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Biol 423L Laboratories in Genetics
Rules: Cell phones off
Computers only for class-related work
No food or drink in lab room
Text Book: Hartwell et al Genetics from Genes to
Genomes, third edition
Web page: www.bio.unc.edu/courses/2009Fall/Biol423L
Goals for course:
Reinforce basic genetic principles
Introduce model organisms commonly
used by geneticists
Learn how genetics is used to understand
Disease
Biochemical pathways
Development
Lab reports:
Abstract
Introduction
Results
Discussion
Course information page has instructions
about preparing your lab reports.
Grading:
Lab Reports: 50% of grade
5% of that is participation
1 day late, 50% off
more than that will only be
graded under special circumstances.
Research Paper: 10% of grade
Topics due Oct. 13.
Outline due Oct. 27.
Paper due Nov. 24.
Midterm: 15% of final grade.
Oct. 26
Final exam: 25% of final grade
comprehensive
Dec. 14.
Genes, Alleles and Epistasis
Genetics starts with observation
Observe variability
Use genetics (patterns of inheritance)
to understand the cause of the variability.
What proteins or RNAs are
responsible for the variability you can see?
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 combination
of genotype and environment.
Genotype: the constitution of alleles at any gene in an individual.
Gene: continuous stretch of DNA sufficient to encode a messenger
RNA 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?
First make sure the types are heritable
and true breeding
(homozygous for flower color alleles)
purple by purple (self)
All uniform
X
Homozygous: a diploid individual with two copies of the same
allele for a given gene.
Heterozygous: a diploid individual with two different alleles for
a given gene.
What are the relationships
between color types?
X
purple is dominant to white
Alleles are distributed as
discrete units
X
Purple W/W
White A wa/wa
F1 W/wa
Punnet square helps to predict genotypes
and phenotypes of the next generation
Two distinct alleles at the same locus
X
F1 W/wa
F1 W/wa
1 W/W: 2 W/wa: 1 wa/wa
3 purple: 1 white
Female gametes
Male
gametes
W
wa
W
W/W
W/wa
wa
W/wa
wa/wa
How many genes are required to make
purple pigment in flowers?
Complementation tests can be made
between recessive alleles.
If plants with recessive alleles are
crossed and the progeny also have
the recessive trait,
The alleles are variants of the same gene
If plants with recessive alleles are
crossed and the progeny have
the dominant trait,
The alleles are variants of different genes
A dominant allele cannot be used.
Why?
Allelism test 1:
Cross different white flowered plants
If the mutations are in the same gene,
The progeny will be white
X
white A wa/wa
white B wb/wb
F1 = wa/wb
Complementation test double check
F2 generation:
Cross white F1 to another white F1
If the mutations are alleles of the same
gene, what is the next generation?
wa/wb
X
wa/wb
1 wa/wa, 2 wa/wb, 1 wb/wb
Allelism test 2:
Cross different white flowered plants
If the mutations are in different genes,
the progeny will be pigmented
X
white A wa/wa;Wc/Wc white C Wa/Wa;wc/wc F1 Wa/wa; Wc/wc
Conclusions
Wa and wa are alleles of the same gene
wa and wc are alleles of different genes.
The dominant allele of wa and
the dominant allele of wc are needed for
purple color to be produced.
Therefore, at least 2 gene products are needed
to produce purple pigment.
To avoid confusion, let’s call Wa and wa: R and r
and wc: p with a dominant allele P.
Allelism test:
Cross different white flowered plants
If the mutations are in different genes,
The progeny will be pigmented
X
white A r/r; P/P
white C R/R; p/p
F1 R/r; P/p
white C
purple
X
white A
rrPP
RRpp
RrPp
Pathway to purple
Precursor 1
R or P
Intermediate
R or P
Purple
Complementation test double check
The discrete alleles of two different genes
Will assort randomly in future generations
X
white A r/r; P/P
white B R/R; p/p
F1 R/r; P/p
Punnet Square:
Predict the genotypes and phenotypes in the
F2 generation when the trait is controlled by
two genes with randomly segregating alleles
F2 after RrPp X RrPp
Male
gametes
Female gametes
Rp
RP
RP RRPP
RRPp
rP
RrPP
rp
RrPp
9R_P_ 3R_pp 3rrP_ 1rrpp
Rp RRPp
RRpp
RrPp
Rrpp
Phenotypes: if both R and
P needed for purple color
rP
RrPP
RrPp
rrPP
rrPp
9 purple and 7 white
rp RrPp
Rrpp
rrPp
rrpp
Using multiple allelism tests with
diverse recessive mutants,
We can identify all the genes specifically
involved in making the purple pigment
Predict the genotypes and phenotypes in the
F2 generation when the traits are independent.
Eg. petal color and leaf size.
Punnet Square:
Predict the genotypes and phenotypes in the
F2 generation when the traits are independent.
Eg. petal color and leaf size.
RrPp X RrPp
Male
gametes
Female gametes
Rp
RP
rP
rp
RP RRPP
RRPp
RrPP
RrPp
Rp RRPp
RRpp
RrPp
Rrpp
rP
RrPP
RrPp
rrPP
rrPp
rp RrPp
Rrpp
rrPp
rrpp
9R_P_ 3R_pp 3rrP_ 1rrpp
Phenotypes: if R and
P affect independent traits
Eg. petal color and leaf size
R- is purple, rr is white
P- is long leaf and
pp is short leaf
9 purple, long;
3 white, long;
3 purple, short;
1 white, short
Calculate ratios with more loci:
probability of RR or Rr is 3/4
probability of rr is 1/4
3 loci; all dominant: ¾ X ¾ X ¾
all recessive: ¼ X ¼ X ¼
one dominant and two recessive: ¾ X ¼ X ¼
Ad-infinitum
Chi-square test for goodness of fit
Null hypothesis: the alleles that control petal color
and leaf size represent two different genes
segregating independently.
Does the data fit your model?
n
Χ2 = Σ (Oi-Ei)2/Ei
i=1
n is the number of types of observations,
ie. the number of different phenotypic classes
Degrees of freedom = n-1
p is probability that the null hypothesis is correct
When the observations are similar to the expected
values, Χ2 is a small number and p is close to 1.0
X2 values for different degrees of freedom
and the probabilities associated with the X2
values
Mendel’s Laws
Mendel's First Law - the law of segregation;
during gamete formation each member of the
allelic pair separates from the other member
to form the genetic constitution of the gamete
Mendel's Second Law - the law of independent assortment:
this says that for two characteristics, the genes are
inherited independently.
Exceptions: Maternal inheritance
Maternal Inheritance
Some traits are encoded by genes in
cytoplasmic organelles
Eg. Mitochondrial traits
Eg. Chloroplast traits in plants
Organelles are transferred to an embryo from
the egg, not the sperm. The organelles are haploid
and (usually) genetically uniform in eggs.
Therefore the trait of the mother will be passed to
all offspring.
Examples of maternally inherited traits?
Mitochondrial:
Mitochondrial myopathy
Diabetes mellitus and deafness
Leber's hereditary optic neuropathy
Chloroplast:
White leaves – loss of chlorophyll, often partial
Yeast complementation test for next week:
Brewers Yeast
Saccharomyces cerevisiae:
16 chromosomes
12,052 kb DNA
6183 ORFs
About 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 cycle
Diploid
Advantages of yeast for identification
of genes in a biosynthetic pathway
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.
a2
a1
a1
Select mutants that are defective in
Adenine synthesiscannot grow without adenine in medium.
Turn red on media with adenine
because an adenine precursor accumulates.
X
a1
a2
X
a1
a1
a2
a1
a1
Which mating results in complementation?
Summary of Lecture 1:
Mendelian Genetics: Mendel’s laws,
Segregation of two alleles at one locus
Segregation of two alleles at two independent loci
Punnet square, calculate expected ratios of phenotypes
Chi-square test to test if observed results can be
explained by the model of choice.
Allelism tests
Yeast as a model, haploid and diploid life-style
End