Transcript Finding genes in the genome

Finding prokaryotic genes and
non intronic eukaryotic genes
Lecture 8
Global Sequence
1
Introduction
•
•
•
•
•
Review structure of prokaryotic genes
What is ORF
Finding open reading frames
Eliminating false ORF
Algorithm for finding ORF.
Prokaryotic coding sequence structure
• The gene coding sequence
in the primary strand begins
an ATG and ends at a stop
codon: TAA, TGA TAG.
• The diagram shows the
template strand (TAC
compliment of ATG) and
three other Amino acids
does not show a stop
codon.
• The codons are contiguous
in ,prokaryotic and nonintronic, protein coding
sequences.
Finding potential OFR
– Translate each reading
frame beginning at:
– Base 1: 5’ 3’ frame 1
– Base 2: 5’ 3’ frame 2
– Base 3: 5’3’ frame 2
• Get the “reverse
compliment of the given
strand” and repeat the
process”; 3’ 5’ frame 1….
• The diagram gives a partial
sequence of the first exon in
this gene.
Global Sequence
4
Finding ORF
• Look for start and stop
codons (amino acids).
• An ORF is the sequence
which begins with the
start codon (ATG in a DNA
strand) and ends with a
stop codon
(TAA/TAG/TGA)
• Which of the translated
reading frames in the
diagram opposite could
contain ORF if you know
that it is a partial
sequence of the exon. ?
True ORF gene
• Not all ORF are “true” genes so one needs to consider:
– “False” start codons An ATG can also exist as part of the
gene sequence where it does not represent a start codon.
• What does it represent?
– length of the ORF should contain at minimum number of
amino acids{consider the smallest protein is about 20 aa in
length.]
• Does an ORF represents a gene or a coding sequence
(CDS) of a gene then:
– An ORF in Prokaryotes can be translated directly into the
amino acid sequence.
– However in eukaryotes the ORF is a mixture of exons and
introns. So the exons sequences must be found in order to
determine the true amino acid sequence (covered in next
lecture).
ORF’s in prokaryotic genes
• In order to make it easier to find true genes or ORF in
prokaryotic cells one needs to consider the structure of
prokaryotic operons such as the “lac operon” below:
• E. G. Within the lac operon there are 3 genes (CDS) all in close
proximity: so the ATG lac Y is close to TAG of LacZ….
Global Sequence
7
Eliminating false positives
• Gene density is about 1 per kilobase, ORF every 1000 bases. In some cases
the genes density can cause the stop codon of one gene to overlap with
the promoter of another [ Zvelebil chapter 9] or even the start codon to
overlap with a stop codon
• The DNA sequence of genes contains different ratio of bases as opposed
to the non “CDS of a gene” or non geneic DNA: (The student is expected to
research this)
• An ORF should have a promoter region a small distance upstream of the
start codon. Promoter sequences can be identified by sequences such as
a TATA box…. (a lecture on the basic of finding promoters will discuss this
in more details).
• The CDS sequence of genes are generally highly conserved so “searching
on line databases, for similar cds sequences can increase the chance of
finding true ORF.
Algorithm to find ORF in prokaryotic Fasta files
1. Open files and convert file sequences (60 per line) into one
string of sequences.
2. Translate the first reading frame of the downloaded strand.
3. Shift one position to the right and translate this sequence;
repeat for reading frame three. (note in Fasta Files there will
be no need to reverse the compliment as it can be assumed
that the CDS is in the sequences given in the files)
4. Mark the start and the stop amino acids
5. Look for sequences with a start followed by a stop if there is
none then there is no ORF in that reading frame.
6. Determine length of ORF and if less than 20 eliminate as it is
a “false positive”
Exercise
• The sequence for TUBAC3 gene can be found
at: TUBAC3 gene complete sequence Note:
the diagram only shows a part of the exon.
And the partial sequence shown in the figure
above begins at position 57 to position 357
• Download the file and analyse it in more detail
to reveal problems that can with polycistronic
and Eukaryotic sequences with muliple
contiguous exons.
Exam question.
• Open reading frames (ORFs) are an essential part
of finding genes in genomes:
• What is an ORF
(2 Marks).
• Using a suitable example describe how you would
predict an ORF
(8 Marks).
• Write a basic algorithm to find ORF in a fasta file;
e.g. (BTEB gene)
(6 marks)
• Describe, by illustrating you answer with a
suitable example, two ways you can eliminate
false ORF.
(6 marks)
•
Reference
• Baxevanis, A.D. 2005 Bioinformatics: a
practical guide to the analysis of genes and
proteins. Wiley; Chapter 5. [book is in the
library]
• Klug, W.A. et al 2010; Concepts of Genetics;
Pearson Education p. 596-p.597
• Zhang, M.Q. 2002 Computational prediction
of eukaryotic coding genes. Nat Rev. Genet. 3
698-709.
• Zvelebil M. “understanding bioinformatics”
chapter 9 {book is in the library]
Global Sequence
12