L13 - Identifying and Analysing Genes Flashcards
Annotating genomic sequence using gene prediction software
Involves scanning sequence for promoters, start and stop sequences and intron splice sites.
Use computer to translate the DNA in all 6 reading frames
Then search for similarity to known proteins (BLAST)
Blast protein alignment method
- Input amino acid sequence of proposed protein
- Blast program searches huge databases for other proteins with similar sequences
- Shows alignment of uncharacterized protein (query) to a protein called zen (subject)
- Similarity between protein sequence suggests proteins evolved from common ancestor
Microarrays role
Allow us to compare the transcriptomes of different tissues to each other
High throughput- small scale, fast and automated
Using microarrays to determine expression profile of liver method
- A precise robot manufactures the array – one sport for every gene
a. Each position in the grid contains one cDNA - as the antisense strand - Purify mRNA from liver tissue and tag with a fluorescent dye
- Put mRNA onto the array – hybridization to cDNA on the array
- Rinse off any excess, unhybridized mRNA
- Reader with a sensitive camera detects which genes are on
Microarray experiments usually involve comparing two samples
Most genes in the sample are the same – liver house keeping gene
Some genes lost in tumour tissue – tumour suppressors
Some genes activated in tumour tissue – potential oncogenes
The three ways to identify genes
Library of cDNA clones from mRNA
Library of genomic clones and make predictions based upon genomic sequence
Microarrays
Gene replacement
Makes small change to endogenous gene
Test whether human mutation causes disease symptoms in mouse by making the same change in corresponding mouse gene
Gene knock-out
Completely remove gene to determine its function
To begin a knock-out project, you must first have a genomic clone of your gene
Gene knock-out in mice method
- Get genomic clone of gene and insert NEO directly into an exon
- Destroys activity of gene
- TK is placed off to one side - Introduce construct into mouse ES cells using cell culture techniques
- Cell’s DNA repair machinery recombines the construct into the mouse genome – inefficient
- Homologous recombination sometime occurs - knock-out - TK gene is lost
What are homologous arms?
Sequences from target gene
- Only sequence with homology to the mouse genome
Double selection to identify the knock-out method
Use selectable markers (Neo/TK) to identify colonies that are the result of homologous recombination
Positive selection
Cells that have integrated Neo gene - grow in Neomycin containing media
Negative selection
Cells that have integrated TK gene and Neo - die when grown in GANC media
After targeting a single gene in mice - 1st generation
Mixture of cells from stem cell line and mother
Their gonads are also mosaic
After targeting a single gene in mice - 2nd generation
Mosaic animals bred to generate non- mosaic carriers of transgene
After targeting a single gene in mice - 3rd generation
Carriers interbred to create homozygous mutant animals
Forward genetics method
- Randomly mutate genome
- Look for interesting phenotypes in offspring
- Identify gene that causes the defect
Random mutagenesis affects the whole genome
Have to analyse many mutagenized animals
- Yeast
- C.elegans
- Drosophila
- Zebrafish
Different types of genetic screens
- Loss of certain cells or tissues
- Disease-like phenotype
- Biochemical abnormalities
- Behaviour
- Drug addiction
Screens can be done for dominant or recessive traits, usually recessive
Forward genetics vs reverse genetics
Forward genetics: function (phenotype) —-> gene
Reverse genetics: gene —-> function (phenotype)
Forward genetic screen in flys method
- Mutagenize male - each sperm has different set of mutations
- EMS - chemical mutagen
- Male is heterozygous for the mutations carried by the parental sperm - Outcross males to wildtype females
- Both offspring heterozygous
- Incross to identify homozygous embryos - 1/4 of offspring
Mutations fail to complement
Alleles of same gene
Mutations complement
Mutations in different genes
Complementation analysis
Allows mutations to be put into groups corresponding to individual genes
Cross two different mutants
- If alleles of same gene - ¼ offspring will have mutant phenotype
- If mutations in different genes – no offspring will have mutant phenotype
Mutations affect gene function by changes in
- Regulatory sequence - affects transcription
- Non-coding sequence - affects RNA splicing, stability or translation
- Coding sequence - alters amino acids affecting protein folding - premature stop codon -> truncated
Transcription factor steps leading to transcriptional activation
- DNA binding
- Dimerisation
- Conformational change
- Transcriptional activation
Loss of function - amorphic
Inactivates DNA binding domain - complete loss of function
Early nonsense or detrimental missense mutation
Recessive
• +/- enough gene product from the one wild-type - haplosufficient
• -/- no transcriptional activation
Loss of function - hypomorphic
Weakens DNA binding domain - reduction of function
Missense or enhancer mutations
Recessive
• +/- enough gene product from the one wild-type copy
- Mutant may dimerize with wild type – transcriptional activation
• -/- poor transcriptional activation
Loss of function - antimorphic
Destroys dimerisation domain - competitive inhibitors
Dominant negative
• +/- mutant form binds DNA but does not dimerise - no transcriptional activation
• -/- mutant form poisons wild type protein - no transcriptional activation
Gain of function - hypermorphic
Activation that is independent of dimerization
Over expression of transcription unit or over activity of gene product
Dominant
• +/- mutant form binds DNA and is active all the time - constitutively active
• -/- the same
