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Rules: Cell phones offComputers only for class-related workNo food or drink in lab room
Text Book: Hartwell et al Genetics from Genes toGenomes, third edition
Web page: www.bio.unc.edu/courses/2009Fall/Biol423L
Biol 423L Laboratories in Genetics
Reinforce basic genetic principles
Introduce model organisms commonly used by geneticists
Learn how genetics is used to understandDiseaseBiochemical pathwaysDevelopment
Goals for course:
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. 13.Outline due Oct. 27.Paper due Nov. 24.
Midterm: 15% of final grade.Oct. 26
Final exam: 25% of final grade comprehensiveDec. 14.
Genetics starts with observation
Use genetics (patterns of inheritance) to understand the cause of the variability.
What proteins or RNAs are responsible for the variability you can see?
Observe variability
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.
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.
Punnet square helps to predict genotypes and phenotypes of the next generation
wa
wa wa/wa
Female gametes
Male gametes
W
W W/W W/wa
W/wa
F1 W/wa
X
F1 W/wa 1 W/W: 2 W/wa: 1 wa/wa
3 purple: 1 white
Two distinct alleles at the same locus
Complementation tests can be madebetween recessive alleles.
How many genes are required to makepurple pigment in flowers?
If plants with recessive alleles arecrossed and the progeny also have the recessive trait,
If plants with recessive alleles arecrossed and the progeny have the dominant trait,
A dominant allele cannot be used. Why?
The alleles are variants of the same gene
The alleles are variants of different genes
Allelism test 1:Cross different white flowered plants
If the mutations are in the same gene,The progeny will be white
white A wa/wa
X
white B wb/wb F1 = wa/wb
F2 generation:Cross white F1 to another white F1If the mutations are alleles of the samegene, what is the next generation?
wa/wb X 1 wa/wa, 2 wa/wb, 1 wb/wbwa/wb
Complementation test double check
Allelism test 2:Cross different white flowered plants
If the mutations are in different genes,the progeny will be pigmented
white A wa/wa;Wc/Wc
X
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 neededto 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
white A r/r; P/P
X
white C R/R; p/p F1 R/r; P/p
The discrete alleles of two different genesWill assort randomly in future generations
white A r/r; P/P
X
white B R/R; p/p F1 R/r; P/p
Complementation test double check
Punnet Square: Predict the genotypes and phenotypes in the F2 generation when the trait is controlled by two genes with randomly segregating alleles
Male gametes
Female gametes
RP
Rp
rP
rp
rprPRP
RRPP
RRPp
RRPp RrPP RrPp
RRpp RrPp Rrpp
RrPP RrPp rrPP
rrppRrPp Rrpp rrPp
rrPp
9R_P_ 3R_pp 3rrP_ 1rrpp
Phenotypes: if both R and P needed for purple color
F2 after RrPp X RrPp
Rp
9 purple and 7 white
Using multiple allelism tests with diverse recessive mutants,
We can identify all the genes specificallyinvolved 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.
Male gametes
Female gametes
RP
Rp
rP
rp
rprPRP
RRPP
RRPp
RRPp RrPP RrPp
RRpp RrPp Rrpp
RrPP RrPp rrPP
rrppRrPp Rrpp rrPp
rrPp
9R_P_ 3R_pp 3rrP_ 1rrpp
Phenotypes: if R and P affect independent traitsEg. petal color and leaf size
R- is purple, rr is whiteP- is long leaf and pp is short leaf
RrPp X RrPp
Rp
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
Does the data fit your model?
Σ (Oi-Ei)2/Eii=1
n
Degrees of freedom = n-1
n is the number of types of observations, ie. the number of different phenotypic classes
p is probability that the null hypothesis is correctWhen the observations are similar to the expected values, Χ2 is a small number and p is close to 1.0
Null hypothesis: the alleles that control petal color and leaf size represent two different genes segregating independently.
Χ2 =
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 incytoplasmic organelles
Eg. Mitochondrial traits
Eg. Chloroplast traits in plants
Organelles are transferred to an embryo from the egg, not the sperm. The organelles are haploidand (usually) genetically uniform in eggs. Therefore the trait of the mother will be passed toall offspring.
Examples of maternally inherited traits?
Mitochondrial:
Chloroplast:
White leaves – loss of chlorophyll, often partial
Mitochondrial myopathyDiabetes mellitus and deafnessLeber's hereditary optic neuropathy
Yeast complementation test for next week:
Brewers YeastSaccharomyces cerevisiae:
16 chromosomes12,052 kb DNA6183 ORFsAbout 5800 expected to encode proteins
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.
Advantages of yeast for identification of genes in a biosynthetic pathway
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
Mendelian Genetics: Mendel’s laws,
Punnet square, calculate expected ratios of phenotypes
Allelism tests
Yeast as a model, haploid and diploid life-style
Segregation of two alleles at one locus
Segregation of two alleles at two independent loci
Chi-square test to test if observed results can beexplained by the model of choice.
Summary of Lecture 1: