identifying and analysing genes

Question 1

how would you find the location of genes in the nucleotide sequence?

Answer 1

• use gene prediction software to scan sequence for promoters, start/stop sequences and intron splice sites
• translate the dna in all 6 reading frames and search for similarity to known proteins using BLAST software

Question 2

what does similarity between protein sequences suggest?

Answer 2

• proteins evolved from the same common ancestor

- proteins have similar functions

Question 3

describe how BLAST is used to find the location of genes in the nucleotide sequence

Answer 3

1. input AA sequence of unknown protein
2. BLAST searches databases for other proteins with similar sequences
3. alignment of unknown protein sequence to all proteins of database

Question 4

what are est = expressed sequence tags?

Answer 4

short sequences from the ends of cDNA

Question 5

DEFINE: transcriptome

Answer 5

all genes expressed in a cell

Question 6

reverse genetics

Answer 6

understanding gene function by analysing phenotype of disrupted gene

Question 7

what techniques are used to perform reverse genetics?

Answer 7

• gene replacement

- gene KO

Question 8

what is gene replacement

Answer 8

making small changes to the endogenous gene

Question 9

describe the process of a gene KO

Answer 9

1. obtain a clone of the gene in a plasmid
2. use genetic recombination to insert:
   - NEO - inserted into exon. destroys gene activity.
   - TK - placed off to one side
3. introduce construct into mouse ES cells using homologous recombination - TK gene lost
4. stimulate mouse to produce embryos
5. insert mutant ES cells into early embryo
6. early embryo partially formed from ES cells
7. reimplant early embryo into recipient mother -> transgenic pups born = 1st generation
8. some mutant cells will give rise to gametes
9. breed 1st gen mice to form heterozygous mice = 2nd generation
10. interbreed 2nd gen mice to create homozygous mutants = 3rd gen

Question 10

during a gene KO, how are colonies with the gene successfully knocked out selected for?

Answer 10

select for colonies arising from homologous recombination instead of non-homologous recombination (involves homologous arms)

1. grow cells on neomycin - no NEO? -> cells die
2. grow cells on GANC - TK integrated? -> cells die

Question 11

forward genetics

Answer 11

identify genes responsible for a phenotype

Question 12

describe the process of performing a forward genetics analysis

Answer 12

1. mutagenise males of p0 generation
2. f1 generation = each sperm has a different set of heterozygous mutations
3. cross mutagenised males with WT females = F2 gen
4. incross f2 gen to obtain homozygous offspring (1/4 of f3 gen)
5. screen f3 gen for interesting phenotypes to identify genes affecting a specific process
6. perform complementation analysis
7. sort mutations into distinct groups corresponding to individual genes

Question 13

what is a missense mutation?

Answer 13

amino acid substitution

Question 14

what is a nonsense mutation?

Answer 14

early stop codon –> truncated protein

Question 15

what is an amorphic mutation?

Answer 15

missense mutation completely inactivating DNA binding domain

Question 16

what is a hypomorphic mutation?

Answer 16

missense mutation weakening DNA binding domain

Question 17

what is an antimorphic mutation?

Answer 17

missense mutation destroying dimerisation domain

Question 18

what is a hypermorphic mutation?

Answer 18

missense mutation resulting in activation independent of dimerisation