Gene mutation origins and repair processes2

The production of a wild type phenotype when two different mutations are combined in a diploid... How do geneticists study gene function?Disrupt the gene and analyze the resulting phenot

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

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 a 1 and b 1

a 1 and b 1are recessive alleles

Both a 1 / a 1 and b 1 / b 1 mice have no fur

She wants to figure out if a 1 and b 1 are alleles of the same gene

a 1 / a 1 X b 1 / b 1

All progeny have fur Therefore a 1 and b 1 complement

This indicates that a 1 and b 1 are alleles of different genes (99.9% of the time)

a 1 and b 1are recessive alleles.

All progeny have fur

More on complementation

Her labmate finds another recessive allele, c 1

The phenotype for homozygous c 1 /c 1 mice is that they have no fur

He tests if c 1 complements a 1 and b 1

c 1 /c 1 X a 1 /a 1 : all progeny have fur

c 1 /c 1 X b 1 /b 1 : no progeny have fur

He concludes that c 1 complements a 1 , but not b 1

Therefore, c 1 and b 1 are alleles of the same gene (99.9% of time)

c 1 and b 1 are in the same complementation group

No progeny have fur

c 1 and b 1 fail to complement; c 1 and b 1 are mutant alleles of the same gene

c 1 is a recessive allele

Using deficiency/deletion chromosomes to map mutations

c 1

c 1

df1

df2

df1 fails to complement c 1 , df2 complements c 1

Therefore, c 1 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.

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 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

3 Scientist showed the coat color genes were linked (on) to the

X-chromosome

4 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

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QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.

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 duplicationsmicroarray/ DNA Chip

DNA from mutant Cy5

DNA from wild type Cy3

Cy5/Cy3=2duplication

Cy5/Cy3=15tandem

duplicationmutant/wild type

Chapter 12

Mutational Dissection

TGF-  gene distribution

in human genome

Internal organ placement in

normal girl and girl affected

with situs inversus

Caused by mutation in

TGF- gene

What does TGF- protein do?

How can we study the function of TGF-?

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)

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

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

Antisense RNA-translation of RNA inhibited

RNAi: Short double-stranded RNA molecules direct an RNA/protein complex to degrade mRNA

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

– 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

Modifier Screen

•Secondary screens

–search for mutations that alter mutant phenotype modifier mutations

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Screen based on gene expression

Enhancer A Enhancer B

“Enhancer Trapping”

Linkage mapping and

• 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