An Introduction to Bioinformatics

Finding genes in prokaryotes

AIMS

To establish the concept of ORFs and their relationship to genes

To describe the features used by software to find ORFs/genes

To become familiar with Web-based programmes used to find

ORFs/genes

OBJECTIVES

To be able to distinguish between the concepts of ORF and gene

Use ORF Finder to find ORFs in prokaryotic nucleotide sequences

Usually the primary challenge that follows the sequencing of
anything from a small segment of DNA to a complete genome
is to establish where the location functional elements such as:

genes (intron/exon boundaries)

promoters,
terminators etc

DNA sequences that may potentially encode proteins are called

Open Reading Frames (ORFs)

The situation in prokaryotes is relatively straightforward since

scarcely any eubacterial and archaeal genes contain introns
FINDING ORFs

The simplest method in prokaryotes is to scan the DNA for

start and stop codons

The DNA is double stranded and each strand has three

potential reading frames (codons are groups of 3 bases)

THE CAT ATE THE RAT Frame 1

T HEC ATA TET HER AT Frame 2

TH ECA TAT ETH ERA T Frame 3

The scan must look at all 6 reading frames

Any region of DNA between a start codon and a stop codon in
the same reading frame could potentially code for a polypeptide
and is therefore an ORF

Start AUG (methionine) Stop UAA UAG UGA

small potential coding sequences like this will occur frequently

by chance, and therefore the longer they are the more likely
they are to represent real coding regions, genes

Problems

Small genes may be missed

The actual start codon may be internal to the ORF

There may be overlapping genes

The simplest tool for finding ORFs is ORF Finder at NCBI

It simply scans all 6 reading frames and shows the position of

the ORFs which are greater than a user defined minimum size

The genetic code used for the analysis can be altered by the
user
This would be important if e.g. mitochondrial or ciliate nuclear
DNA were being analysed
To overcome the limitations of ORF finder, more sophisticated
programmes detect compositional biases and increase the
reliability of gene detection

These compositional biases are regular, though very diffuse,

And arise for a variety of reasons:

many organisms there is a detectable preference for G or C

over A and T in the third ("wobble") position in a codon

all organisms do not utilize synonymous codons with the same

frequency - consequently there is a codon bias

there is an unequal usage of amino acids in proteins sufficient to

cause a bias in all three positions of codons and increase the
overall codon bias
the %GC content of the first two codon positions of the
universal genetic code is approximately 50%, therefore,
organisms which have a low or high %GC content will exhibit
a marked bias at the third position of codons to achieve their
overall %GC content

The most recent approaches to using compositional features

to distinguish coding from non-coding regions employ Markov
models

such approaches include the popular GENEMARK and

GLIMMER programs
An Introduction to Bioinformatics

Finding Genes in Eukaryotes

AIMS To establish the concept of ORFs and their relationship to genes
To describe the features used by software to find ORFs/genes
To become familiar with Web-based programmes used to find
ORFs/genes

To describe the complications of the eukaryote signals

To be aware of the Web-based programmes

OBJECTIVES
To be able to distinguish between the concepts of ORF and gene
Use ORF Finder to find ORFs in prokaryotic nucleotide sequences

To be able to use the eukaryote programmes for a number of

organisms
Organisms whose cells have a membrane-bound
nucleus and many specialised structures located within
their cell boundary.

In these organisms, genetic material is organized into

chromosomes that reside in the nucleus.
 Principles

Content - codon usage

often species or class specific

Signals - PWMs
principle is the same, signals are different

Complication of introns/exons
 Eukaryotic promoter

-110 -40 -25 +1mRNA

5 3
CAAT box GC box TATA box

In addition - transcription factor binding sites

Genes can be enormous!

Controlled by distant enhancers

 Signals on the mRNA

Polyadenylation sequence
AAUAA
AUG STOP AAAAA...

~ 12bp polyA
Kozak sequence

At translational start
 Introns and Exons

Chicken 12 collagen gene

has - 38 kb > 50 Introns

Muscular Dystrophy gene is 2.5 Mb and has

? Exons!
 Splicing signals

5Exon 3Exon

C
A
A
AGGT AGT
G ()
T
C
C
N AGG
>11
T

GT-AG rule
 Exon finding

Initial exons, from the initiation codon to the first

splice site;

Internal exons from splice site to splice site;

Terminal exons from splice site to stop codon;

Single introns corresponding to uninterrupted,

intronless genes, i.e., running from initiation codon to
stop codon.
Intergrated Gene Parsing

Search for signals

Perform a content analysis

Define the intron/exon boundaries

 Gene finding web sites

>25 listed sites

GENSCAN

FGENES

http://www.tigr.org/~salzberg/appendixa.html