Download Chapter 5 part I

Bioinformatics, Genomics, and
Proteomics
Bioinformatics
 Bioinformatics involves the building and
use of biological databases
 Some of these databases contain the
DNA sequences of genomes
 Essential for mining the massive
amounts of biological data for meaningful
knowledge about gene structure and
expression
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Molecular Databases
• The GenBank databases was established
in 1982 in anticipating of the increasing
availability of DNA sequences.
• Its purpose was the collection,
management, storage, and distribution of
sequence data.
• Initially, it was linked to the NSFnet
(National Science Foundation Network.)
Molecular Databases
• Increasing of large-scale projects required
additional databases and the expansion of
the existing databases for storing and
retrieving information.
• NSFnet was replaced by the Internet
(World Wide Web.)
• Submissions, access, and especially
retrieval (data mining) became rapid and
easy.
Molecular Databases
Molecular Databases
Foundation of - omics
• Large-scale, whole-genome experimentation,
with the analysis of many samples at one time.
• Proteomics – study of entire protein population
of various cell types and tissues and numerous
protein-protein interactions.
• As new methods were implemented and
research targets become more focused, other –
omics emerged, such as metagenomics,
functional genomics (transcriptomics), and
metabolomics.
Foundation of Genomics
• The term genomics came into used with the
development of large-scale sequencing in the
1980s.
• Research strategies are designed based on
knowledge of the complete genetic information
of an organism.
• Using computer to search for features in the
genome or to analyze the expression patterns of
tens of thousands of genes simultaneously.
Foundation of Genomics
• The invention of DNA sequencing in the 1970s.
• DNA sequence information in the GenBank
database rose rapidly.
• The genomic sequence data drove the design of
new experiments and interpretation of gene
function and of molecular processes in the cell.
• The genomic sequence data drove the design of
new experiments and interpretation of gene
function and of molecular processes in the cell.
Metagenomics
• Identification, characterization of biological functions
of microorganisms required cultivating of each strain
in the laboratory.
• In the 1990s, DNA can be directly extracted from
environmental samples, such as soil and sea water.
• Researcher began to examine the sequence
diversity of microorganisms using the universal 16S
ribosomal RNA gene as taxonomic marker.
• It is revealed that less than 1 % of bacteria species
could be cultured.
Metagenomics
• The study of the collective genomes in the
environmental samples is known as metagenomics.
• The primary objective of a metagenomic project is to
construct a comprehensive DNA library from all the
microorganisms of a particular system or location.
• The metagenomic clones can be characterized in
various ways, such as shotgun sequencing strategy.
• Metagenomic project is supported by development
of high-efficiency cloning, robotic work stations,
inexpensive DNA sequencing, databases, and
bioinformatics resources.
Metagenomics
• A massive study including 50 ocean samples yielded
6.3 x 109 bases of sequence.
• Analysis of the sequences indicated that there might
be as many as 400 new bacterial species.
• The analysis also revealed sequences encoding
potentially novel forms of many proteins, including
proteins for repair of UV light-induced DNA damage
and RuBisCO.
• Sequence-based metagenomic projects are
especially effective with microbial communities that
have relatively few species.
Metagenomics
• The toxic runoff often continues long after the mining
operation has been abandoned.
• It is interesting to learn more about the metabolic
pathways of the microorganisms found in these
environments and how they survived under such
conditions.
• In one metagenomic study, the nearly complete genomes
of two major bacterial species and partial genomes of
three other microbes were cloned and assembled.
• Leptospirillum group III plays a critical role in fixing
atmospheric nitrogen in this environment.
Metagenomics
• Metagenomic libraries are frequently screened for
enzyme activity to identify novel enzymes with
biotechnological potential.
• Selection for growth of transformed E. coli cells on
particular substrates, complementation tests, and, most
often, simple indicator systems are used for these studies.
• A metagenomic library was screened for cloned lipase
genes by growing transformed cells on agar plates that
were supplemented with various triglyceride substrates,
such as tricaprylin.
• The halo indicated that the colony produced and secreted
an enzyme that digested tricaprylin.
Metagenomics - Limitation
• Selection schemes depends on transcription and
translation of the cloned gene in the host cell, E. coli.
• Based on computer modeling using codon usage and
other transcription and translation features from the genes
of many different organisms suggests that only 40% of the
heterologous genes will be expressed in E. coli.
• Broad-host-range vectors and other host cells are being
used for constructing and maintaining metagenomic
libraries.
• Specialized gene expression systems aid in detecting
metagenomic clones that carry genes with certain
functions.
Metagenomics - SIGEX
• SIGEX – substrated-induced gene expression.
• This procedure identifies catabolic genes that are
expressed when their promoters are activated in the
presence of particular substrates.
• It also relies on the cloning of regulatory elements that are
often found upstream of the catabolic genes that they
control.
• The system utilizes a vector that contains the green
fluorescent protein (gfp) gene under the control of lac
promoter (plac) in a pUC-based plasmid, designated
p18GFP.
Metagenomics - SIGEX
• The cloning site lies between the lac promoter and the gfp
gene. DNA from a microbial community is fragmented
and cloned into p18GFP.
• The cells are grown in selective media containing
ampicillin, to prevent the growth of untransformed cells,
and IPTG to induce the expression of gfp gene from the
lac promoter.
• Cells that produced green fluorescent protein in the
presence of IPTG are those that carry plasmids without
inserts, plasmids with inserts that do not prevent
transcription of gfp from the lac promoter (no
transcriptional terminator), or plasmids with inserts
containing constitutively active promoters.
Metagenomics - SIGEX
• The cloning site lies between the lac promoter and the gfp
gene. DNA from a microbial community is fragmented
and cloned into p18GFP.
• The cells are grown in selective media containing
ampicillin, to prevent the growth of untransformed cells,
and IPTG to induce the expression of gfp gene from the
lac promoter.
• Cells that produced green fluorescent protein in the
presence of IPTG are those that carry plasmids without
inserts, plasmids with inserts that do not prevent
transcription of gfp from the lac promoter (no
transcriptional terminator), or plasmids with inserts
containing constitutively active promoters.
Metagenomics - SIGEX
• Transformed cells of interest are those that do not
produce green fluorescent protein in the presence of
IPTG because they carry plasmids with inserts that do not
include expression of gfp under these conditions.
• The transformed cells are subjected to fluorescentactivated cell sorting (FACS) to remove cells that produce
green fluorescent protein.
• These cells will fluoresce green when exposed to blue
light and are separated from the cells that do not
synthesize green fluorescent protein, and therefore, do
not fluoresce.
Metagenomics - SIGEX
• The gfp-negative cells are then grown in the presence of
a low-molecular weight substrate, for example, benzoate.
• A second round of FACS is carried out, and the cells that
expressed green fluorescent protein in the presence of
substrate are retained.
• These cells contain substrate-inducible regulatory
elements. The inserts are analyzed to identify full or
partial sequences encoding catabolic enzymes.
• Additional experiments may be required to isolate entire
catabolic operons.
Functional Genomics
 Completion of genomic sequences of many organisms
led to new experimental tools to study the functions of
genomes on a large scale.
 “High-throughput biology” includes microarray, largescale genetic analysis, methods for identifying and
assaying huge numbers of DNA sequence variants.
 These evolving methods advanced the studies of
protein-DNA and protein-protein interactions, cis-acting
transcriptional regulatory sequences, the location of
mRNAs in cells and tissue, and protein expression
levels.
Dot blot hybridization
 Dot blot hybridization has been developed to determine
the presence, absence, or relative amounts of a gene
or its mRNA in a cell.
 DNA fragments are denatured and then attached to a
filter in a simple array either by manual spotting or by
using an inexpensive apparatus.
 DNA or mRNA from a biological sample is labeled and
then hybridized to the filter, just as in Southern blot, to
determine whether the sequence is present.
 However, it did not provide the scale required to cope
with the flood of sequences.
Microarrays
 In the early 1990s, scientists developed microarrays for
hybridizing tens or even hundreds of thousands of
nucleic acid fragments in a single experiment.
 The principle is similar to that of dot blot, but instead of
just a few dozen dots of DNA, several million different
DNA fragments are deposited in an ordered array on a
glass slide using automated methods.
 It can be used to analyze the transcript levels as well
as the copy number of essentially all the genes from an
organism, genotyping the millions of DNA sequence
variants, and identifying the genomic DNA segments
bound by proteins in the cell.
Making Microarrays
 A quartz wafer of the kind used to make computer chips
is treated with a photolabile chemical that prevents
nucleotides binding to the surface.
 A photolithographic mask is overlaid on the chip and
illuminated. The holes in the mask correspond to the
position on the chip where the first nucleotide is to be
added (in this case, adenosine.)
 The light destroys the blocking chemical and adenosine
is added so that it binds to the unprotected locations
and they are once again protected.
 Three different masks are used in turn to deprotect the
positions for thymidine, guanosine, and cytidine.
Making Microarrays
 Three different masks are used in turn to deprotect the
positions for thymidine, guanosine, and cytidine.
 At this stage, the first position of every oligonucleotide about one million – on the chip has been put in place
and all carry a blocking group.
 The process is repeated for the four nucleotides that
will occupy the second place in the oligonucleotide.
 By the end of the second cycle, there are dinucleotide
chains at every location.
 This cycle of reproduction and addition of a nucleotide
is repeated until the array carries oligonucleotides of
20-25 nucleotides in length.
Microarrays
 For this type of microarray, the probes are typically 10
to 40 nucleotides, and several probes with different
sequences for each gene will be synthesized on the
microarray.
 Longer oligonucleotides up to 100 nucleotides can also
be used.
 A complete whole-genome oligonucleotide array may
contain more than 500,000 probes representing as
many as 30,000 genes.
 The design of the probes (probe set) for a microarray
depends on the objective of the experiment and the
degree of resolution that is required.
Microarrays
 Computer programs determine probe sequences that
are specific for their target sequences, are least likely
to hybridize with nontarget sequences (crosshybridize).
 These probes have no secondary structure (foldback)
that would prevent hybridization with the target
sequence.
 They have similar melting (annealing) temperatures ,
so that all target sequences can bind to their
complementary probe sequences under the same
conditions.
Gene Expression Microarrays
 The gene expression microarrays contain a set of 22
different oligonucleotides (25-mers) for each gene to be
tested.
 Eleven of the 25-mers have the exact sequence of
different segments of the mRNA transcribed from the
gene. The other 11 are identical except for an incorrect
base at nucleotide number 13.
 The 11 mismatched oligonucleotides help to establish
the background signal for each correct oligonucleotide
in the hybridization step.
 These signals are subtracted from the signals of each
of the perfectly match during the data analysis.
Gene Expression Microarrays
 The mRNAs to be measured are fragmented and the
fragments are labeled with a fluorescent dye.
 In some experiments, the mRNAs are directly labeled
or are made into cDNA and then labeled.
 The microarray is bathed with the labeled mixture and
each mRNA fragment finds its complementary mate at
a feature on the microarray.
 After hybridization and washing, the microarray is
placed in a special detector, which scans and
measures the amount of fluorescent signal.
 The amount of the signal is corresponding to the
amount of each mRNA being studied.
Gene Expression Microarrays
 mRNA is extracted from two samples (sample 1 and sample
2), and during reverse transcription, the first cDNA strands
are labeled with fluorescent dyes (Cy3 and Cy5,
respectively.
 The cDNA samples are mixed and hybridized to an ordered
array of either gene sequences or gene-specific
oligonucleotides.
 Probe cells that produce only a green or red emission
represent genes that are transcribed only in sample 1 and 2,
respectively.
 The yellow emissions denote genes that are active in both
samples, and black represent genes that are not transcribed
in either sample.
Gene Expression Microarrays
 mRNA is purified with a poly(dT) sequence that has a T7
RNA polymerase primer sequence extension.
 After two-stranded cDNA synthesis, the second cDNA
strand acts aas a template for synthesis of cRNA by T7 RNA
polymerase in the presence of biotinylated CTP and UTP.
 The biotynilated cRNA is purified, fragmented into pieces
from 50-100 nucleotides in length, and hybridized to an
oligonucleotide microarray.
 The microarray is treated with streptavidin-phycoerythrin,
and the probe cells are scanned for emission (yellow) from
the biotin-bound streptavidin-phycoerythrin.
Spotted cDNA Microarrays
 The cDNA clones are generated using plasmid library
and sequences are available from the expressed
sequence tag (EST).
 PCR is used to amplify the cDNA inserts, and the
double-stranded DNA fragments are denatured and
then “spotted” onto a glass microscope slide.
 The DNA fragments on the array are large (several
hundred to several thousand base pairs), hybridization
is more specific so that only one feature per gene is
needed.
 A single spotted cDNA microarray can assay all the
genes in an organism.
Spotted cDNA Microarrays
 The “two-color” experiment design is used to determine
the set of genes that are expressed in higher level in
different tissue.
 Two different sets of complex mRNA sample are used.
 Each set, a control and the test sample, are labeled
separately with a different fluorescent label, usually
Cy3 (green) or Cy5 (red.)
 The two labeled samples are mixed together and
hybridized to the microarray.
 The ratio of the amounts of each mRNA in the
experimental to control samples is determined by
relative fluorescent level at each spot.
mRNA Profiling Reveals New Relationship
 Microarrays can examine the expression of thousands
of genes following different treatments at multiple time
points. Extensive statistical analysis and innovative
ways of displaying data are needed.
 Genes are ranked in a table according to how similar
their gene expression levels are in a single experiment
or across different experiments in different cell types or
under different physiological conditions.
 The “heat-map” is produced, in which each data point is
shown as a shade of green or red or other pairs of
colors.
 Positive log ratios represent more Cy5 than Cy3 and,
therefore, greater expression of the gene in the test sample
than in the reference sample.
 Negative values (more Cy3 than Cy5) indicate a lower level
of expression in the test sample than the reference sample.
 Red denotes over-expression and green denotes underexpression.
Gene expression profile of cirrhotic liver tissue.
Gene expression profile of lymphocyte-specific genes.
SAGE Technique
 Serial Analysis of Gene Expression (SAGE) technique
replaced hybridization in microarray.
 Though it is powerful, microarrays rely on hybridization,
a complex process subject to experimental variation.
 First, it is difficult, if not impossible, to have excess
probe on the microarray.
 Second, cross-hybridization is another source of error
with microarrays.
 SAGE infers the abundance of an mRNA by counting
the number of times it is represented in a sample.
 Gene expression can be measured by counting short
segments of mRNAs.
SAGE Technique
 In one version of SAGE, a tag and its attached
restriction sites are 17-bp long, so a plasmid insert of
700 bp will contain tags for about 40 different mRNAs,
and it can be obtained in one read.
 More than 500 genes were found to differ significantly
in their expression levels in normal compared to
cancerous cells.
 In some cases, the differences were greater than 10
fold, leading for investigating the causes of cancer.
 As the cost of sequencing falls, greater numbers of
tags and larger variety of samples can be analyzed.
Additional SAGE protocols, such as LongSAGE and
SuperSAGE, have been developed to produce longer tags.