Announcements 1. Lab this week: a. Bring your results from Lab 9/10 - human PCR lab. Were you homozygous +/- Alu repeat or heterozygous? We will use our class data for Hardy-Weinberg problem solving this week. b. Problem set 8, questions 1,2 due start of lab. c. Review/problem solving; quest. 3-6 problem set 8, end of chapter problems; lab report questions, etc… 2. Reminder to check your t-test data with Jason or myself. Be sure you determine your unknowns correctly.

Review of lecture 37 I. Tumor suppressors - normally arrest cell cycle II. Proto-/Oncogenes - normally promote cell cycle III. Translocations and Genomic instability IV. Colon cancer results from series of mutations

Learning Check Recall that Rb (retinoblastoma) protein must be modified/ phosphorylated (+P) in order for the cell cycle to proceed. Modification of Rb results in release of E2F. At the end of mitosis, Rb is de-phosphorylated (-P) and binds E2F again. 1. What 2 types of proteins work together to modify/+P Rb? 2. What phenotype would you expect when cells have a mutation such that Rb is severely truncated and can not bind E2F? 1. Cyclins and kinases (cdks) 2. E2F will be “free” continuously to bind and activate genes for transition to S phase - uncontrolled cell division results.

Overview of lecture 38 I. Cancer overview - Viruses and Carcinogens - Genetic testing for cancer II. Population genetics - Calculating allele frequencies - Hardy-Weinberg law

Tumor Progression: Evolution at the Cellular Level Benign tumor (polyp in epithelial cells) is confined by basal lamina; then additional mutation occurs. Malignant tumor (carcinoma in epithelial cells) grows very fast, becomes invasive, and metastasizes.

Cancer Cells Evade Two “Safety” Mechanisms Built into the Cell Cycle 1. Once p53 is inactivated, cells with DNA damage don’t arrest from G1 and don’t undergo apoptosis. 2. Telomerase enzyme is activated, avoiding the limit to cell divisions imposed by telomere shortening.

HBV - hepatitis B virus Normal liver cell Infected cell - HBV genome integrates into human genome 1. Oncogenes activated 2. Chromosome instability 3. Cyclin genes disrupted Tumor cell - hepatocellular carcinoma Example of virus in humans causing cancer If infected with HBV, risk of liver cancer is increased 100x

Carcinogens Chemicals are responsible for more cancers than viruses. Most are pro-carcinogens - must be converted metabolically to become active carcinogens; then they bind DNA and cause point mutations Historically, first seen in 1700’s - scrotal skin cancer in people who worked as chimney sweeps as children. Now, radiologists and farmers develop skin cancer; insulation workers develop lung cancer, etc.. Chemical carcinogens (tobacco smoke and diet) responsible for 50-60% of cancer-related deaths. -30% of cancer deaths related to smoking (cigarettes) - the polycyclic hydrocarbons in cigarette smoke are converted within cells and cause mutations to DNA

Genetic testing and predicting/treating cancer Predictive testing Do you want to know if you have a mutation in a tumor-suppressor gene or proto-oncogene? Could mean an increased chance of developing cancer, but no clear answer if you will or will not get cancer. - what if it involves predisposition to a cancer where medical surveillance could detect cancer early? breast cancer vs. pancreatic cancer??? Testing for treatment/prognosis Is there a difference in how you view a small breast tumor depending on whether it has a mutation in p53 or not??? Know limitations and utilities of these tests

Learning Check 1) In chickens, rous sarcoma virus can cause sarcomas (cancer) by converting the c-src proto-oncogene into v-src, an oncogene. List 3 ways a proto-oncogene can be converted into an oncogene? 2) In humans, hepatitis B virus can cause liver cancer. In this case, the virus can cause cancer by: Point mutation, translocation, overexpression a. Converting proto-oncogenes into oncogenes b. Causing chromosome instability c. Disrupting cyclin genes d. a and b e. b and c f. All of the above

II. Population genetics Studying changes in the frequencies of alleles in populations, a subdiscipline within evolutionary biology Linking Darwin’s theory of evolution to Mendel’s genes: key insight = changes in relative abundance of phenotypic traits can be tied to changes in the relative abundance of alleles that influence traits Key to understanding genetic evolution is to focus on populations, not individuals

Key Concepts Population: a group of individuals, of the same species and location, that can actually or potentially interbreed with each other. Genotype frequency: fraction of the population with a particular genotype. Gene pool: all the gametes made by all the breeding members of a population in one generation. Populations are dynamic: birth, death, migration, merging populations - all lead to changes in the genetic structure

First Step in Population Genetics Analysis: Calculate Frequencies of Alleles Counting alleles from known genotypes is the easiest way. In simple example, there are 2 AA, 4 AB, and 2 BB genotypes (8 total individuals, 16 total alleles): fr(A) = (2 x 2) + (4 x 1) = 8 A/16 total = 0.5 = 50% fr(B) = (4 x 1) + (2 x 2) = 8 B/ 16 total = 0.5 = 50% Measurement of allele frequencies: –Genotypes inferred directly from phenotypes –Genotypes from DNA samples - comparing nt sequences –Genotypes from protein samples (allozymes, next slide)

Allozyme Analysis to Detect Genetic Variation In protein gel electrophoresis, gel is prepared of starch, polyacrylamide, or agarose. Samples move through gel based on electric charge (no detergent). Enzyme substrate added to reveal presence of enzyme bands. Allozymes are different alleles of enzymes that can be detected on protein gels. Gel showing monomorphism: Gel showing polymorphism: ABAB

Real-life example - calculating allele frequencies CCR5 Function, Genotypes and Phenotypes A small number of individuals seem to be resistant to acquiring HIV, even after repeated exposure. How? Breakthrough all have mutations in CC-CKR-5 gene CC-CKR-5 gene encodes chemokine receptor, CCR5. Chemokines are signaling molecules used by the immune system. HIV-1 uses CCR5 receptors to enter host immune cells.

 32/  32 genotype associated with resistance to HIV-1 infection. +/  32 genotype is susceptible, but may progress to AIDS slowly. +/+ genotype is susceptible to HIV-1. Allelic variation in the CCR5 gene RFLP analysis 32 bp deletion in exon of CCR5 gene results in non-functional protein, and therefore resistance to HIV infection

Determine Allele Frequencies from Genotypes How common is ∆32 allele and where is it present? A sample of 100 French individuals in Brittany revealed the following genotypes. Genotype: +/+ +/  32  32/  32Total No. of individuals ) Determining the allele frequencies by counting alleles: No. of + alleles No. of  32 alleles Frequency of CCR5+ in sample = 178 / 200 = 0.89 = 89% Frequency of CCR  32 in sample = 22 / 200 = 0.11 = 11% 2) Determining the allele frequencies from genotype frequencies: No. of individuals Genotype frequency79/100 20/100 1/ (0.79) (0.20) (0.01) Frequency of CCR5+ in sample = (1/2) 0.20 = 0.89 = 89% Frequency of CCR  32 in sample = (1/2) = 0.11= 11%

Conclusions and more questions Highest frequency of ∆32 allele is in Northern Europe; populations without European ancestry = no ∆32 Why is the  32 allele present in this distribution? Where did it originate? Would we expect the allele to become more common where it is presently rare? Use tools developed to model answers to such questions: Godfrey H. Hardy, a mathematician, and Wilhelm Weinberg, a physician, independently proposed a simple algebraic equation for analyzing alleles in populations. –Under certain conditions, one can predict what will happen to genotype and allele frequencies

Assumptions of Hardy-Weinberg 1. No natural selection; equal rates of survival, equal reproductive success. 2. No mutation to create new alleles. 3. No migration in or out of population. 4. Population size is infinitely large. 5. Random mating. If these assumptions are true, then: 1. The allele frequencies in the population will not change from generation to generation. 2. After one generation of random mating, the genotype frequencies can be predicted from the allele frequencies.

How does such a strict law, where there is NO change from generation to generation, help in studying evolution? KEY POINT: By specifying ideal conditions when allele frequencies do NOT change, H-W law identifies forces of evolution (forces that cause allele frequencies to change). Know these five forces of evolution and H-W law.

Demonstration of H-W Law Suppose the gene pool for a population for two alleles is fr(A) = 0.7 and fr(a) = 0.3 in eggs and sperm. (Note = 1) If random mating occurs, then what are the probabilities that each of the following genotypes will occur? AA, Aa, aa. You can solve using a Punnet square:

Calculating Genotype Frequencies from Allele Frequencies Eggs Sperm Total fr(Genotypes): 0.49 AA Aa aa = 1

What are the allele frequencies in the next generation? Determine allele frequencies from genotype frequencies: Genotype:AAAaaaTotal Frequency Frequency of A in sample = /2 (0.42) = 0.7 Frequency of a in sample = 1/2 (0.42) = 0.3 So after one generation of random mating, the allele frequencies can be predicted and have not changed. We’re back where we started. No evolution of population.

General Allele and Genotype Frequencies under H-W Assumptions Total fr(Genotypes): p 2 + 2pq + q 2 = 1

Summing up H-W Equations Gene Pool Equation: p + q = 1 where p = frequency of the dominant allele in the population, q = frequency of the recessive allele in the population. Genotype Equation: p 2 + 2pq + q 2 = 1 where p 2 = frequency of dominant homozygotes, 2pq = frequency of heterozygotes, q 2 = frequency of recessive homozygotes. KEY POINT: When population has constant allele frequencies from generation to generation, and when genotype frequencies can be predicted from allele frequencies, then population is in Hardy - Weinberg equilibrium.

Three important consequences of H-W law 1. Dominant traits do NOT automatically increase in frequency from generation to generation 2. Genetic variation can be maintained 3. Knowing the frequency of one genotype can allow for calculation of other genotypes