Genomics-study of the
molecular organization of genomes, their information content, and the
gene products they encode
Structural
genomics-study of the physical nature of genomes (i.e., determine and
analyze the DNA sequence of the genome)
Functional
genomics-study of the way the genome functions
Comparative
genomics-compares genomes from different organisms; helps discern
patterns in function and regulation and provides information about
microbial evolution
Genomics will decrease
the amount of time microbiologists spend cloning genes; instead they will
generate new questions and hypotheses by computer analyses of genome data
Determining DNA Sequences
Sanger Method
Uses
dideoxynucleoside triphosphates (ddNTP) in DNA synthesis; these lack a
3'-hydroxyal and terminate DNA synthesis
Single strands of DNA
are mixed with a primer, DNA polymerase I, four deoxynucleoside
triphophates (one is labeled), and a small amount of one of the ddNTP;
DNA synthesis begins with primer but terminates each time a ddNTP is
added to the chain
Four reactions are
run, each with a different ddNTP; these reactions generate DNA fragments
of different length, because the site at which the ddNTP is inserted is
random
Newly synthesized DNA
fragments are separated electrophoretically, the gel is autoradiographed
and the sequence read
Automated systems now
exist, which make rapid sequencing possible
Whole-Genome Shotgun
Sequencing
Sequencing a genome by
the whole-genome shotgun approach is a multi-step process
Library
construction-chromosomes are broken into gene-sized fragments, inserted
into plasmids, and transformed into special E. coli strains
Random sequencing-the
cloned fragments are sequenced
Fragment alignment
and gap closure-DNA fragments are clustered and assembled into longer
stretches of sequence by comparing nucleotide sequence overlaps between
fragments; this produces larger contiguous sequences called contigs; the
contigs are aligned in the proper order to form the completed genome
sequence; gaps in the sequence are filled
Editing-sequence is
proofread to resolve any ambiguities in the sequence
Annotation is done
once the sequence is obtained; annotation involves identifying open
reading frames (ORFs), determining potential amino acid sequences and
comparison to known proteins; such comparison allows tentative assignment
of gene function as well as identification of transposable elements, operons,
and repeat sequences, and the detection of various metabolic pathways
Bioinformatics
The field concerned
with the management and analysis of biological data using computers
DNA sequence data is
stored in large databases such as the International Nucleic Acid Sequence
Data Library (GenBank)
General Characteristics of
Microbial Genomes
Analysis of a number
of microbial genomes has enabled scientists to: develop hypothese about
minimal genome size, identify large numbers of genes for which function
is unknown, and formulate hypotheses regarding the evolutionary
relationships among the three domains of life
Findings of particular
interest
Deinococci-bacteria
with remarkable resistance to radiation-have the same array of DNA
repair genes as other bacteria; they just have more copies of the repair
genes
Rickettsia
prowazekii-a bacterium thought to be related to the ancient bacterium
that gave rise to eucaryotic mitochondria-has sequences consistent with
this hypothesis
Chlamydiae-bacteria
that have a unique life cycle, are often referred to as energy
parasites, and lack peptidoglycan-have a genome that is similar to many
other bacteria and even contains some genes for ATP synthesis and
peptidoglycan synthesis; however, these bacteria lack a gene long held
to be required for septum formation during cell division
Treponema
pallidum-the causative agent of syphilis, which has not been cultured
outside the human body-has a genome that shows it is metabolically
crippled, both anabolically and catabolically; it also has a family of
genes that encode surface proteins, suggesting that this bacterium may be
able to change its surface proteins and thereby avoid attack by the host
immune system
Mycobacterium
tuberculosis-the causative agent of tuberculosis-has a very large genome
with more than 250 genes devoted to lipid metabolism; it also has a
large number of regulatory genes, suggesting that the infection process
is very complex; other genes may enable the bacterium to change its
antigens and thus elude the host immune system
Mycobacterium
leprae-the causative agent of leprosy-has a much smaller genome and
about half of its genome is devoid of functional genes
General Patterns
Despite the
conservation of protein sequences, genome organization is quite variable
in the Bacteria and Archaea
Considerable
horizontal gene transfer, especially of housekeeping genes, has occurred
in the evolution of these microbes
Functional Genomics
Genome annotation-used
to tentatively identify genes; allows analysis of the kinds of genes and
functions present in the microorganism
Evaluation of
RNA-level gene expression
DNA microarrays (DNA
chips)-solid supports (e.g., glass) that have DNA attached in highly
organized arrays; in commercial chips, the array consists of many
expressed sequence tags (a partial gene sequence unique to the gene that
can be used to identify and position the gene during genomic analysis)
The mRNA or cDNA to
be analyzed (target mixture) is isolated, labeled with fluorescent
reporter groups, and incubated with the DNA chip; fluorescence at an
address on the chip indicates that the DNA probe on the chip is bound to
a mRNA or cDNA in the target mixture; analysis of the hybridization
pattern shows which genes are being transcribed
Using this procedure
the characteristic expression of whole sets of genes during
differentiation or in response to environmental changes can be observed;
patterns of gene expression can be detected and functions can be
tentatively assigned based on expression
Evaluation of
protein-level gene expression
Proteome-entire
collection of proteins that an organism produces; proteomics is the
study of the proteome
The traditional
method for studying the proteome is two-dimensional electrophoresis,
which can resolve thousands of proteins in a mixture
Differences in the
proteins produced under various conditions can be detected
Although functional
genomics already has provided valuable information, there are still
problems that must be solved
The Future of Genomics
New methods are needed
for the large-scale analysis of genes and proteins so that more organisms
can be studied
All new information
about DNA and protein sequences, variations in mRNA and protein levels,
and protein interactions must be integrated in order to understand genome
organization and the workings of a cell
Genomics can be used
to provide insights into pathogenicity and suggest treatments for
infectious disease.
Pharmacogenomics
should produce many new drugs to treat disease
The nature of
horizontal gene transfer and the process of microbial evolution can be
studied by comparing a wide variety of genomes
There are numerous
industrial applications (e.g., identification of novel enzymes)
Genomics will probably
impact agriculture (e.g., identification of new biopesticides)