Genome Analysis & Gene Prediction
Overview about Genes
Gene : whole nucleic acid sequence necessary for the synthesis of a
functional protein (or functional RNA)
A human cell contains approximately 23,000 genes.
 Some of these are expressed in all cells all the time. These socalled housekeeping genes are responsible for the routine metabolic
functions (e.g. respiration) common to all cells.
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Some are expressed as a cell enters a particular pathway of
differentiation.
Some are expressed all the time in only those cells that have
differentiated in a particular way. For example, a liver cell expresses
continuously the genes for the metabolizing enzymes.
Some are expressed only as conditions around and in the cell change.
For example, the arrival of a hormone (due to environmental factors
or others) may turn on (or off) certain genes in that cell.
How Gene Expression is Regulated?
To Know about gene expression, first we look for the
basic structure of a gene.
Genomic DNA
Genomic DNA
5’….
Upstream
Primary Transcript
Downstream
…3’
About Upstream region of a Gene
Genomic DNA
Upstream
5’….
Upstream
Primary Transcript
Upstream promoter/Regulatory region
Regulatory Locus
Distal
Distal (GC box)
Central
Downstream
…3’
Promoter
Proximal
Central (CAAT box)
Core/basal Promoter (TATA Box)
About Core Promoter
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basal or core promoter located within about 40 base pairs (bp) of the
transcription start site (TSS)
It is found in all protein-coding genes. This is in sharp contrast to the
upstream promoter whose structure and associated binding factors differ
from gene to gene.
It contains a sequence of TATA box (either canonical TATA box or TATA
variant). It is bound by a large complex of some 50 different proteins,
including
- Transcription Factor IID (TFIID) which is a complex of
 TATA-binding protein (TBP), which recognizes and binds to the TATA box
 14 other protein factors which bind to TBP — and each other — but not to
the DNA.
- Transcription Factor IIB (TFIIB) which binds both the DNA and pol II.
About Upstream Promoter/Regulatory Regions
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an "upstream" promoter, which may extend over as many as 200 bp farther
upstream
It has three regions
- Proximal region: insulators are possibly present in this region. Insulators are
stretches of DNA (as few as 42 base pairs) and located between the
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enhancer(s) and promoter or
silencer(s) and promoter
of adjacent genes or clusters of adjacent genes. Their function is to prevent a
gene from being influenced by the enhancer (or silencer) of its neighbors.
- Central Region: Silencers are possibly present in this region. Silencers control
regions of DNA that may be located thousands of base pairs away from the
gene they control. However, when transcription factors (Silencers) bind to them,
expression of the gene they control is repressed.
- Distal Region: Enhancers may be present in this region. Enhancer bind to
regions of DNA that are thousands of base pairs away from the gene they
control. Binding increases the rate of transcription of the gene. Enhancers can be
located upstream, downstream, or even within the gene they control.
About Upstream Promoter/Regulatory Regions
About Primary Transcript
Genomic DNA
5’….
Upstream
Primary Transcript
Downstream
TSS
Exon
Intron
ATG….
Start codon
mRNA
Exon
GT……..AG ………... GT…..AG
Donor site
Exon
Intron
…......TGA
Acceptor site
ATG…………………………………………TGA
Stop codon
…3’
About Primary Transcript
Primary transcript consists of
 Cap region: 5' cap is a specially altered nucleotide on the 5'
end of precursor messenger RNA.
 5’-UTR: Regions of the gene outside of the CDS are called UTR’s
(untranslated regions), and are mostly ignored by gene finders,
though they are important for regulatory functions.
 Coding sequence (CDS): CDS of a gene is delimited by four types
of signals: start codons (ATG in eukaryotes), stop codons (usually
TAG, TGA, or TAA), donor sites (usually GT), and acceptor sites (AG).
 3’-UTR: three prime untranslated region (3' UTR) is a particular
section of messenger RNA (mRNA).
 Poly-A tail: Polyadenylation is the addition of a poly(A) tail to
an RNA molecule. The poly(A) tail consists of multiple adenosine
monophosphates.
About Intron and Exon
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Intron: It is derived from the term intragenic region,
i.e. a region inside a gene. these are sometimes
called intervening sequences which refer to any of
several families of internal nucleic acid sequences
that are not present in the final gene product
Exon: these sequences are present in the mature
form of an RNA molecule after removing of introns.
The mature RNA molecule can be a messenger
RNA or a functional form of a non-coding RNA such
as rRNA or tRNA.
More about Exon
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Three types of exons are defined:
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initial exons extend from a start codon to the first donor site;
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internal exons extend from one acceptor site to the next donor site;
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final exons extend from the last acceptor site to the stop codon;
single exons (which occur only in intronless genes) extend from the
start codon to the stop codon.
Structure of a Gene
An Hypothetical Example Gene Parse Tree
Gene Prediction
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Analysis by sequence similarity can only reliably
identify about 30% of the protein coding genes in
a genome
50-80% of new genes that are identified, have a
partial, marginal, or unidentified homolog
Frequently expressed genes tend to be more easily
identifiable by homology than rarely expressed
genes
Gene finding is species-specific
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Codon usage patterns vary by species
Functional regions (promoters, translation initiation
sites, termination signals) vary by species
Common repeat sequences are species-specific
Gene finding programs rely on this information to
identify coding regions
Protein Coding Gene
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ab initio using computational methods is the
most suited to protein-coding genes
Protein-coding genes have recognizable features
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open reading frames (ORFs)
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codon bias
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known transcription and translational start and stop
motifs (promoters, 3’ poly-A sites)
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splice consensus sequences at intron-exon boundaries
ab initio gene discovery
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Protein-coding genes have recognizable features
We can design software to scan the genome and
identify these features
Some of these programs work quite well,
especially in bacteria and simpler eukaryotes
with smaller and more compact genomes
It’s a lot harder for the higher eukaryotes where
there are a lot of long introns, genes can be
found within introns of other genes, etc.
ab initio gene discovery—Validating
predictions and refining gene models
Standard types of evidence for validation of predictions
include:
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match to previously annotated cDNA
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match to EST from same organism
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similarity of nucleotide or conceptually translated protein
sequence to sequences in GenBank
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protein structure prediction match to a PFAM domain
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associated with recognized promoter sequences, ie TATA
box, CpG island
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known phenotype from mutation of the locus
Finding Non–protein Coding Genes
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Non-protein coding genes (tRNA, rRNA, snoRNA,
siRNA, miRNA, various other ncRNAs) are harder
to find than protein-coding genes. Because
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often not poly-A tailed—don’t end up in cDNA
libraries
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no ORF
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constraint on sequence divergence at nucleotide not
protein level, so homology is harder to detect
Finding Non–protein Coding Genes
To find out, Non-protein coding genes, we have
identify…..
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secondary structure
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homology, especially alignment of related species
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experimentally
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isolation through non-polyA dependent cloning methods
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microarrays
ab initio gene discovery—approaches
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Most gene-discovery programs makes use
form of machine learning algorithm. A
learning algorithm requires a training set
data that the computer uses to “learn” how
pattern.
of some
machine
of input
to find a
Two common machine learning approaches used in
gene discovery (and many other bioinformatics
applications) are
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Dynamic programming model
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Artificial neural networks (ANNs) and
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Hidden Markov models (HMMs)
Control of Gene Expression—Transcription Factors
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Transcription factors (TFs) are proteins that bind to the DNA and help
to control gene expression. The sequences to which they bind are
transcription factor binding sites (TFBSs), which are a type of cisregulatory sequence
Most transcription factors can bind to a range of similar sequences.
These can be found in either of two ways, as a consensus sequence, or
as a position weight matrix (PWM).
Once we know the binding site, we can search the genome to find all
of the (predicted) binding sites
Evidence based Approaches
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Comparative or similarity based gene
prediction
Combine gene models with alignment to
known ESTs & protein sequences
Gene Prediction Tools
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SNAP
TwinScan
Gnomon (NCBI)
GeneWise
Jigsaw
GLEAN
Grail
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BLAST
FASTAX
BLAT
WABA
MZEF,
MZEF-SPC
FGENESH
Genome Annotation-Much work remains
Despite good progress in identifying both
protein coding and non-protein coding
genes, much work remains to be done
before even the best-studied genomes are
fully annotated.
 For the higher eukaryotes, only a tiny
percentage of features such as TFBSs and
other non-gene features have so far been
indentified.
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References
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http://users.rcn.com/jkimball.ma.ultranet/Biolog
yPages/P/Promoter.html