L13 - Identifying and Analysing Genes

Question 1

Annotating genomic sequence using gene prediction software

Answer 1

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)

Question 2

Blast protein alignment method

Answer 2

1. Input amino acid sequence of proposed protein
2. Blast program searches huge databases for other proteins with similar sequences
3. Shows alignment of uncharacterized protein (query) to a protein called zen (subject)
4. Similarity between protein sequence suggests proteins evolved from common ancestor

Question 3

Microarrays role

Answer 3

Allow us to compare the transcriptomes of different tissues to each other
High throughput- small scale, fast and automated

Question 4

Using microarrays to determine expression profile of liver method

Answer 4

1. A precise robot manufactures the array – one sport for every gene
   a. Each position in the grid contains one cDNA - as the antisense strand
2. Purify mRNA from liver tissue and tag with a fluorescent dye
3. Put mRNA onto the array – hybridization to cDNA on the array
4. Rinse off any excess, unhybridized mRNA
5. Reader with a sensitive camera detects which genes are on

Question 5

Microarray experiments usually involve comparing two samples

Answer 5

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

Question 6

The three ways to identify genes

Answer 6

Library of cDNA clones from mRNA
Library of genomic clones and make predictions based upon genomic sequence
Microarrays

Question 7

Gene replacement

Answer 7

Makes small change to endogenous gene

Test whether human mutation causes disease symptoms in mouse by making the same change in corresponding mouse gene

Question 8

Gene knock-out

Answer 8

Completely remove gene to determine its function

To begin a knock-out project, you must first have a genomic clone of your gene

Question 9

Gene knock-out in mice method

Answer 9

1. Get genomic clone of gene and insert NEO directly into an exon
   - Destroys activity of gene
   - TK is placed off to one side
2. Introduce construct into mouse ES cells using cell culture techniques
3. Cell’s DNA repair machinery recombines the construct into the mouse genome – inefficient
   - Homologous recombination sometime occurs - knock-out - TK gene is lost

Question 10

What are homologous arms?

Answer 10

Sequences from target gene

- Only sequence with homology to the mouse genome

Question 11

Double selection to identify the knock-out method

Answer 11

Use selectable markers (Neo/TK) to identify colonies that are the result of homologous recombination

Question 12

Positive selection

Answer 12

Cells that have integrated Neo gene - grow in Neomycin containing media

Question 13

Negative selection

Answer 13

Cells that have integrated TK gene and Neo - die when grown in GANC media

Question 14

After targeting a single gene in mice - 1st generation

Answer 14

Mixture of cells from stem cell line and mother

Their gonads are also mosaic

Question 15

After targeting a single gene in mice - 2nd generation

Answer 15

Mosaic animals bred to generate non- mosaic carriers of transgene

Question 16

After targeting a single gene in mice - 3rd generation

Answer 16

Carriers interbred to create homozygous mutant animals

Question 17

Forward genetics method

Answer 17

1. Randomly mutate genome
2. Look for interesting phenotypes in offspring
3. Identify gene that causes the defect

Question 18

Random mutagenesis affects the whole genome

Answer 18

Have to analyse many mutagenized animals

• Yeast
• C.elegans
• Drosophila
• Zebrafish

Question 19

Different types of genetic screens

Answer 19

• 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

Question 20

Forward genetics vs reverse genetics

Answer 20

Forward genetics: function (phenotype) —-> gene

Reverse genetics: gene —-> function (phenotype)

Question 21

Forward genetic screen in flys method

Answer 21

1. Mutagenize male - each sperm has different set of mutations
   - EMS - chemical mutagen
   - Male is heterozygous for the mutations carried by the parental sperm
2. Outcross males to wildtype females
3. Both offspring heterozygous
4. Incross to identify homozygous embryos - 1/4 of offspring

Question 22

Mutations fail to complement

Answer 22

Alleles of same gene

Question 23

Mutations complement

Answer 23

Mutations in different genes

Question 24

Complementation analysis

Answer 24

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

Question 25

Mutations affect gene function by changes in

Answer 25

• 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

Question 26

Transcription factor steps leading to transcriptional activation

Answer 26

1. DNA binding
2. Dimerisation
3. Conformational change
4. Transcriptional activation

Question 27

Loss of function - amorphic

Answer 27

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

Question 28

Loss of function - hypomorphic

Answer 28

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

Question 29

Loss of function - antimorphic

Answer 29

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

Question 30

Gain of function - hypermorphic

Answer 30

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