated gene families Mobile DNAMobile DNA Simple-sequence DNA Simple-sequence DNA Tandemly repeated short sequencesTandemly repeated short sequences Found in centromeres and telomeres (and others)Found in centromeres and telomeres (and others) Used in DNA fingerprinting to identify individualsUsed in DNA fingerprinting to identify individuals

29. Mobile DNA Move within genomes Move within genomes Most of moderately repeated DNA sequences found throughout higher eukaryotic genomes Most of moderately repeated DNA sequences found throughout higher eukaryotic genomes L1 LINE is ~5% of human DNA (~50,000 copies)L1 LINE is ~5% of human DNA (~50,000 copies) Alu is ~5% of human DNA (>500,000 copies)Alu is ~5% of human DNA (>500,000 copies) Some encode enzymes that catalyze movement Some encode enzymes that catalyze movement

30. Transposition Movement of mobile DNA Movement of mobile DNA Involves copying of mobile DNA element and insertion into new site in genome Involves copying of mobile DNA element and insertion into new site in genome

31. Why? Molecular parasite: “selfish DNA” Molecular parasite: “selfish DNA” Probably have significant effect on evolution by facilitating gene duplication, which provides the fuel for evolution, and exon shuffling Probably have significant effect on evolution by facilitating gene duplication, which provides the fuel for evolution, and exon shuffling

32. Mitochondrial genome (mtDNA) Number of mitochondria in plants can be between 50- 2000 Number of mitochondria in plants can be between 50- 2000 One mitochondria consists of 1 – 100 genomes (multiple identical circular chromosomes. They are one large and several smaller One mitochondria consists of 1 – 100 genomes (multiple identical circular chromosomes. They are one large and several smaller Size ~15 Kb in animals Size ~15 Kb in animals Size ~ 200 kb to 2,500 kb in plants Size ~ 200 kb to 2,500 kb in plants Mt DNA is replicated before or during mitosis 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 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. Over 95% of mitochondrial proteins are encoded in the nuclear genome. Often A+T rich genomes Often A+T rich genomes

33. Chloroplast genome (ctDNA) Multiple circular molecules, similar to procaryotic cyanobacteria, although much smaller (0.001-0.1%of the size of nuclear genomes) 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 Cells contain many copies of plastids and each plastid contains many genome copies Size ranges from 120 kb to 160 kb 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 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 Some of plastid genomes contain introns Many chloroplast proteins are encoded in the nucleus (separate signal sequence) Many chloroplast proteins are encoded in the nucleus (separate signal sequence)

34. “Cellular” Genomes VirusesProcaryotesEucaryotes Viral genome Bacterial chromosome Plasmids Chromosomes (Nuclear genome) Mitochondrial genome Chloroplast genome Genome: all of an organism’s genes plus intergenic DNA Intergenic DNA = DNA between genes Capsid Nucleus

35. Estimated genome sizes 1e1 1e2 1e3 1e4 1e5 1e6 1e7 1e8 1e9 1e10 1e11 1e12 viruses (1024) bacteria (>100) fungi mitochondria (~ 100) plants mammals Size in nucleotides. Number in ( ) = completely sequenced genomes

36. What Did These Individuals Contribute to Molecular Genetics? Anton van Leeuwenhoek Anton van Leeuwenhoek Discovered cells Discovered cells BacteriaBacteria ProtistsProtists Red bloodRed blood

37. Gregor Johan Mendel Gregor Johan Mendel Discovered genetics Discovered genetics What Did These Individuals Contribute to Molecular Genetics?

38. Walter Sutton Walter Sutton Discovered Chromosomes Discovered Chromosomes What Did These Individuals Contribute to Molecular Genetics?

39. Thomas Hunt Morgan Thomas Hunt Morgan Discovered how genes are transmitted through chromosomes Discovered how genes are transmitted through chromosomes What Did These Individuals Contribute to Molecular Genetics?

40. Rosalind Elsie Franklin Rosalind Elsie Franklin Research led to the discovery of the double helix structure of DNA Research led to the discovery of the double helix structure of DNA What Did These Individuals Contribute to Molecular Genetics?

41. James Watson and Francis Crick James Watson and Francis Crick Discovered DNA Discovered DNA What Did These Individuals Contribute to Molecular Genetics?

42. DNA’s History DNA’s History 1866 Gregore Mendel Law of Heredity Law of Heredity 1900 Carl Correns, Hugo de Vries& Eric von Tschermak Mendelian Law re-invention 1944 Avery, Macleod & McCarty Gene consists of DNA 1952 Hersey dan Chase DNA as genetic matarials 1953 Watson & Crick Double helix DNA 1971 Cohen & Boyer Transformation Technology 1972Berg DNA Recombinant Technology 1973 Arber, smith & Nathans Restriction Enzyme

43. Chromosome parts Chromatid Chromatid sister strands aftersister strands afterreplication still joined at centromerestill joined at centromere Centromere Centromere ~ “middle” of Chromosomes ~ “middle” of Chromosomes spindle attachment sitesspindle attachment sites Telomeres Telomeres ends of chrmends of chrm important for the stability of chromosomes tips.important for the stability of chromosomes tips.

44. Chromosomal Regions  Heterochromatin  compact;  few genes;  largely structural role  Euchromatin  contains most of the genes.

45. Chromosome

48. Gene The hereditary determinant of a specified difference between individual The hereditary determinant of a specified difference between individual The unit of heredity The unit of heredity The unit which passed from generation to generation following simple Mendelian inheritance The unit which passed from generation to generation following simple Mendelian inheritance A segment of DNA which encodes protein synthesis A segment of DNA which encodes protein synthesis Any of the units occurring at specific points on the chromosomes, by which hereditary characters are transmitted and determined, and each is regarded as a particular state of organization of the chromatin in the chromosome, consisting primarily DNA and protein Any of the units occurring at specific points on the chromosomes, by which hereditary characters are transmitted and determined, and each is regarded as a particular state of organization of the chromatin in the chromosome, consisting primarily DNA and protein

49. Gene classification coding genes non-coding genes Messenger RNA Proteins Structural RNA Structural proteinsEnzymes transfer RNA ribosomal RNA other RNA Chromosome (simplified) intergenic region

50. Gene Molecular definition: DNA sequence encoding protein What are the problems with this definition?

51. Gene Some genomes are RNA instead of DNA Some genomes are RNA instead of DNA Some gene products are RNA (tRNA, rRNA, and others) instead of protein Some gene products are RNA (tRNA, rRNA, and others) instead of protein Some nucleic acid sequences that do not encode gene products (noncoding regions) are necessary for production of the gene product (RNA or protein) Some nucleic acid sequences that do not encode gene products (noncoding regions) are necessary for production of the gene product (RNA or protein)

52. 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

53. Noncoding regions Regulatory regions Regulatory regions RNA polymerase binding siteRNA polymerase binding site Transcription factor binding sitesTranscription factor binding sites Introns Introns Polyadenylation [poly(A)] sites Polyadenylation [poly(A)] sites

54. Gene Molecular definition: Entire nucleic acid sequence necessary for the synthesis of a functional polypeptide (protein chain) or functional RNA

55. Bacterial genes Most do not have introns Most do not have introns Many are organized in operons: contiguous genes, transcribed as a single polycistronic mRNA, that encode proteins with related functions Many are organized in operons: contiguous genes, transcribed as a single polycistronic mRNA, that encode proteins with related functions Polycistronic mRNA encodes several proteins

56. What would be the effect of a mutation in the control region (a) compared to a mutation in a structural gene (b)? Bacterial operon

57. Eukaryotic genes Most have introns Most have introns Produce monocistronic mRNA: only one encoded protein Produce monocistronic mRNA: only one encoded protein Large Large

58. Eucaryotic genes Exon 1 90 bp Exon 2 222 bp Exon 3 126 bp Intron A 131 bp Intron B 851 bp Hemoglobin beta subunit gene Introns: intervening sequences within a gene that are not translated into a protein sequence. Collagen has 50 introns. Exons: sequences within a gene that encode protein sequences Splicing: Removal of introns from the mRNA molecule. Splicing

59. Alternative splicing Splicing is the removal of introns Splicing is the removal of introns mRNA from some genes can be spliced into two or more different mRNAs mRNA from some genes can be spliced into two or more different mRNAs