Microbial Genomics

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• 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
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• 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.

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• 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 - gt300 kb can be
cloned
6.7 kb
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YAC (10kb) 200-800 kb can be cloned
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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.

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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.

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• 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.

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Microbial Genomes  Prokaryotic Genomes Sizes
and ORF Contents
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• 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.

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• 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).

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Prokaryotic Genomes Bioinformatic Analyses and
Gene Distributions

• Bioinformaticsthe use of computational tools to
  acquire, analyze, store, and access DNA and
  protein sequencesplays 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.

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

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• 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.

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Eukaryotic Microbial Genomes

• The complete genomic sequence of the yeast
  Saccharomyces cerevisiae and of many other
  microbial eukaryotes has been determined.

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• 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.

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• Table 15.3 shows some eukaryotic nuclear genomes.

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

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• Figure 15.10 shows a map of a typical
  chloroplast genome, and Table 15.4 lists some
  chloroplast genomes.

Large single copy region
Typical chloroplast genome 120 to 160 kb
Inverted repeats 6 to 76 kb
Small single copy region
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• 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)
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• 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
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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.

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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.

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

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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.

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• 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.

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