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: Biol 423L Laboratories in Genetics

Reinforce basic genetic principles Introduce model organisms commonly used by geneticists Learn how genetics is used to understand Disease Biochemical pathways Development Goals for course:

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 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

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) X purple by purple (self) All uniform 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? purple is dominant to white X

Alleles are distributed as discrete units X Purple W/W White A w a /w a F1 W/w a

Punnet square helps to predict genotypes and phenotypes of the next generation wawa wawa w a /w a Female gametes Male gametes W W W/WW/w a F1 W/w a X 1 W/W: 2 W/w a : 1 w a /w a 3 purple: 1 white Two distinct alleles at the same locus

Complementation tests can be made between recessive alleles. How many genes are required to make purple pigment in flowers? If plants with recessive alleles are crossed and the progeny also have the recessive trait, If plants with recessive alleles are crossed 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 w a /w a X white B w b /w b F1 = w a /w b

F2 generation: Cross white F1 to another white F1 If the mutations are alleles of the same gene, what is the next generation? w a /w b X1 w a /w a, 2 w a /w b, 1 w b /w b w a /w b 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 w a /w a; W c /W c X white C W a /W a ;w c /w c F1 W a /w a ; W c /w c

Conclusions W a and w a are alleles of the same gene w a and w c are alleles of different genes. The dominant allele of w a and the dominant allele of w c 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 W a and w a : R and r and w c : 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/pF1 R/r; P/p

white A white C X purple rrPPRRpp RrPp

Precursor 1 Intermediate R or P Purple Pathway to purple

The discrete alleles of two different genes Will assort randomly in future generations white A r/r; P/P X white B R/R; p/pF1 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 rP RP RRPP RRPp RrPPRrPp RRpp RrPpRrpp RrPPRrPprrPP rrppRrPpRrpprrPp 9R_P_3R_pp3rrP_ 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 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. Male gametes Female gametes RP Rp rP rp rP RP RRPP RRPp RrPP RrPp RRpp RrPpRrpp RrPPRrPprrPP rrppRrPpRrpprrPp 9R_P_3R_pp3rrP_ 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 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 ( O i -E i ) 2 /E i i=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 correct When the observations are similar to the expected values, Χ 2 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. Χ 2 =

X 2 values for different degrees of freedom and the probabilities associated with the X 2 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: Chloroplast: White leaves – loss of chlorophyll, often partial Mitochondrial myopathy Diabetes mellitus and deafness Leber's hereditary optic neuropathy

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

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

a1 a2 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

a2 a1 X X a2 Which mating results in complementation?

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 be explained by the model of choice. Summary of Lecture 1:

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