細菌基因體學

1. 細菌基因體學

2. 內容包含 • 15.1 Introduction • 15.2 Determining DNA Sequences • 15.3 Whole-Genome Shotgun Sequencing • 15.4 Bioinformatics • 15.5 General Characteristics of Microbial Genomes • 15.6 Functional Genomics Genome Annotation • Evaluation of RNA-Level Gene Expression • Evaluation of Protein-Level Gene Expression • 15.7 The Future of Genomics

3. 15.1 Introduction 緒言 • Genomics基因體學: 研究基因體的分子組成包含其訊息內容與基因產物 • Structural genomics結構基因體學: 研究基因體的自然物理性質-序列 • Functional genomics功能基因體學: 轉錄產物和蛋白質的功能性 • Comparative genomics比較基因體學: 比較不同基因體的不同處和相同的意義 • 重要性、保留性、功能性、調控 • 可提供演化上重要的訊息 • Whole-genome: new starting point for biological research new questions, hypotheses

4. 15.2 Determining DNA sequences • 去氧核醣核酸序列的決定 • 1975 Frederick Sanger – • dideoxynucleoside triphosphates (ddNTPs)

5. dNTP dNTP dNTP dNTP ddATP ddTTP ddCTP ddGTP P32 or S35 P32 or S35 P32 or S35 P32 or S35

6. Figure 15.2 The Sanger Method for DNA Sequencing. A sequencing gel with four separate lanes. The sequence begins, reading from the bottom, CAAAAAACGGACCGGGTGTAC.

7. DNA定序效率的進展 (samples/person/week/average read length) 1977 4 x 50 bp 1980 20 x 100 bp M13 cloning 1987 30 x 250 bp partial automation 1990 60 x 300 bp automated improved 1997 180 x 300 bp complete sequence determined 1999 500 x 650 bp 2000 5000 x 600 bp Automated system: 350,000 bases/day Today over 1,000,000 bases/day/one machine

11. 15.3 Whole-genome shotgun sequencing 全基因體散彈槍定序 • The Institute of Genomic Research (TIGR) • Haemophilus influenzae • Mycoplasma genitalium • Process • Library construction • Random sequencing • Fragment alignment and gap closure: lamda phage libraries • Editing • Less than 4 months • Celera Genomics: Human Genome Project, Drosophila genome )

15. Annotation begins註解的開始 • Open reading frame (ORF): a sequence not interrupted by a stop codon • Computer programs predicted ORF • Information about transposable elements, operons, repeat sequences, Metabolic pathways, and others • 15.4 Bioinformatics生物資訊學 • management and analysis of biological data using computer • Focus on DNA or protein sequences

16. 15.5 General Characteristics of Microbial Genomes

18. 2007.11.22

19. 真核生物

20. 哺乳動物

21. 真菌

22. 昆蟲

23. 植物

25. 15.5 General Characteristics of Microbial Genomes • Mycoplasma genitalium- 580 kilobases：one of the smallest genomes of any free-living organism • 517 genes • 480 protein-encoding genes • 37 genes for RNA species • 90 proteins in translation • 29 proteins for DNA replication • 140 genes for membrane proteins • 25 genes in host immune responses • 5 genes have regulatory functions • Transposon (108-121) 265-350 genes • 100 unknown function

28. Escherichia coli,Haemophilus influenzae, cyanobacterium Synechocystis sp. Mycoplasma genitalium • These four bacteria have only 111 proteins in common • Methanococcus jannaschii, eucaryotic yeast Saccharomyces cerevisiae • Only 16 proteins are essentially the same in all six organisms. Many gene losses and changes during the course of evolution

29. Deinococci are soil bacteria 耐輻射奇異球菌 • two circular chromosomes of different size (2.6 Mb and 0.4 Mb) • a megaplasmid (177,466 bp), and (45,704 bp) • They survive by stitching together their splintered chromosomes after radiation exposure. • Have quite different DNA repair genes? • 20 MutT-like genes (oxidized nucleotides) • Why resistance to radiation? • The genome also possesses many repeat sequences • Many genes are unknown functions

32. In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.

33. Rickettsia prowazekii 立克次體 • Obligate intracellular parasite of lice and humans • Many microbiologists established an endosymbiotic relationship • Its proteins show similarities to mitochondrial genes • Glycolysis is absent, but genes for the TCA cycle and electron transport are present, and ATP synthesis is similar to that in mitochondria • Lack many genes for biosynthesis • Thus aerobic respiration in eucaryotes may have arisen from an ancestor of Rickettsia

34. Chlamydia trachomatis 披衣菌 • Life cycle is so unusual, its genome to be somewhat atypical? • “Energy parasite” has the genes to make ATP • Peptidoglycan? cell walls lack peptidoglycan • Why antibiotic penicillin is able to inhibit chlamydial growth?The purpose of peptidoglycan synthesis? • Absence of the FtsZ gene, during cell division unknown functions proteins play a major role in cell division • The genome contains at least 20 genes obtained from eucaryotic (most bacteria have no more than 3 or 4 genes) • Some of these genes are plantlike; originally Chlamydia may infected a plantlike host and then moved to animals.

35. Treponema pallidum 梅毒螺旋體 • It has not been possible to grow Treponema outside Know little about metabolism, avoids host defenses, no vaccine • Has glycolysis, lacks TCA cycle, oxidative phosphorylation • Lacks many biosynthetic pathways (e.g., for enzyme cofactors, fatty acids, nucleotides, and some electron transport proteins) • The genes for surface proteins are of particular interest! • Have not provided how Treponema causes syphilis? • About 40% of the genes have unknown functions (avoiding host defenses, toxins, virulence factors)

36. Mycobacterium tuberculosis 肺結核桿菌 (kills about 3 million people annually) (4.40 Mb) • E. coli (4.60 Mb), Pseudomonas aeruginosa (6.26 Mb) • More than 250 genes are devoted to lipid metabolism (E. coli has only about 50 such genes) • May obtain much of its energy by degrading host lipids? • Large number of regulatory elements in the genome? • Infection process is much more complex? • Glycine-rich proteins with unknown functions 10% • Antigenic variation,defense against host immune system? • Lack of a good vaccine? Multiply drug resistant! • Mycobacterium leprae

37. 15.6 Functional Genomics • Postgenomics • How the genome operates? • RNA- and protein-level expression • Evaluation of RNA-Level Gene Expression • DNA microarrays (DNA chips) • Evaluation of Protein-Level Gene Expression • Proteins- itsproteome • Proteomics is the the large-scale effects of gene activity • Two-dimensional electrophoresis is even more powerful

41. 分 子 量 pH

42. 15.7 The Future of Genomics • Develop new methods for analysis of genes and proteins • How mRNA and protein would respond to environmental changes (computer and make predictions) • Pathogenicity and suggest treatments for infectious disease. ( virulence genes, host responses to pathogens, more sensitive diagnostic tests, new antibiotics, and different vaccines ) • The field of pharmacogenomics should produce many new drugs to treat disease • Comparative genomics will study of microbial biodiversity • The industrial applications are numerous. ( novel enzymes, enhance the bioremediation, other fuels) • Genomics will profoundly impact agriculture. (biopesticides……)