1. CHAPTER 15 Microbial Genomics Genomic Cloning Techniques Vectors for Genomic Cloning and Sequencing MS2, RNA virus- 3569 nt sequenced in 1976 X17, ssDNA virus 5386 nt “ in 1977 Fredrick Sanger H. influenzae bacteria 1,830,137 bp 1995 Human genome draft 2000

2. Specialized cloning vectors have been constructed that are useful for the sequence and assembly of genomes. Some, such as the M13 derivatives (Figure 15.1a), are useful both for cloning and for direct DNA sequencing.

3. Others, such as artificial chromosomes (Figures 15.2, 15.3), are useful for cloning fragments of DNA approaching a megabase in size. M13 – 5 kb Lambda – 20 kb BAC - >300 kb can be cloned 6.7 kb

4. YAC (10kb) – 200-800 kb can be cloned

5. Sequencing the Genome Virtually all genomic sequencing projects today employ shotgun sequencing. Shotgun techniques use random cloning and sequencing of relatively small genome fragments followed by computer-generated assembly of the genome using overlaps as a guide to the final sequence.

6. Annotating the Genome After major sequencing is through, computers search for open reading frames (ORFs) (Figure 15.4) and genes encoding protein homologues as part of the annotation process.

7. Figure 15.5 shows a genetic map constructed by computer from shotgun sequencing of the 4.4-Mbp genome of Mycobacterium tuberculosis, the causative agent of tuberculosis.

8. Microbial Genomes Prokaryotic Genomes: Sizes and ORF Contents

9. Sequenced prokaryotic genomes range in size from 0.49 Mbp to 9.1 Mbp. Table 15.1 lists a few representative examples of species of Bacteria and Archaea containing circular as well as linear genomes.

12. The smallest prokaryotic genomes are the size of the largest viruses, and the largest prokaryotic genomes have more genes than some eukaryotes. In prokaryotes, ORF content is proportional to genome size (Figure 15.6).

14. Prokaryotic Genomes: Bioinformatic Analyses and Gene Distributions Bioinformatics—the use of computational tools to acquire, analyze, store, and access DNA and protein sequences—plays an important role in genomic analyses. Many genes can be identified by their sequence similarity to genes found in other organisms. However, a significant percentage of sequenced genes are of unknown function. On average, the gene complement of Bacteria and Archaea are related but distinct.

15. Figure 15.7 summarizes some of the metabolic pathways and transport systems of Thermotoga maritima that have been derived from analysis of its genome. ATP-binding cassette (ABC) transporters

16. Table 15.2 gives an analysis of the division of genes and their activities in some prokaryotes.

18. Analyses of gene categories have been done on several prokaryotes beyond the three species of Bacteria shown in Table 15.2, and the results are compared in Figure 15.9.

20. Eukaryotic Microbial Genomes The complete genomic sequence of the yeast Saccharomyces cerevisiae and of many other microbial eukaryotes has been determined.

21. Yeast may encode up to 5570 proteins, of which only 877 appear essential for viability. Relatively few of the protein-encoding genes of yeast contain introns.

22. Table 15.3 shows some eukaryotic nuclear genomes.

24. Other Genomes and the Evolution of Genomes Genomes of Organelles

25. Chloroplasts and mitochondria have small genomes independent of nuclear genomes. These genomes encode rRNAs, tRNAs, and a few proteins involved in energy metabolism. Although the genomes of the organelles are independent of the nuclear genome, the organelles themselves are not. Many genes in the nucleus encode proteins required for organellar function. These genes have various phylogenetic histories.

26. Figure 15.10 shows a map of a typical chloroplast genome, and Table 15.4 lists some chloroplast genomes. Large single copy region Small single copy region Typical chloroplast genome – 120 to 160 kb Inverted repeats – 6 to 76 kb

28. Figure 15.11 shows a map of the human mitochondrial genome. Size – 16,569 bp 16S and 12S (23 and 16S in bacteria) rRNA and 22 tRNA NAD dehydrogenase (NA1-6) Cytochrome oxygenase (COI-III)

29. RNA editing involves the insertion or deletion of nucleotides into the final mRNA that were not present in the DNA transcribed. Figure of the Microbial Sidebar, RNA Editing, illustrates RNA editing. Trypanosoma brucei, a protozoan cytochromosome oxidase

30. Genomic Mining Often it is necessary to search carefully through a genomic database to find a particular gene, a process called genomic mining. The search for the DNA polymerase of the cyanobacterium Synechocystis is a good example (Figure 15.12). This can be done to find novel genes or to find genes that one predicts must be present.

32. Gene Function and Regulation Proteomics The proteome encompasses all the proteins present in an organism at any one time. The aim of proteomics is to study these proteins to learn their structure, function, and regulation.

33. Figure 5.14 shows why differences in DNA sequence do not necessarily lead to differences in the amino acid sequence.

34. Microarrays and the Transcriptome Microarrays are genes or gene fragments attached to a solid support in a known pattern. These arrays can be used to hybridize to mRNA and analyzed to determine patterns of gene expression.

35. The arrays are large enough and dense enough that the transcription pattern of the entire genome (the transcriptome) can be analyzed. Figure 15.16 shows a method for making and using microarrays.