forward genetics

Question 1

the forward genetics approach seeks to..

Answer 1

find the genes encoded by DNA that are responsible for a phenotype of interest

Question 2

is forward or reverse genetics the classical genetic strategy

Answer 2

forward

Question 3

forward genetics starts with the..

Answer 3

phenotype

Question 4

mutants and forwards genetics

Answer 4

mutated genes allow us to figure out which genes responsible for specific phenotypes. e.g. if specific genes are artificially mutated then this will show up as a faulty phenotype and can be identified

Question 5

simple process of forward genetics (4)

Answer 5

1. isolate mutants with a phenotype of interest 2. define gene responsible for these phenotypes 3. clone.identify genes 4. analyse the genes to predict encoded proteins

Question 6

1.isolate mutants with a phenotype of interest

Answer 6

using model organism

mutagenesis:

• physical mutagens (UV)
• chemical (base analogies)
• biological mutagen

-screen for desired phenotype

Question 7

screening for desired phenotype

Answer 7

for essential cell function there are conditional lethal point mutants e.g. temp sensitive mutants -mutant protein is functional at permissive temp -non-function at non-permissive temp

Question 8

2.define genes responsible for these phenotypes

Answer 8

gene complementation test an determine if a phentope arises from mutations on the same or separate genes

e. g. white mutants of a normally purple diploid organism -two diff mutant organisms white 1x purple and white 2 x purple both causes a 3:1 purple ration
- however are the mutations present on the same chromosomes e.g. the genes will be on the same chromosome if they do not complement eachother

Question 9

3. clone./identify the genes

Answer 9

cloning by mutant rescue e.g. using a gene library to transform cells. Then isolate transformants which rescue or restore a wild-type phenotype e.g. identify cells that have the mutation and clone

Question 10

mutant rescue

Answer 10

using a gene library to transform cells. Then isolate transform-ants which rescue or restore a wild-type phenotype

Question 11

cell cycle and gene determination

Answer 11

we can look at cell cycle and determine the role of specific genes- since diff genes have diff roles in the cycle e.g. if one turns off the nda3 gene, G1 will not begin

Question 12

gene library

Answer 12

A genomic library is a collection of the total genomic DNA from a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA.

Question 13

4. analyse the genes to predict encoded proteins

Answer 13

-compare predicted sequence with database sequences

BLAST-basic local alignment search tool- gives clues as to function, which can be tested for

e. g. if you look at the proteins encoded for by genes found at specific points of cell cycle gene libraries can then be used to compare known DNA sequence for genes in one organism to compare to genes in another organism
e. g. when they used s.pombe to find the CDC2 mutant, they also discovered it in the human cDNA library

Question 14

CDC2

Answer 14

found in all eukaryotes snd produces cyclin dependent kinases, which control cell cycle

Question 15

s.pombe experiment and gene library and the discovery of CDC2

Answer 15

s.pombe double stranded DNA is cleaved by restriction endonuclease–> cut into millions of tiny little segments and inserted into plasmids. -these recombinant plasmids are then introduced into bacteria–> genetic library for s.pombe mutant rescue in S.pombe to discover CDC2 gene

Question 16

mutant rescue in s.pombe

Answer 16

conditional lets s.pombe (temp sensitive) is crossed with s.pombe wild top library in plasmid replicating vector. next: transform and selection those cells which survive at 36 degrees have their plasmids recovered (in e.coli) and this gives us a cloned CDC2 gene

Question 17

what principally control cell cycle

Answer 17

protein phosphorylation–> therefore controlled by CDKs (cyclin dependent kinases)

Question 18

cdk

Answer 18

cyclin dependent kinases

Question 19

how were CDKs discovered

Answer 19

by genetic analysis of the cell cycle

Question 20

what can we use to estimate relatedness of species

Answer 20

phylogenetics

Question 21

phylogenetics can be used to

Answer 21

estimate the relatedness between species based on sequence data

Question 22

forward genetics has been used to..

Answer 22

understand relationships e.g. using BLAST similarities between organismic can be found and similarity often infers homology e..g. if genes are similar then similar proteins will be coded for

Question 23

divergence between two homologues sequences is proportional to

Answer 23

mutation/recombination rate

Question 24

phylogenetic can be examined using

Answer 24

phylogeny trees

Question 25

BLAST

Answer 25

BASIC LOCAL ALIGNMENT SEARCH TOOL -BLAST finds regions of similarity between biological sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance.

Question 26

orfs found in mutant rescue

Answer 26

(ORF) is the part of a reading frame that has the potential to be translated