Lec 21

Question 1

Forward genetic

Answer 1

using phenotypes to start, don’t need to know the gene. They are unbiased.

Question 2

Reverse genetics

Answer 2

using DNA to start

Question 3

Forward genetic screens begin with?

Answer 3

mutagenesis, an organism is treated with a mutagen to create mutations randomly throughout the genome

Question 4

Typical goal of forward genetics?

Answer 4

to achieve saturation mutagenesis meaning that every gene has undergone at least one mutation

Question 5

A good model organism for forward genetics

Answer 5

must be able to:

• progress through its life cycle in a laboratory
• have a short generation time
• produce a reasonable number of progeny
• be amenable to crossing
• organisms that are diploid usually have a starting genotype that is homozygous at all loci.

Question 6

choosing a mutagen for forward genetics

Answer 6

• the choice of mutagen depends on both the research organism and the type of mutant alleles desired
• chemical mutagens can induce many mutations, allowing saturation to be reached with only a few thousand individuals
• transposons can be used to induce mutations but result in fewer mutations, so saturation is difficult. However, they provide a DNA “tag” that makes finding and cloning the affected genes relatively easy

Question 7

In all mutagenesis used for forward genetic screens…

Answer 7

mutants of interest must be crossed to WT to ensure that the collected mutants have only the mutation of interest

Question 8

How can newly induced dominant mutations be identified in forward?

Answer 8

can be identified in the F1 generation produced by breeding the mutagenized males with WT females. Dominant mutations are relatively rare and they are usually gain of function.

Question 9

Recessive mutations in forward

Answer 9

loss of function mutations are much more common and usually recessive; therefore further breeding is required to make each mutation homozygous

Question 10

Screending for a dominant mutation

Answer 10

• Individual mutants in F1 are likely all different from another, this results in different genes or different mutations in the same gene.
• segregate in 1:1 ration

Question 11

Screening for recessive mutation

Answer 11

with saturation mutagenesis, most F1 progeny will carry at least one mutation contributed by a mutant sperm but you cant “see” it. You mate each of these F1 progeny with a WT female to obtain F2.
If the F1 male does carry a recessive mutation, 1/2 of F2 progeny will carry the mutation but you can’t see it. Need to interbreed F2 and screen F3.
-F3 will be a 3:1 ration of WT

Question 12

Screening for a recessive mutation in self-fertilizing organism

Answer 12

recessive mutations in plants or self-fertilizing animals are easier to identify, as self-fertilized F1 individual produce an F2 generation from which recessive mutations can be identified

Question 13

How can inefficiency of F3 screen be circumvented?

Answer 13

by using chromosomes that are marked and can be followed through generation. Balancer chromosomes in Drosophila allow specific chromosomes to be transmitted intact through multiple generations.

Question 14

Features of balancer chromosomes

Answer 14

1. one or more inverted segments to prevent transmission of chromosomes that have undergone crossing over (no homologous recombination)
2. a recessive allele resulting in lethality so that individuals cannot be homozygous for the balancer
3. A dominant mutation producing a visible phenotype so that segregation of the chromosome can be followed through generations

Question 15

Advantage of using haploid organisms in a forward genetic screen

Answer 15

both recessive and dominant mutations can be identified directly

Question 16

disadvantage of using haploid organisms in a forward genetic screen

Answer 16

mutations that cause lethality cannot be obtained. This problem can be circumvented by screening for conditional mutant alleles

Question 17

conditional alleles

Answer 17

• in conditional mutants, the enconded gene product is either functional or not needed under one condition- the permissive condition- but it is required and either absent or inactive under another condition- the restrictive condition
• in some lethal conditions, the mutant phenotype can be rescued by addition of a needed substance to the growth medium, or another condition, such as temperature, can be altered

Question 18

Null alleles

Answer 18

cells can’t divide so no colony of yeast with this allele/mutation can be recovered

Question 19

Temperature sensitive allele

Answer 19

gene product works fine at permissive temperature (23˚C). Yeast make colonies even if they contain a mutation in an essential gene for cell division

• the temp-sensitive mutants fall into categories based on the phenotype of the yeast after shifting them to be restrictive temp. (36˚C).

Question 20

Two approaches to physically identify specific genes

Answer 20

• certain genes can be identified by complementation of recessive mutant phenotype by introduction of a WT gene
• using DNA sequencing technology, genes identified in genetic screens may be found by comparing mutant and WT genome sequences

Question 21

how to determine the number of genes identified in the screen

Answer 21

complementation test

Question 22

if progeny produced by crossing two mutant alleles are WT the mutant alleles…

Answer 22

complement, affect two different genes

Question 23

If progeny of two mutant alleles are mutant…

Answer 23

the mutations affect the same gene, they fail to complement

Question 24

Genomic libraries

Answer 24

collections of cloned DNA fragments that represent the entire genome of an organism, including repetitive and noncoding sequences

Question 25

Complementary DNA libraries (cDNA)

Answer 25

represent only a portion of the genome and are collections of cloned DNA fragments that represent mRNA produced by an organism or cell type

Question 26

in both types of libraries, howre fragments carried?

Answer 26

cloning vectors

Question 27

genomics approach to gene identification following mutagenesis -1

Answer 27

• the mutant line is backcrossed with the WT strain

- F1 individuals are interbred and homozygous mutantts selected from the F2 generation