Finding genes

ORF is Open Reading Frame that are the Exon sequences which are to be translated in the process of translation

1. An Introduction to Bioinformatics
Finding genes in prokaryotes

2. 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

3. 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

4. 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

5. 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

15. 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

16. 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

21. Finding Genes in Eukaryotes
An Introduction to Bioinformatics

22. 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
To describe the complications of the eukaryote “signals”
To be aware of the Web-based programmes
To be able to use the eukaryote programmes for a number of
organisms

23. 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.

24. Principles
• Content - codon usage
– often species or class specific
• Signals - PWMs
– principle is the same, signals are different
– Complication of introns/exons

25. Eukaryotic promoter
TATA boxGC boxCAAT box
5’ 3’
-110 -40 -25 +1mRNA
In addition - transcription factor binding sites
Genes can be enormous!
Controlled by “distant” enhancers

26. AAUAA
~ 12bp polyA
AAAAA…...
Kozak sequence
At translational start
Polyadenylation sequence
AUG
Signals on the mRNA
STOP

27. Introns and Exons
Chicken 1a2 collagen gene
has - 38 kb > 50 Introns
Muscular Dystrophy gene is 2.5 Mb and has
? Exons!

28. Splicing signals
C A T C
A G C T
AGGT AGT N AGG()
>11
5’Exon
3’Exon
GT-AG rule

29. 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.

30. Intergrated Gene Parsing
• Search for signals
• Perform a content analysis
• Define the intron/exon boundaries

31. Gene finding web sites
http://www.tigr.org/~salzberg/appendixa.html
>25 listed sites
GENSCAN
FGENES