Transcript Freeman 1e: How we got there

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
• 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.
• 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
YAC (10kb) – 200-800 kb can be cloned
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.
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.
• 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.
Microbial Genomes
Prokaryotic Genomes: Sizes
and ORF Contents
• 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.
• 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).
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.
• 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
• Table 15.2 gives an analysis of the division
of genes and their activities in some
prokaryotes.
• 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.
Eukaryotic Microbial Genomes
• The complete genomic sequence of the
yeast Saccharomyces cerevisiae and of many
other microbial eukaryotes has been
determined.
• 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.
• Table 15.3 shows some eukaryotic nuclear
genomes.
Other Genomes and the
Evolution of Genomes
Genomes of Organelles
• 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.
• 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
• 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)
• 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
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.
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.
• Figure 5.14 shows why differences in DNA sequence do not
necessarily lead to differences in the amino acid sequence.
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.
• 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.