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Analysing Genes:
Bioinformatics
Gene prediction software
Homology modelling
Experimental
Gene inactivation
Gene over-expression
Localisation studies
ORF Scanning:
ORF = Open Reading Frame
A stretch of DNA without translational termination (stop) codons
Software can be used to search for these within a genome (can find up to 90% of ORFs)
In prokaryotes, ORFs will often contain gene products and will be translated into protein
In eukaryotes, ORFs can be found in exons and cDNA, but rarely in large fragments of genomic
DNA (there are introns)
In a given sequence of DNA there will be 3 possible reading frames, only one will contain the
correct ORF.
Lecture 10 [Page 1]
Molecular Biology II Gene Cloning 5: Analysing Gene Function
Codon Bias
Not all codons are used equally frequently in genes of a given organism
Leu has 6 codons, of these CTG is the most common in humans
Some organism have high / low GC content
GC in thermophilic bacteria (higher Tm)
GC in Plasmodium falciparum (20%) – the parasite that causes malaria
This codon bias has to be introduced into the ORF scanning software
Exon-intron Boundaries
Have distinctive features
GT is invariant, but in other positions alternative nucleotides can be found
If this position is known then we can ‘splice’ out the intron using a computer
ORF scanning mistakes are often made at this boundary
Homology searching:
Homology searching – related genes have similar sequence
Due to natural selection, there is greater sequence similarity within genes than outside
Intergenic DNA is less conserved than coding DNA
Homologous genes between species are conserved
Genes are kept functional by natural selection
If the common ancestor of a gene between 2 species is unknown, it can be predicted using the
sequences of the related species
A short ORF present in only one of a few related species is unlikely to be functional
If the ORF is found in multiple related species, then it probably has function
Lecture 10 [Page 2]
Molecular Biology II Gene Cloning 5: Analysing Gene Function
A high sequence identity (% matching) does not necessarily mean gene products will be
homologous.
Some very similar DNA sequences will give vastly different AA sequences when translated. This
will suggest there is no functional relatedness.
Software programs like BLAST and PSI-BLAST can be used to investigate sequence homology
Some genes are linked to diseases in humans. By looking for homologous sequences in other
species we can often find the biochemical basis for the disease.
I) Gene Inactivation
Results in a loss of function
The conventional approach is to obtain a phenotype and locate the mutant gene (Mendel)
In genomic analysis we start with the gene.
Gene is inactivated
A change in phenotype should be observed (this can be difficult)
Homologous recombination can be used to interrupt a gene
Vector DNA contains a portion of DNA homologous to the gene, with an interrupted region
that will result in no expression.
We can’t interrupt essential genes (death)
a) Deletion Cassettes:
Can be used in yeast to create a library in which each culture has one gene specifically deleted.
Yeast cells are inclined to undergo homologous recombination
A specific deletion cassette is generated for each gene
Each deletion cassette is introduced into yeast cells, where it will rapidly replace the target gene
by homologous recombination
Lecture 10 [Page 3]
Molecular Biology II Gene Cloning 5: Analysing Gene Function
b) Transposition
Many species contain transposons – mobile genetic elements
Transposition (transposon hopping) is normally rare
It is also a random process, so the transposons can enter any gene
Transposition can be induced in response to a stimulus
Recombinant derivatives of transposons can be constructed, and are incorporated with a marker
gene such as antibiotic resistance
This process can be used for the random disruption of genomes, which can then be screened for
a phenotype (e.g. antibiotic resistance)
c) Site-directed mutagenesis:
We suspect a gene has a known function
Firstly we know out the gene, function should cease.
To investigate further we can mutate parts of the gene and see how function changes.
This could be looking for active site. A change in a single AA could stop catalytic function.
We use modified PCR primers to carry out site-directed mutagenesis (Lecture 7, Pages 4-5)
Lecture 10 [Page 4]
Molecular Biology II Gene Cloning 5: Analysing Gene Function
Lecture 10 [Page 5]