Rules: Cell phones offComputers only for class related workNo food or drink in lab room

Text Book: Hartwell et al., 2nd Edition 2004

Genetics from Genes to Genomes Mc Graw-Hill, Boston.

Web page: www.bio.unc.edu/courses/2006Fall/Biol423L

Biol 423L Laboratories in Genetics

Goals for course:

Reinforce basic genetic principles

Introduce model organisms commonly used by geneticists

Learn how genetics is used to understandDiseaseBiochemical pathwaysDevelopment

Lab reports:

AbstractIntroductionResultsDiscussion

Course information page has instructions about preparing your lab reports.

Grading:

Lab Reports: 50% of grade5% of that is participation1 day late, 50% offmore than that will only be graded under special circumstances.

Research Paper: 10% of gradeTopics due Oct. 9.Outline due Oct. 23.Paper due Nov. 29

2 quizzes: 10% each of final grade.Oct. 2 and Nov. 13.

Final exam: 20% of final grade comprehensiveDec. 11.

Genes Alleles and Epistasis

Genetics starts with observation

Use genetics to understand the cause of the variability.

What proteins or RNAs are responsible for the variability you can see?

Observe variability

Easy example, flower color

How many genes affect flower color?

How variable are the proteins encoded by those genes?

What is the pathway to make flower color?

List of terms:

Trait: some aspect of an organism that can be observed, measured

Phenotype: the way a trait appears in an individual, the combinationof genotype and environment.

Genotype: the constitution of alleles at any gene in an individual.

Gene: continuous stretch of DNA sufficient to encode a messengerRNA or a functional RNA.

Locus: A region of a chromosome, usually for a single gene.

Messenger RNA: the RNA message for a single protein.

Allele: a variant of the sequence of a given gene.

Diploid: an individual with two copies of each chromosome.Haploid: an individual with one copy of each chromosome.

How many genes affect flower color?

How variable are the proteins encoded by those genes?

What is the pathway to make flower color?

First make sure the types are heritable and true breeding

(homozygous for flower color alleles)

X

purple by purple (self) All uniform

Homozygous: a diploid individual with two copies of the sameallele for a given gene.

Heterozygous: a diploid individual with two different alleles for a given gene.

What are the relationships between color types?

Purple is dominant to Red

X

What are the relationships between color types?

Purple is dominant to white

X

Complementation tests can be madebetween recessive alleles.

A dominant allele cannot be used.

Why?

1. How many genes are required to makepurple pigment in flowers?

Allelism test:Cross different white flowered plants

If the mutations are in the same gene,The progeny will be white

White 1

X

White 1

Allelism test:Cross different white flowered plants

If the mutations are in different genes,The progeny will be pigmented

White 1

X

White 2

Allelism or complementation test

If plants with recessive alleles arecrossed and the progeny also have the recessive trait, The alleles are variants of the same gene

If plants with recessive alleles arecrossed and the progeny have the dominant trait, The alleles are variants of different genes

Precursor 1

Intermediate

White1

White2

Purple

Pathway to purple

White 1

White 2

X

Purple

rrAA RRaa RrAa

Using multiple allelism tests with diverse recessive mutants,

We can identify all the genes specificallyinvolved in making the purple pigment

Genetics can be used to determine the Order of steps in a biological pathway

Epistasis tells which gene productsact earlier or later in a process.

What are the relationships between color types?

Purple is dominant to White 1

X

F2 1 RR, 2Rr and 1rr

X

Purple is dominant to White1

PurpleRR

White1rr

F1X

PurpleRr

Punnet square

r

r rr

Female gametesMale gametes

R

R RR Rr

Rr

What are the relationships between color types?

Purple is dominant to Red

XPP

Pp

pp

Epistasis

Two genes for flower color

Two steps in a pathway to make pigment

Where are the two genes in the pathway?

Purple is either a mixture of blue and red pigments

or

Purple results from modification of the same precursor from a white precursor to a red intermediate and finally a purple pigment.

We can use genetics to distinguish the two possibilities.The effect of variant alleles in multiple genes that affect pigment in combination will answer the question.

Precursor 1 Precursor 2

Blue Red

Precursor 1

RP

Red

R

P

Purple

Pathway 1 Pathway 2

Coexpression of Blue and red pigmentderived from different precursorsMakes purple

Modification of the sameprecursor leads to first a red pigment and then a purple pigment

Relationship between White1 and Red

X

X

White1rrPP

RedRRpp

F1 is all PurpleRrPp

F2

9 43

Punnet Square: two genes with randomly segregating alleles

Male gametes

Female gametes

RP

Rp

rP

rp

rprPRpRP

RRPP

RRPp

RRPp RrPP RrPp

RRpp RrPp Rrpp

RrPP RrPp rrPP

rrppRrPp Rrpp rrPp

rrPp

9R_P_ 3R_pp 3rrP_ 1rrpp

RrPp X RrPp

9R_P_ 3R_pp 3rrP_ 1rrppPhenotypes:

Purple WhiteRed White

Precursor 1

Red

R

P

Purple

No R - get no red precursorNeither purple nor red pigment can be made

White - no pigment

No P – get red pigment but not purple

Precursor 1 Precursor 2

Blue Red

RP

If Pathway 1

Coexpression of Blue and red pigmentderived from different precursorsMakes purple

9R_P_ 3R_pp 3rrP_ 1rrppPhenotypes:

Purple WhiteRed Blue

R no P would make red pigment only

No R but P would make blue pigment,

Fact that loss of R changes phenotype to rr even if Functional P is expressed indicates that P and R affecta common pathway and R is before P in the pathway to make pigment.

Yeast complementation test for next week:

Brewers YeastSaccharomyces cerevisiae:

16 chromosomes12,052 kb DNA6183 ORFsAbout 5800 expected to encode proteins

Yeast is a very useful model for genetics because of its life cycle

Haploid life cycle

Yeast is a very useful model for genetics because of its life cycle

Mating cycleDiploid

We can isolate mutants as haploids

We can test the mutations for allelism by a complementation test

Two haploids are mated. The resulting Diploid has both mutations.

Either the mutations are allelic and do not complement,or they are mutations in two different genes and they do complement.

a1a2

Select mutants that are defective in Adenine synthesis- cannot grow without adenine in medium.

Turn red on media with adenine because an adenine precursor accumulates.

a1

a1

a2

a1

a1

X

X

a1

a1

a2

Which mating results in complementation?

Lab experiment: Corn kernels

R and P

RR or Rr is full color

rr is no color

PP or Pp are full color

pp is weak color

Question is pprr weak color?P epistatic to R or

Is pprr no color? R epistatic to P

End