Gene Mutation: Origins and Repair Processes2

Review of some concepts from Chapter 11
Chromosome mutation


This chart describes chromosome constitutions for a normally diploid animal.
An individual that is normally diploid and has only one chromosome set is called monoploid to distinguish it from individuals that are normally haploid.
The terms, monosomic, disomic and trisomic are used to describe aneuploid conditions.

Aneuploid: an individual organism whose chromosome number differs by part of a chromosome set.

The terms haploid, monoploid, diploid and triploid are used to describe multiples of the basic chromosome set.


Complementation:
The production of a wild type phenotype when two different mutations are combined in a diploid.
A scientist has identified two new alleles she calls a1 and b1.

a1 and b1are recessive alleles.

Both a1/ a1 and b1 / b1 mice have no fur.

She wants to figure out if a1 and b1 are alleles of the same gene.

a1/ a1 X b1 / b1
All progeny have fur. Therefore a1 and b1 complement.
This indicates that a1 and b1 are alleles of different genes (99.9% of the time).

a1
a1
b1
b1
b+
X
a1/ a1 X b1 / b1
a1
b1
a1 and b1 complement. a1 and b1 are mutant alleles of different genes.
a1 and b1are recessive alleles.
All progeny have fur
b+
a+
a+
a+
b+
More on complementation

Her labmate finds another recessive allele, c1.
The phenotype for homozygous c1/c1 mice is that they have no fur.

He tests if c1complements a1 and b1
c1/c1 X a1/a1 : all progeny have fur
c1/c1 X b1/b1 : no progeny have fur

He concludes that c1 complements a1, but not b1.
Therefore, c1 and b1 are alleles of the same gene (99.9% of time).
c1 and b1 are in the same complementation group.
b1
b1
X
c1
c1
c1/c1 X b1/b1 : no progeny have fur
c1
b1
No progeny have fur
c1 and b1 fail to complement; c1 and b1 are mutant alleles of the same gene.
c1 is a recessive allele
Using deficiency/deletion chromosomes to map mutations
c1
c1
df1
df2
df1 fails to complement c1 , df2 complements c1.
Therefore, c1 is contained in the region deleted in df1.
red square indicates the region of chromosome deleted.
More on Deletion/Deficiency Mapping
pn (prune): only deletion 264-38 fails to complement
fa (facet): all but 258-11 and 258-14 fail to complement
pn/df 264-38 ;see pruned phenotype. pn/ any of the other df; see wild type phenotype.
Therefore, pn contained in 2D4-3A2, or 3E1-3E2.
fa is in 3C7
Mechanisms of Dosage Compensation
mammals
XX (female) XY (male)
One X chromosome is inactive in females.
Called X inactivation. The inactive X is called a Barr body.
C. elegans (nematode)
XX (hermaphrodite) XO (male)
Both X chromosomes produce 1/2 the gene product (hypotranscription) in hermaphrodites as compared to males.
Hermaphrodites have both male and female internal genitalia and produce both eggs and sperm.
Drosophila
XX (female) XY (male)
The one male X chromosome is hypertranscribed.
Historical perspective on the discovery of X chromosome inactivation in mammals
1. In 1953 Dr. Mary Lyon made this observation about mouse coat color: only females of certain strains showed spotting or mottling, not males.
Assume genes for blue and yellow coat are on the X
yellow+
blue +
yellow -
blue -
In female mice some of the cells will have the maternal X and some will have the paternal X active. Therefore, some cells will produce yellow fur and some blue.
Males will either have only the maternal X active; therefore no spotting.
Hypothetical fictional example:
Historical perspective 2

2. Dr. Barr and colleagues in 1949 were staining neurons with dyes that bind DNA and noticed that there was a densely stained structure in neurons that were derived from females, but not in neurons derived from males. That is why the inactive X is called the Barr body.

Scientist showed the coat color genes were linked (on) to the X-chromosome.

Found a mouse that was XO. She was viable and fertile. This suggested that only one X chromosome was required for normal development. Therefore, it was postulated that normal female mice have only one X chromosome active (in XX females, only one X active per cell, and it can be either the maternal X or paternal X).
Many years of work have substantiated this postulate.
Image from
Developmental Biology
Gilbert
6th edition
Image from Akhtar Group web page
(b-d) Xist RNA-light blue
pgk mRNA-red
Using genomic approaches to identify deletions and duplications
microarray/ DNA Chip
DNA from mutant Cy5
DNA from wild type Cy3
A. Mutant has a deletion (chr. 1)
B. Mutant has a duplication
(chr 4)
Cy5/Cy3=1
normal chromosome
Cy5/Cy3=.5
deletion
Cy5/Cy3=2
duplication
Cy5/Cy3=15
tandem duplication
mutant/wild type
Chapter 12
Mutational Dissection
TGF- b gene distribution
in human genome
Internal organ placement in normal girl and girl affected with situs inversus.

Caused by mutation in TGF-b gene
What does TGF-b protein do?

How can we study the function of TGF-b?

Can study in model systems like Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, and Mus musculus.

Scientist have completed genome sequence, have tools to map genes, and introduce genes into genome.

Genes are conserved. Organisms have genes from a common ancestor.
How do geneticists study gene function?

Disrupt the gene and analyze the resulting phenotype
Forward genetics: Classical approach to genetic analysis. Genes are first identified by their mutant phenotype and mutant alleles, and then subsequently cloned and analyzed.
Reverse genetics: Scientist begins with cloned gene and sequence information. He/she introduces engineered mutations into the genome to investigate the function.
Reverse genetics
Forward genetics
Designing a genetic screen (forward genetics)
Different mutagens give rise to different DNA changes (chapter 10)
Genes have different mutational target sizes (forward genetics)
Directed Mutations: You start with a gene and want to know the gene’s mutant phenotype (reverse genetics)
Gene knockouts are generated by homologous recombination
Site directed mutagenesis (reverse genetics)
Shown here is a scheme for mutating a gene cloned into a circular plasmid.
An example of when you want to perform site directed mutagenesis
clone a gene that shares sequence identity to a DNA binding protein
ADRTSVCGNSVTNPIL

AGRTSVTILNSVTNRA
Amino acid sequence of known hypothetical DNA binding protein
arrow indicates residue know to be important for DNA binding
Amino acid sequence of your new cloned gene
With site directed mutagenesis can determine if that residue is important for DNA binding in the product of the gene you cloned
Antisense RNA-translation of RNA inhibited
mRNA is considered sense strand;
antisense is complementary strand
phenocopy: mimicking a mutant phenotype by manipulating something in the interior environment of the cell.
RNAi: Short double-stranded RNA molecules direct an RNA/protein complex to degrade mRNA
RNAi will phenocopy a mutation at a DNA locus.
Scientist introduces double-stranded RNA (dsRNA) into cell that is homologous to a gene/transcript.

dsRNA is cleaved into small RNAs (siRNAS). Enzyme called Dicer.

These serve as templates for the RNAi pathway directing cleavage of the mRNA via RISC complex.

Cells have endogenous small RNAs that can also silence genes (miRNAs)
Chemical Genetics
Somatic vs Germline mutation
In haploid organisms or on the sex-chromosome in diploids, both dominant and recessive alleles can be identified in the F1
Specific Locus Test: Want to identify new recessive mutations in gene c.
Genetic Screen vs
Genetic Selection
Types of genetic selection
auxotroph:
a strain that will proliferate only when the medium is supplemented with a specific-substance not required by wild type
Can be applied to any problem, depending upon ingenuity and resources
Biochemical mutations
screening for auxotrophs from mutagenized prototrophs

Morphological mutations
change in shape or form

Lethal mutations
premature death
recessive lethals are more useful than dominant lethals that are difficult to maintain
Genetic screens
A geneticist can screen for a mutation affecting any phenotype.
As long as you can score the phenotype you can screen for mutations that affect the biological process
Morphological Mutation
Conditional mutations
display wild-type under permissive (nonrestrictive) conditions
display mutant phenotype under restrictive conditions
e.g., temperature-sensitive mutations

Behavioral mutations

Behavioral screen
Temperature sensitive mutation:
An example is a mutation in a protein required for cell division that becomes unstable at high temperature
18°C-no phenotype
29°C-cell division phenotype observed
Modifier Screen
Secondary screens
search for mutations that alter mutant phenotype

modifier mutations
Screen based on gene expression
Enhancer A
Enhancer B
“Enhancer Trapping”
Linkage mapping and complementation analysis.
Sort through the mutations identified
Both gain-of-function and loss-of-function can be dominant or recessive

Loss-of-function
partial or complete elimination of activity of gene’s encoded product
Gain-of-function
hypermorph: more gene activity
neomorph: novel gene activity

A single gene can have both loss-of-function and gain-of-function alleles.
Remember can have different alleles of same gene
Determining the type of allele generated
Distinguishing between loss- and gain-of-function mutations
Null
Hypomorph
Gain-of-function
Neomorph
not always true