Molecular Biology II Gene Cloning 5: Analysing Gene Function

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.

1. Identify all STOP codons in each reading frame (UAA/UAG/UGA)

2. Identify all possible START codons (AUG)
3. Identify all ORFs
4. Analysis of each possible ORF to decide probability of being expressed
 There will be many possible ORFs, many will be short  not containing genes
 Promoters are AT rich – this helps to identify correct ORFs
 Look for RBS and promoter (-35,-10) upstream of START in prokaryotes
o The RBS (Ribosome Binding site) is homologous to 16s RNA (in ribosomes)
Computers are not very good at this and does not give a definitive answer, just a likelihood.

Gene annotation – Assigning each ORF within a genome

This is difficult, in all genomes sequenced today, up to half the ORFs have unknown function

Much more difficult to carry out in eukaryotes than prokaryotes :

 Much more intergenic DNA (introns) ~60%
 The introns mean ORFs do not appear as contiguous sequences within genes
 If an ORF is continued into an intron it will usually lead to termination of translation
 Introns need to be spliced out first to ensure accurate ORF scanning

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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

Locating genes for functional RNA:

Proteins are not the only product of DNA
Functional (r/t)RNAs are not in ORFs, so we cannot use ORF scanning to locate their genes
Some RNAs have distinctive features such as intramolecular base paring
 Secondary structure elements such as stem loops and hairpins
Software can predict RNA structure from DNA sequence

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

Homologous genes are either homologs or paralogs (Lecture 9, Page 2)

 Generally do not have an identical sequence (due to mutations during evolution)
 Sequences are still very similar though as changes have been made to a common
ancestor

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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.

Assigning gene function:

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

It is also possible to load a deletion cassette with

another gene in order to replace the WT gene.

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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)

II) Gene over-expression

By over-expressing a gene we can observe the ‘gain of function’ in the phenotype
This is easier than knockout and can be used on essential genes

1. Insert gene under the influence of a strong promoter into a vector

2. Insert vector into cells
3. Transgenic organisms are produced (Lecture 11, Page???)
4. Gene is expressed at higher levels
5. Investigate phenotype

There are many possible phenotype screens

Saccharomyces cerevisiae – Protein synthesis and stress responses (2/15)
Caenorhabditis elegans – Egg size (1/25)

III) Localisation of gene product

Find out where in the organism / cell a gene product is active
Use homologous recombination to replace a target gene with a reporter (GFP, X-Gal)
The reporter gene should be under the same regulatory controls that would usually dictate the
expression of the target gene. This relies of the regulatory elements being upstream of the gene –
this is not always the case in eukaryotes.

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Molecular Biology II Gene Cloning 5: Analysing Gene Function

We can do this in vivo

Probe with a labelled antibody
Use ‘tagged’ derivatives of proteins (e.g. GFP) – the sequence for the tag is placed next to
the gene. Sequence for a linker (spacer) is incorporated between the two to allow each
gene product to fold independently of one another. The linker is glycine rich.

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