Genetics Final Exam

Populations

- Local groups of organisms belonging to a single species, sharing a common gene pool • Populations can be described by age structure, geography, birth and death rates, and allele frequencies

The Hardy-Weinberg Equation

p2 +2pq+q2 =1 - 1 = 100% of genotypes in the new generation - p2 and q2 are the frequencies of homozygous dominant and recessive genotypes, respectively - 2pq is the frequency of the heterozygous genotype in the population

Analyzing Gene Function

• *Forward Genetics* - Start with mutant phenotype individual - Map mutations to specific gene - Isolate and sequence gene - Predict and isolate protein from gene - Biochemically analyze function of protein and role in phenotype • *Reverse Genetics* - Start with isolated gene sequence - Alter gene sequence or inhibit expression of gene to determine the effect on phenotype

Finding Genes of Interest

• 1) DNA Libraries - Construct either a Genomic or cDNA Library - Then screen library for presence of gene • 2) In situ Hybridization • 3) Positional Cloning - Chromosome Walking or Jumping

Cloning Animals • Two Techniques:

• 1) Embryo Splitting • 2) Somatic Cell Nuclear Transfer (SCNT)

• Genetically-engineered animals can be used to:

• 1) Generate animal with symptoms that mirror those in humans • 2) Use the model to study the development & progress of the disease • 3) Test treatments for the disease

Types of Restriction Enzymes (REs)

• Type I and III REs cut DNA at sites *outside* the recognition sequences • Type II REs cut DNA *within* the specific recognition site - Primarily used for all molecular genetics work

Portable DNA Sequencer

• Unveiled in 2015, the palm-sized MinION from Oxford Nanopore Technologies contains thousands of wells • Each well can hold a single strand of DNA • Has 30% error rate • Upon testing, could detect DNA from E. coli and several species of poxviruses • The need to amplify DNA before sequencing remains a stumbling block

Silencing Genes with RNAi

• Using siRNA (small interfering RNA) and miRNA (microRNAs) to temporarily silence genes to study their function • siRNAs and miRNAs combine with proteins (Dicer protein) to form the RNA-induced silencing complex (RISC) - Pairs with complementary sequences on mRNA and results in cleaving the mRNA or preventing its translation • Referred to as RNA interference (RNAi)

Two Theories Differ on How and Where Homo sapiens Originated

• 1. Out-of-Africa hypothesis (speciation and replacement) 2. Multiregional hypothesis (evolution and transition) • The fossil record shows a gradual transition that took place at multiple sites outside of Africa

Codominant Allelic Frequencies

• Codominant allelic frequencies can be measured directly by counting phenotypes - Phenotypes are equivalent to genotypes • Example) The MN blood group - LM and LN alleles are codominant and produce three phenotypes, M, N, and MN

DNA Fingerprinting

• Every human's DNA is 99.9% identical to every other person's DNA • However certain parts of the genome are highly variable • Provides distinctive patterns that allows for identification • Most now involve micro satellites, or also called short tandem repeats (STRs) - Short repeats found in tandem (side-by-side) throughout human genome • Individuals vary in the number of copies of these repeat sequences • Detect micro satellites using PCR • Primers flank repeat to be amplified • Length of amplified segment depends on the number of repeats in that person - More repeats = longer segment • Separate PCR fragments by gel electrophoresis

Isolating the Gene for Cystic Fibrosis

• Gene for Cystic Fibrosis (CF) was the first gene for a disease to be identified entirely by positional cloning • Autosomal Recessive (Chapter 6) - About 5% of all Caucasians are carriers for CF • Studied families with several members having CF • CF gene closely linked to MET & D7S8 markers on 7q - Separated by 1.5 mu (1,500,000 bp) • Narrowed down area using more markers - Markers D7S122 & D7S340 are closely linked to CF • Used combination of chromosome walking and jumping in human genomic libraries that completely covered the region of interest • Four genes were present in region encompassed by markers • Three genes were eliminated as candidates as they did not have appropriate DNA sequence or were not closely linked with CF inheritance • Remaining gene showed high levels of expression in lung, pancreas & sweat glands - Sites where CF affects • After sequencing, CF allele has 3-bp deletion - Absence of Phenylalanine from chloride channel, channel remains closed • Named gene CFTR

Transformation & Selection

• Gene inserted inside lacZ gene • Competent bacteria are transformed by the plasmids - Uptake of DNA from the environment • Bacteria without plasmid will not grow on media containing ampicillin • Bacteria with plasmid lacking inserted gene (lacZ+) will be blue colonies • Bacteria with recombinant plasmid (lacZ-) will be white colonies

Calculating Genotypic Frequency in a New Generation

• Given alleles in the parental generation are a (q = 0.4) and A (p = 0.6) • In new generation, 36% (p2)of offspring AA • 48% (2pq) are Aa • 16% (q2) are aa

Genetic Variation in Human Populations

• The biological concept of race changed from an emphasis on phenotypic differences to an emphasis on genotypic differences • Mutation introduces genetic variation • Natural selection and genetic drift are the primary mechanisms that spread alleles through local population groups

2. Multiregional hypothesis (evolution and transition)

- After H. erectus spread through the Middle East and Asia, H. sapiens developed from an interbreeding network of H. erectus

Analyzing Gene Function • Site-Directed Mutagenesis

- Change specific codon in DNA/ amino acid in protein to observe effect on protein function - Take single-stranded DNA target sequence in plasmid - Hybridize single stranded complementary DNA with single base change - Fill in strand - After transfection of bacterial cells and replication of plasmid, some plasmids will retain original sequence & some will have substitution - Bacteria can be screened for altered gene

Next Generation DNA Sequencing • C) Ion Torrent Sequencing

- DNA fragmented into millions of short overlapping pieces - Attached to beads and placed in single well of chip - Each time a nucleotide is added, a H+ is released, which can be detected and recorded

Next Generation DNA Sequencing • B) Illumina Sequencing

- DNA fragmented into millions of short overlapping pieces - Attached to slide and amplified into clusters • 1000 copies in close proximity - Similar to Sanger sequencing • Each nucleotide has unique fluorescent tag - However, after nucleotide is added and identified, it can be removed and process repeated

Next Generation DNA Sequencing • A) Pyrosequencing

- DNA is fragmented and then ligated to adaptor sequences - DNA made single stranded - PCR amplicons for every DNA library molecule are attached to a bead and spatially clustered in arrays - Alternate cycles of enzyme reactions and fluorescent imaging - Release of pyrophosphate (PPi) reacts to generate light, which can be detected

Next Generation DNA Sequencing • D) Third Generation Sequencing

- Example) Nanopore sequencing • Single strand of DNA passed through a nanopore in a membrane • Electrical charge causes disruption of DNA structure • Each of four bases causes a characteristic disruption • Hundreds of thousands of nanopores can be created in a single chip

• 1. Out-of-Africa hypothesis (speciation and replacement)

- H. sapiens arose from H. erectus in Africa about 200,000 years ago from population of about 10,000 individuals - H. sapiens spread from Africa, displacing and driving populations of H. erectus to extinction • Genetic evidence supports this model - African populations have greatest amount of genetic diversity, as measured by mtDNA • Members of Non-African species show much less diversity - Also markers on Y-chromosome support origin of H. sapiens about 200,000-270,000 years ago

Effects of Genetic Drift

1) Produces changes in allelic frequencies • Large populations have relatively constant allelic frequencies • Small populations show significant changes in allelic frequencies • 2) Reduces genetic variation within populations • 3) Different populations diverge genetically with time - Figure 25.13= all 5 populations begin with the same allelic frequency (q = 0.5) - Each gradually acquires genetic differences

Natural Selection Interactions

1. Competition 2. Coevolution 3. Sexual Selection

• 3) Sexual selection

A type of selection that favors traits that help an organism acquire a mate - Examples: • Conspicuous features (bright colors, long feathers or fins, elaborate antlers) • Bizarre courtship behaviors • Loud, complex courting songs

- 1) Competition

An interaction among individuals who attempt to utilize a limited resource • The competition may be among individuals of the same species or of different species • It is most intense among members of the same species because they all require the same things

• Predation

An interaction in which one organism (the predator) kills and eats another organism (the prey) - Coevolution between predators and prey is akin to a "biological arms race" • Wolf predation selects against slow, careless deer • Alert swift deer select against slow, clumsy wolves

• 3) Underdominance (Disruptive selection)

Favors individuals at both extremes of a trait (homozygotes), and individuals with intermediate values are selected against - The population divides into two phenotypic groups over time - Disruptive selection adapts individuals within a population to different habitats • Beak size in black-bellied seedcrackers is subject to disruptive selection - Birds with large, stout beaks are able to crack the hard seeds found in their habitat - Birds with small, pointy beaks are better adapted to process the soft seeds of the habitat - Birds with intermediate-size beaks have a lower survival rate than individuals with either large or small beaks

• 1) Directional selection

Favors individuals with an extreme-value trait and selects against both average individuals and individuals at the opposite extreme - If environmental conditions change in a consistent way, a species may respond by evolving in a consistent direction • For example, if the climate becomes colder, mammal species may evolve thicker fur

• 2)Overdominance (Stabilizingselection)

Favors individuals with the average value of a trait, such as intermediate body size, and selects against individuals with extreme values - Stabilizing selection commonly occurs when a trait is under opposing environmental pressures from two different sources - For example, Aristelliger lizards of intermediate body size are favored over the extremes • The smallest lizards have difficulty defending territory • The largest lizards are more likely to be eaten by owls

• 2) Coevolution

The constant, mutual feedback between two species - When two species interact extensively, each exerts strong selective pressures on the other - When one evolves a new feature or modifies an old one, the other typically evolves new adaptations in response - Predator-prey relationships generate strong coevolutionary forces

Concerns About Gene Therapy

• 1) Difficulty in delivering foreign gene into human cells and having it expressed correctly • 2) Immune response against the foreign gene product or the vector • 3) Safety? - 1999 = Jesse Gelsinger had a fatal immune reaction to the adenovirus vector used during a clinical safety trial for OTC-deficiency - 2002 = two children receiving gene therapy for SCIDs developed leukemia due to the retroviral vector DNA inserting into cancer-causing genes • 4)Ethical Issues - A) Off-label Use = Using a treatment designed to cure a disease for some other purpose - B) Designer Babies = Introducing genes into a developing embryo/fetus to produce desired traits - C) Germ-Line Gene Therapy = introducing foreign genes into gamete-producing germ cells • Genes and traits can be passed onto offspring - D) Eugenics = engineering individuals with desired traits to produce an ideal population - E) Evolutionary Consequences = how will these foreign genes affect the ongoing evolution of the human species

Types of Natural Selection

• 1) Directional Selection - favors one allele over another • Eventually population becomes fixed for favorable allele • 2) Overdominance (Stabilizing Selection) - leads to stable equilibrium where both alleles maintained - Also called "heterozygote advantage" • 3) Underdominance (Disruptive Selection) - unstable equilibrium because heterozygote is less fit either two homozygotes

Concerns About Genetic Testing

• 1) Is it ethical to test for genetic disorders when there is no cure or treatment? - Do you abort pregnancy? - Can you prepare for an affected child? • 2) Who should have access to the results of genetic testing? • 3) Should insurance companies be allowed to deny coverage for healthy individuals who may be at risk for a genetic disorder? • 4) Should relatives who may also be at risk be informed of the testing results? • 5) Some tests only identify a mutation the predisposes an individual to a disease. Person may not actually develop symptoms (false positives)

Limitations of PCR

• 1) Need to know part of sequence to be amplified in order to design primers • 2) Minute traces of contaminated DNA could also be amplified • 3) Taq polymerase can not proofread, unlike other DNA polymerases - Incorrect base incorporated in 1 in 20,000 bp - Important consideration for gene cloning • 4) Size of amplified fragment usually needs to be less than 2,000 bp

Concerns About GE-Crops

• 1) Unintended Harm to Other Organisms - Death of monarch butterfly caterpillars • Feed on milkweed contacted by pollen from Bt corn • 2) Reduced Effectiveness of Pesticides - Some insects may become resistant to Bt toxin or related pesticides made by GE-crops • 3) Gene Transmission to Non-Target Species - Potential crossbreeding of HT plants with weeds or other non-resistant crops • Could generate "superweeds" that would be extremely difficult to kill - To prevent, plants may need to be engineered to be "male-sterile" or so that pollen doesn't contain the transgene • 4) May Disrupt Ecosystem - They are free of predators and other natural control mechanisms - A National Academy of Science study published in 2002 found that the United States lacks an adequate system to monitor the changes in the ecosystem wrought by GMOs • 5) May Decrease Biodiversity - Of other plants and insects • 6) Allergenicity - People might develop an allergic response to the protein made from the transgene • 7) Unknown Effects on Human Health - Currently under study in Europe & US - A toxicology study of GM plants in 2003 concluded that ingestion of current transgenic crops poses no significant health dangers • 8) Cost of GE Seeds - May become too high for poorer farmers and third world countries to afford - Engineering, testing and licensing GE crops is expensive • 9) Patent Enforcement May Be Difficult - Farmers may cross-pollinate with non-modified crops • 10) Will bacteria acquire the antibiotic-resistance genes used as makers in the GE crops?

Mechanisms of Evolution

• 4) Mating within a population is almost never random - Non-random mating will not change the overall frequency of alleles in a population - Non-random mating, however, will change the distribution of genotypes and, therefore, of phenotypes in a population

Evolution Of Homo sapiens

• A combination of anthropology, paleontology, archaeology, and genetics is being used to reconstruct the dispersal of human populations around the globe • According to fossil and artifact evidence, the ancestral species Homo erectus originated in Africa and began migrating 1 to 2.5 million years ago, spreading through parts of Middle East and Asia

DNA Fingerprinting in Forensics

• A sample from a crime scene is taken: blood, semen, bones, cigarette butts, shirt collars, hats, weapons, bottles and envelopes, etc. • PCR can be used to amplify DNA in sample if sample is very small • Collect DNA from one or more suspects • Compare crime scene patterns with those of suspects for a match

Genetic Assortative Mating?

• A study analyzing 825 Caucasian American couples has shown that, after comparing 1.7 million SNPs, couples were more likely to be genetically similar to one another than to the people with whom they were randomly paired • Interestingly, another similar study showed that we are more likely to be friends with those who are more genetically similar to us - We share about 1 percent of our genes with friends, which is a large number for geneticists

Next Generation DNA Sequencing Advantages and Disadvantages

• Advantages - 1) In vitro DNA library construction and clonal amplification bypasses need for transforming E. coli and colony sampling - 2) Millions of DNA sequences can be read in parallel - 3) Dramatically lower cost per bp sequenced • Disadvantages - 1) Much shorter read lengths than Sanger sequencing • In many cases as small as 35 bp - 2) Much less accurate (at least 10-fold) than Sanger sequencing

Identifying Target Gene

• After chromosome walking or jumping has identified region with gene of interest, must identify all genes in region • Need to determine the function and pattern of expression of gene • Positional cloning has allowed the identification of genes for important human diseases, even without an understanding of the biochemical role of the gene product

5) Natural Selection

• Alfred Wallace and Charles Darwin identified natural selection as the primary force that leads to evolutionary divergence and the formation of new species • Natural selection increases the reproductive success of fitter genotypes - Differential reproduction shown by some members of a population that is the result of differences in fitness - Individuals with adaptive traits produce greater number of offspring - Adaptive traits appear with greater frequency in next generations - Makes population better suited to its environment (Adaption)

Causes of Genetic Drift

• All drift arises from sampling error, however there are different ways this can occur • A) Decrease in Population size over generations due to limitations in space, food or resources. • B) Founder Effect • C) Genetic Bottleneck

Hardy-Weinberg Law

• Allelic and genotypic frequencies remain constant from generation to generation when the population meets certain assumptions: - 1) Population is large enough that there are no errors in measuring allele frequencies - 2) No genotype is better than any other; all have equal ability to survive & reproduce - 3) Mating in the population is random - 4) Other factors that change allelic frequency, such as mutation, migration or natural selection, are absent or rare events and can be ignored • There is a difference between how a trait is inherited and the frequency of recessive and dominant alleles in a population

Lethal Alleles

• Almost all individuals with Duchenne muscular dystrophy die before reproducing • The mutation rate for DMD is high (1 x 10-4), introducing more DMD alleles • The frequency of the DMD allele in a population is balanced between alleles introduced by mutation and those removed by deaths

Chromosome Jumping

• Alternative for chromosome walking for very large regions • Cut DNA by partial digestion into 80 - 150 kb pieces • Ligate ends of fragment to form circular molecule • Then circles cut with another restriction enzyme into small fragments • Some fragments contain both beginning and end of original fragment • Used to create DNA library • Select probes to find genes that are distantly linked

Transgenic Animals

• An animal that permanently carries a foreign gene in its genome • While fertilized egg still contains two pronuclei (prior to fusion), inject foreign DNA into one of the pronuclei - A few hundred copies of cloned linear DNA • In a few of the injected eggs the DNA randomly integrates into one of the chromosomes through non- homologous recombination • Embryos implanted into pseudopregnant female - Female prepared for pregnancy by mating with a vasectomized male • Only 10-30% of eggs survive • Of those only a few have the transgene stably integrated into a chromosome - Implant several hundred embryos into multiple mice to improve chances • Transgenic mice can pass gene onto offspring - Can generate homozygous mice by interbreeding • Can be used to study gene function - Example) Injecting SRY gene into XX embryos, develop male • Can be used to study human diseases - Example) Alzheimer's Disease • Can be used to treat disease - Example) Sheep expressing human insulin in their milk - Example) Chicken eggs with human protein in egg whites

Nonrandom Mating

• An assumption of Hardy-Weinberg Law is that mating is random • Non random mating alters the genotypic frequencies of a population • Two Types of Nonrandom Mating: • 1) Positive Assortative Mating - Tendency for like individuals to mate - Example) two tall people • 2)Negative Assortative Mating - Tendency for unlike individuals to mate - Example) tall and short people would preferentially mate • Assortativematingisusuallyforaparticulartraitand would affect only those genes that encode the trait

Bowen-Conradi Syndrome

• Autosomal recessive disorders • Almost exclusively found in Hutterites - Branch of Anabaptists which originated in Austria in 1500's and moved to South Dakota in 1870's • Gene pool can be traced to no more than 100 ancestors • Founder Effect (will discuss in Chapter 25) • Intermarry within community • 1 in 10 Hutterites are heterozygous carriers • Child born with prominent nose, small head, unusual curvature of little finger, fail to thrive, usually die in first year • Results from single base pair mutation of EMG1 gene on chromosome 12 • Plays essential role in processing 18S rRNA and assembling small subunit • Protein synthesis is universally affected

Humans Have Spread Across the World

• Available molecular and fossil evidence suggests that - H. sapiens emigrated from Africa about 137,000 years ago - H. sapiens spread through Southeast Asia and Australia 40,000 to 60,000 years ago - H. sapiens replaced Neanderthals in Europe 40,000 to 50,000 years ago - North America and South America were populated in waves 15,000 to 30,000 years ago

Human Genome Project

• Began in 1990 by National Institutes of Health (NIH) • Completed in 2003 • Cost $3 billion • Project goals were to: - 1) identify all the approximately 20,000-25,000 genes in human DNA, - 2) determine the sequences of the 3 billion chemical base pairs that make up human DNA - 3) store this information in databases, - 4) improve tools for data analysis, - 5) transfer related technologies to the private sector, - 6) address the ethical, legal, and social issues (ELSI) that may arise from the project. • Analysis of data still continues

Fitness

• Better-adapted individuals have an increased chance of leaving more offspring • Fitness(W) - A measure of the relative survival and reproductive success of a specific individual or genotype - W ranges from 0 to 1 - Example) A1A1 = 10 offspring; A1A2 = 5; A2A2 =2 • Divide each by offspring produced by most prolific genotype • A1A1 =10/10=1.0=W11;A1A2 =5/10=0.5=W12; A2A2=2/10= 0.2 = W22 • Selection Coefficient(s) - Relative intensity of selection against a genotype - s=1-W - For A1A1 s=0; For A1A2 s=0.5; For A2A2 s=0.8

Are There Human Races?

• Biologists use term "Race" to describe groups of individuals within a species that were phentoypically different from other groups in that species • Studies of variations in proteins, microsatellites, and nuclear genes show more genetic variation within populations than between populations - Genetic variation within groups from 85-95%, between groups around 10% • Conclusion: There is no clear genetic basis for dividing our species into races

Cosmid Vectors

• Can hold DNA fragments up to 44 kb • Combine properties of plasmids and bacteriophage vectors • Plasmids that carry phage λ cos sites, which allows cosmids to be packaged into phage particles to infect cells • Have plasmid origin of replication - Can replicate like a plasmid inside cell • Have several unique restriction sites • Have selectable markers

2. In situ Hybridization

• Can hybridize fluorescently-labeled DNA probe to cells • Can check for location of certain genes on chromosomes (a) or tissue distribution of specific mRNA molecules (b), providing insight into gene expression

Genomic Libraries

• Cells are ruptured to obtain DNA - Shake cells with phenol - Precipitates most components - DNA remains soluble - Extract and ethanol precipitate DNA - Treat with RNAse to get rid of RNA • Do partial digestion (limited time) with a restriction enzyme • Get set of overlapping genomic fragments • Fragments ligated into a vector - Plasmid, bacteriophage or cosmid • Insert into bacteria • A few cells have gene of interest, a few have fragments of gene of interest, most do not have gene of interest

Cloning Endangered Animals

• Cloning attempts are being made to preserve endangered species - Gaur, Banteng, Mouflon, Water Buffalo • Cross-species nuclear transfer = fusing somatic cells from an endangered species into enucleated eggs from another organism - Example) Gaur (Bos gaurus) cell into cow (Bos taurus) egg • Benefit= Proponents hope to increase the number of individual organisms • Concerns: - Detractors afraid that cloning will decrease donations to preserve natural habitats and wild animal population - Cost of cloning organisms - Lack of genetic diversity

Embryo Splitting

• Collect an unfertilized egg and fertilize it with sperm in culture dish - In vitro fertilization (IVF) • Embryo allowed to develop to 8 or 16 cell stage • Cells carefully separated to yield single embryonic cells • Each develops into genetically identical embryos • Implant into surrogate cow • Give birth to identical cloned calves

Stacked Traits

• Combination of more than one GE trait per organism • Use of corn with both Bt and HT traits rose to 40% of corn-planted acres in 2008 (1% in 2000) • Cotton with stacked traits also rose to 45% in 2008

Combined DNA Index System (CODIS)

• Computer database established by FBI as part of National DNA Index System (NDIS) • More than 170 public law enforcement laboratories participate • Categories of Profiles stored: - 1) Convicted Offenders - 2) Forensic Samples from Crime Scenes - 3) Arrestees (state-law permitting) - 4) Missing Persons - 5) Unidentified Human Remains - 6) Biological Relatives of Missing Persons (voluntarily contributed) • Helps different law enforcement agencies to coordinate efforts to identify suspect perpetrators or to identify missing people • Store 13 core STR loci in nuclear DNA - Odds that two individuals will have the same 13-loci DNA profile is about one-in-a-billion • Also, for missing persons only, mitochondrial DNA - Maternally inherited

Bacterial Artificial Chromosomes (BACs)

• Constructed from F factor plasmid - Conjugative plasmid= encodes for sex pilus • Can hold up to 300 kb

Artificial Selection

• Controlled breeding modifies organisms - Artificial selection is selective breeding to produce plants and animals that possess desirable traits • Modern dogs are descended from wolves - In only a few thousand years, humans artificially selected for all breeds of modern dogs • Native Americans breeding teosinte to produce corn

Calculating the Frequency of Autosomal Dominant and Recessive Alleles

• Count the frequency of individuals in the population with the recessive phenotype, which is also the homozygous recessive genotype (aa) - The frequency of genotype aa = q2 - The frequency of the a allele is √q2 = q - The frequency of the dominant allele (A) is calculated p = 1 -q • Example) 1 in 2,500 people are affected by cystic fibrosis (autosomal recessive) - q2 = 1/2,500 = 0.0004 - q = √0.0004 = 0.02 (1/50) - p=1-0.02=0.98 - 98% of CF alleles are dominant, 2% are recessive

Estimating the Frequency of Heterozygotes in a Population

• Count the number of homozygous recessive individuals in the population (q2) and calculate the frequency of the recessive allele q • Calculate the frequency of the dominant allele p (p=1- q) • Calculate the frequency for the heterozygote genotype 2pq • Example) cystic fibrosis in US occurs at rate of 1/2,500 white Americans (European descent) - q = √q2 = √0.0004 = 0.02 (2%) - p=1-0.02=0.98(98%) - f(Aa) = 2pq = 2(0.98 x 0.02) = 0.039 = 3.9% - 3.9% of white Americans, approximately 1 in 25, are heterozygous for cystic fibrosis allele

Engineering Plants With Pesticide

• Cut Bt gene into fragments of various sizes • Ligated fragments to neo+ gene (kanamycin resistance) • Inserted constructs into expression vectors • Transformed A. tumefaciens with vectors • Homologous recombination transferred Bt gene constructs (with promoter and poly-A sequence) into Ti plasmid • Recombinant Ti plasmid inserted into tobacco chromosome

Inbreeding Depression

• Decreased fitness of population arising from inbreeding • Due to increased expression of lethal and deleterious recessive traits • Example) Allele a causes a genetic disease with frequency (q) of 0.01 - If mating is random, f(aa) = q2 = 0.0001 (only 1 in 10,000 individuals) - If F = 0.25 (brother & sister), then expected homozygous recessive = q2 + Fpq = (0.01)2 + (0.25)(0.99)(0.01) = 0.0026 • Genetic disease is 26-times more frequent with this level of inbreeding

DNA Sequencing

• Determines sequence of bases in DNA • Developed by Frederick Sanger (1975) • DNA to be sequenced serves as template - Often amplified using PCR • Mix DNA with DNA Pol, buffer, primers, all 4 dNTPs, and a small amount of specific dideoxyribonucleoside (ddNTP) - ddNTPs are missing both 2' and 3'-OH - Have separate reaction tubes containing different ddNTP (ddATP, ddGTP, ddCTP, ddTTP) - Primer usually radioactively or chemically labeled for detection • When ddNTP is incorporated into new strand, chain elongation stops

What is Gene Therapy

• Direct transfer of genes into humans to treat disease • Somatic Gene Therapy =treating cells or organs of affected individual • Catholic Church supports gene therapy with the intent of curing disease • 1990 = Dr. W. French Anderson treated Ashi DeSilva, who had adenosine deaminase (ADA) deficiency • Form of severe combined immunodeficiency (SCIDs) • Lack functional B- & T-cells, often die young due to infection - Removed hematopoietic stem cells (HSCs) from Ashi - Treated HSC cells with retrovirus vector containing a recombinant adenosine deaminase gene - Cultured the treated HSC cells in the laboratory - Implanted the treated HSC cells back into the patient - Several repeated treatments alleviated disease symptoms

Restriction Enzymes

• Discovered in the late 1960's • Naturally produced by bacteria - For defense vs. bacteriophage (viruses) - Bacteria protects its own DNA thru methylation • Make double-stranded cuts at specific nucleotide sequences • More than 800 restriction enzymes have been isolated, which cut DNA at more than 100 sites

Eukaryotic Vectors

• Eukaryotic proteins expressed in other eukaryotic cells have similar modifications (glycosylation, etc.) • 1) *Shuttle Vectors* - Can be cloned and manipulated in bacteria, then transferred to yeast cells for study - Contain origins and selectable markers for both hosts • 2) *Yeast Artificial Chromosomes* (YAC) - Has yeast origin, telomeres and centromere - Can attach to spindle fibers and segregate like other chromosomes - Can hold up to 1,000 kb of DNA • 3) *Ti plasmid* - Plasmid from Agrobacterium tumefaciens, which infects plant wounds and causes crown galls (tumors) - Part of the Ti plasmid gets transferred to the plant during infection - Bacterial enzymes help the plasmid insert in the plant chromosome and be expressed - Transfer to the plant requires two 25 bp sequences that flank the Ti DNA (TR & TL)

Evolution

• Evolution is the change over time in the characteristics of a population • Evolution by natural selection is a unifying theme for all of biology • Evolution results from changing allelic frequencies within a population • Can a Catholic believe in evolution?

Mapping with RFLP

• Example) Huntington's Disease • Autosomal dominant • Father heterozygous for both Huntington (Hh) and for a restriction pattern (AC) • Child can inherit either H or h allele, and either A or C sites • All children who inherit C pattern also have Huntington's disease (Hh) - Closely linked

Migration

• Example) Unidirectional Model - Frequency of a in Pop I=qI ; frequency of a in II= qII - In each generation, portion of Population I migrates to Pop II • Adding genes to II's gene pool - Have migrants (m) from Pop I and residents (1-m) of Pop II - q'II = qI(m) + qII(1-m) - When difference in allelic frequency between two populations is large, effect of migration will be large - Δq = m(qI - qII)

Bacteriophage Vectors

• Example) phage λ • Cut out non-essential phage genes (EcoRI) • Insert DNA fragment • Advantages - 1) High efficiency at transferring DNA into bacterial cells - 2) Can insert as much as 23 kb of DNA - 3) Only phage DNA will insert into virions, ensuring DNA will be replicated after infection

Population Diversity

• Extensive genetic variability exists in populations that sexually reproduce • Populations are more diverse than individuals - Only a group can carry all the alleles for traits such as blood types A, B, AB, and O - All the alleles in a population are the gene pool • The set of genetic information carried by the members of a sexually reproducing population • Basis of all evolution • Extent of variability within population affects ability of adapt to environmental changes

Plasmid Vectors

• Extrachromosomal circular DNA molecules in bacteria • Ex) pUC19 (ampicillin) resistance gene - lacZ gene = encodes for β- galactosidase enzyme • Normally cleaves lactose • Can cleave X-gal in medium, turning colony blue • Cut fragment & vector with same enzyme • Generate complementary sticky ends, which align • Use DNA ligase to join pieces

Inbreeding Population

• F = 1 • AA genotypes produce all AA offspring • aa genotypes produce all aa offspring • Aa genotypes produce 1/4 AA, 1/2 Aa, 1/4 aa • Selfing therefore reduces heterozygote population by half with each generation • Eventually all genotypes would be homozygous

Genetically-Engineered (GE) Crops Generations

• First Generation = crops with enhanced input traits (herbicide tolerance, insect resistance, drought resistance, etc.) • Second Generation = have added-value output traits (enhanced nutrition) • Third Generation = crops that produce pharmaceuticals (such as edible vaccines), enhance bio-fuel production, or produce products beyond food or fiber

Pharmaceuticals

• First commercial products to be developed with use of genetic engineering • 1977 - Lilly company began selling recombinant human insulin - Expressed by bacteria - Prior - used bovine or porcine insulin • Problemswithcontaminatingviruses • Now available products include human growth hormone (HGH), clotting factors, tissue plasminogen activator, among others • In 1980's clotting factors were isolated by concentrating serum from donated blood - HIV pandemic started in early 1980's - Donated blood was not tested for HIV or hepatitis at that time - About 60% of all hemophiliacs became infected by HIV at that time • Recombinant human clotting factor VIII for hemophilia A & recombinant human clotting factor IX for hemophilia B became available in 1990's - No risk of HIV or hepatitis contamination

Calculating the Frequency of Alleles for X-Linked Traits

• For X-linked traits, females (XX) carry 2/3 of the alleles and males (XY) carry 1/3 of the alleles • The number of males with the mutant phenotype equals the allele frequency for the recessive trait - Frequency of an X-linked trait in males is q - Frequency of the trait in females is q2 - Example) In the US, 8% of males are red-green colorblind • q = 0.08 • We would expect colorblindness in females to be q2 = 0.0064 (0.64%)

Calculating Frequencies of Heterozygotes

• For a genetic disorder inherited as a recessive trait, most disease-causing alleles are carried by heterozygotes • The frequency of heterozygous carriers of deleterious recessive alleles in a population is used to calculate risk of having an affected child • Estimating the frequency of heterozygotes in a population is an important part of genetic counseling

Mathematics of the Hardy-Weinberg Law

• For a population, p+q=1 - p = frequency of the dominant allele A - q = frequency of the recessive allele a • The chance of a fertilized egg carrying the same alleles is p2 (AA) or q2 (aa) • The chance of a fertilized egg carrying different alleles is pq (Aa)

3. Positional Cloning

• For many important, disease-causing genes, a protein function is still unknown • Determine general location of gene on the chromosome by recombination frequencies • Then clone any genes in that area of chromosome • A large number of gene loci have been established as molecular markers in mapping • *Step one*: By genotyping one or more families with the disease, demonstrate linkage of the disease phenotype with one or more molecular markers - Provides info about which chromosome carries disease locus and its general location on the chromosome • *Step two*: Use additional molecular markers clustered near locus to pinpoint its location within 1 map unit (mu) - In humans 1 mu represents 1,000,000 bp of DNA - Still large area • *Step three*: Chromosome Walking • Genomic library consists of overlapping clones • Start with cloned gene marker close to gene of interest • Make probe complimentary to end of Clone A (marker) • Use probe to find second clone (B) that overlaps with first clone • Isolate & prepare probe to end of Clone B, to find Clone C • Eventually "walk" to clone that has gene of interest

Expression Vector

• For when you not only want to clone a gene, but express the protein • Has origin, selectable marker, restriction sites • Also contains sequences necessary for transcription and translation in bacteria - Promoter, etc.

Knockout Mice

• Fully disable a specific gene in the mouse • Phenotype of mouse is helpful in determining the function of the gene • Common technique: - Insert neo (antibiotic G418 resistance) gene inside of gene to be disabled • Disrupts gene function - Herpes virus Thymidine Kinase (tk) gene also linked to disabled gene - Transfect construct into cultured mouse embryonic stem cells - Look for homologous recombination - Screen by adding G418 to medium • Only cells with disabled gene with neo insert survive • Common technique: - Frequency of non-homologous recombination is higher than homologous • Results in cells with both normal gene copies and inactivated copy - Need to select for only homologous recombination • No functional gene copies - tk+ gene makes cell sensitive to gangcyclovir - Add gangcyclovir to medium with G418 - Cells that grow have neo insertion but not TK gene • Have homologously recombined with chromosome - Non-homologous recombination have both neo & TK • Killed by gangcyclovir

Measuring Gene Flow Between Populations

• Gene flow between populations is used to reconstruct the origin and history of populations • Example) Gene flow into the American black population from Europeans - West African populations have blood group FY*O(close to 100%) - Europeans have blood groups FY*A and FY*B - In northern US cities, about 20% of genes in the black population are derived from Europeans

1) Mutation

• Generates new alleles and allelic frequencies • Example) 25 diploid individuals, each has 2 copies of allele, so gene pool has 50 allele copies - 2 Alleles = G1 (frequency p) + G2 (frequency q) - Begin with 45 copies of G1, 5 copies of G2 (p=0.9,q=0.1) - Mutate G1 to G2 (forward mutation) • p decreases, q increases • Rate of change depends on amount of G1 (more G1, higher rate) and rate of mutation - Some reverse mutations also occur - Eventually will reach equilibrium

Populations Can Be in Genetic Equilibrium

• Genetic (Hardy-Weinberg) Equilibrium - When the allele frequency for a particular gene remains constant from generation to generation - Equilibrium in a population explains why dominant alleles do not replace recessive alleles • In equilibrium populations, Hardy-Weinberg law can be used to measure allele and genotype frequencies from generation to generation

Human Accelerated Regions (HARs)

• Genome regions conserved across most animals, but with notable differences in humans - Evolutionary signature predicts a loss or modification of function in humans • Short — on average just 227 base pairs long, much smaller than a gene • Includes nearly 3,000 genome segments • Nearly all HARs are outside genes, some quite far away from any gene in the genome. • Most HARs are enhancers, increasing or decreasing expression of genes involved in development

Transgenic Animals With GFP

• Green Fluorescent Protein (GFP) from Aequorea victoria, a fluorescent jellyfish from northwest Pacific Ocean • Genetic sequence of GFP is often fused to target genes in a cell to determine if the targeted gene is being expressed (reporter gene) • Transgenic animals have been engineered using GFP fused to target genes

Genetic Testing

• Identification and cloning of many disease- causing genes has allowed for probes to detect their mutations • Prenatal Genetic Testing (Chapter 6) can detect several hundred genetic disorders • Several postnatal tests are also available for children and adults • Newborn screening tests infants within 48 to 72 hours after birth for a variety of genetically- controlled metabolic disorders • All states require newborns to be tested - Most states screen for 3 to 8 disorders - New methods can scan for 30 to 50 disorders

Speciation

• If members of the same species are exposed to different forces (environment, population size, food sources, etc.), natural selection may cause them to evolve into different species • A new species occurs when the females of that species no longer recognize or mate with the males of the original species

Calculating the Frequency of Multiple Alleles

• In ABO blood types, six different genotypes are possible (AA, AO, BB, BO, AB, OO) - Allele frequencies: p (A) + q (B) + r (O) = 1 - Genotype frequencies: (p + q + r)2 = 1 • Expanded Hardy-Weinberg equation: - p2 (AA)+2pq(AB)+2pr(AO)+q2 (BB)+2qr (BO)+r2 (OO)=1

2) Migration (Gene Flow)

• Influx of genes from other populations • Hardy-Weinberg Law assumes migration does not take place • Effects: • 1) Prevents genetic divergence between populations - Causes gene pools to become more similar • 2) Increases genetic variation within populations

Neanderthals & Modern Humans

• Initial studies indicate that humans from non-African origin share as much as 4% of our genes in common with Neanderthals, who went extinct 30,000 years ago - European average = 2.7% - Newer study suggests as much as 20% of Neanderthal genes live on in entire human genome today - Africans have no Neanderthal DNA • Human ancestors migrating out of Africa likely mated with existing Neanderthal populations in the areas • Neanderthal genes appear to be concentrated in areas that control appearance of skin and hair, and immune system - Genes were beneficial to humans likely because they helped them adapt to colder climates (example - thicker body hair) - Also genes that cause diabetes, lupus, Crohn's disease and allergies • However, human X chromosomes and genes controlling testes had no Neanderthal DNA - Perhaps they reduced the fertility of male hybrids, so they were not passed on

Cloning Animals

• July 5, 1996= Dolly the sheep was born at the Roslin Institute of Scotland • Before Dolly, only cells from early embryos could be cloned • Most experts thought you could not clone from adult cells as they have differentiated during development • Dolly was cloned using the Somatic Cell Nuclear Transfer (SCNT) technique • Dolly is NOT completely identical to her DNA donor mother • They ARE identical for nuclear genes • They are NOT identical for cytoplasmic genes, such as mitochondrial genes - Donated by both the donor cell and the enucleated egg cell

Gene Therapy For LCA

• Leber Congenital Amaurosis (LCA) is a rare form of blindness • Autosomal recessive disorder • Begin losing sight at birth, most are completely blind by age 40 • One type of LCA is mutation in RPE65 gene, which encodes an enzyme that helps convert vitamin A to rhodopsin, light-absorbing pigment • Without rhodopsin, photoreceptor cells atrophy over time • In 2007, four patients treated with a genetically-modified virus carrying functional RPE65 gene - All patients showed dramatic improvement in vision, though still legally blind - Researchers predict even more dramatic improvements if the treatment is used on younger patients

cDNA Libraries

• Make library only of DNA sequences transcribed into mRNA • cDNA = complementary DNA • Advantages: - 1) Don't copy DNA that doesn't encode for anything - 2) Enriched with fragments from actually transcribed genes - 3) Introns are not present, can express cDNA in bacteria • Disadvantages: - 1) Gene is missing introns, promoters, enhancers, etc. - 2) Library contains only cDNA from genes being actively transcribed in the tissue that was isolated • Not all genes

Automated DNA Sequencing

• Manual technique is tedious, labor intensive and expensive • New automated DNA sequencers are much easier and faster • Can do all 4 ddNTPs in same reaction tube using thermocycler - Each ddNTP has a different colored fluorescent label • Machine runs products through gel-containing capillary tubes • Laser lights fluorescent labels of bands as they go through gel • Computer interprets peak distribution on graph • Can read 50-60 kb in a few hours

Ethics of Human Cloning

• Many nations and Catholic Church are against reproductive cloning of humans • 1) Artificial Conception • 2) Reason for Creation • 3) Would the Clone Have a Soul? • 4) Rights of Cloned Individual • 5) Cloning Individuals for Organs • 6) Who Gets to be Cloned? • 7) Involuntary Cloning • 8) Success Rate of Cloning Experiments - Potentially many sacrificed embryos • 9) Undue Expectations of Clone • 10) Age of Clone? - Is the clone genetically a young individual or the age of the DNA donor • 11) Safety of Cloning - Many clones have had genetic defects and died prematurely • 12) Reproducing Without Males - Dolly had three mothers (DNA donor, egg, surrogate) • 13) Lack of Genetic Diversity

Genetic Engineering of Plants with Pesticides

• Mark Vaeck and colleagues (1987) at Plant Genetic Systems (Belgium) engineered tobacco plants to express Bt toxin - Toxin made by Bacillus thuringiensis that is lethal to insect larvae - Non-toxic to humans, specific to certain insects and breaks down quickly in the environment • Cloned Bt toxin (Bt) gene into an E. coli plasmid for easy manipulation

Inbreeding Coefficient (F)

• Measure of the probability that two alleles are identical by descent • F ranges from 0 to 1 • F=0 indicates random mating • F=1 indicates all alleles are identical by descent • When inbreeding occurs, the proportion of heterozygotes decreases by 2Fpq - Half of this value (Fpq) is added to homozygous dominant, half to homozygous recessive • f(AA) = p2 + Fpq • f(Aa)=2pq-2Fpq • f(aa) = q2 + Fpq

Cutting Sites

• Most restriction enzymes recognize sequences between 4-8 bp long • Often *palindromes* = same sequence read in opposite direction

Mutations

• Mutations are the source of new alleles and can be beneficial, harmful, or neutral • Mutations arise spontaneously, not as a result of or in anticipation of, environmental necessity - Cells without beneficial mutation die under adverse conditions - Only cells that already possess a beneficial mutation will survive under adverse conditions - Example) Antibiotic resistance in bacteria • Antibiotic kills off sensitive bacteria, leaving resistant bacteria to survive and flourish

Using RNAi To Treat Hypercholesterolemia

• Mutations in Apo B protein can raise lipoprotein/cholesterol levels • Encapsulated apoB-siRNAs inside of lipids, creating stable nucleic-acid-lipid particles (SNALPs) to prolong time in circulation • Injected cynomolgus monkeys • Monkeys receiving treatment had dramatically lowered cholesterol levels and no apparent side effects

Southern Blot

• Named after Edwin Southern • Transfer DNA bands from agarose gel to nitrocellulose membranes via capillary action • Treat membrane with radioactively (or chemically) labeled probe - Complementary sequence to gene or fragment of interest • Expose to photographic film to identify bands of interest • Also Northern Blot for RNA, Western Blot for Proteins

B) Founder Effects

• Occasionally, populations start with a small number of individuals (founders) • Founder Effects - Allelic frequencies established by chance in a population that is started by a small number of individuals - By chance, the allele frequencies of founders may differ from those of the original population • Although the population may eventually grow to be quite large, all genes were derived from limited number of individuals

Screening DNA Libraries

• Once a genomic or cDNA library is created, must screen to find bacterial clones that contain gene of interest • If plasmid or cosmid used, dilute & plate bacteria to get isolated colonies • If bacteriophage is used, plate on a lawn of bacteria to view phage plaque • Each bacterial colony or phage plaque came from one original bacterial cell = single cloned DNA fragment present • Transfer some bacteria/phage from colonies/plaques onto a nitrocellulose filter,creating a *Replica Filter* • Disrupt the cells/ phage to release and denature DNA • Hybridize a labeled probe to filter - 32P or fluorescent • Rinse of excess probe • Expose to film to identify colonies containing gene of interest • Note: could also probe for the protein product using biochemical tests or labeled antibodies

What Have We Learned So Far About the Human Genome?

• Only about 5% of our 3.2 billion nucleotides of DNA encode genetic information • Genes are distributed unequally on chromosomes - Clusters are separated by gene-poor bands • Humans have 20,000 to 25,000 genes - Far fewer than the predicted 80,000 to 100,000 • There are more proteins in the body than genes - mRNAs are processed in many ways so 20,000 to 25,000 genes can produce 300,000 proteins • Genomes of humans and other higher organisms are similar - We share half our genes with the fruit fly and more than 90% with mice

4) Non-Random Mating

• Organisms within a population rarely mate randomly - Many organisms have limited mobility and tend to remain near their place of birth, hatching, or germination, thus increasing the likelihood of inbreeding - In animals, non-random mating can also arise if individuals have preferences or biases that influence their choice of mates • Example - snow geese, which favor mates of the same color • Example - guppies where females prefer males with bright colors - A preference for mates that are similar is known as positive assortative mating • Non-random mating may lead to inbreeding, increasing the chance of producing homozygous offspring with two copies of harmful alleles

Bighorn Sheep

• Ovis canadensis • Live in Rocky mountains • Before 1900, 2 million • Today, fewer than 70,000 • 1922, a population of 12 sheep (4 males, 8 females) was established by wildlife biologists at the National Bison Range • No further animals introduced in next 60 years • Number grew to 90 animals within 8 years, but then fell to 50 by 1985 • Genetic variation was low, reproduction rates dropped, and size & survival were lower • Introduced 5 new rams in 1985 and 10 new sheep between 1990-94 • Population recovered to 69 animals in 2003

Genotypic Frequency

• Percentage of individuals with a particular genotype • Frequency (f), N = total individuals • f(AA) = # of AA individuals/N • f(Aa) = # of Aa individuals/N • f(aa) = # of aa individuals/N • f(AA) + f(Aa) + f(aa) = 1

Benefits of GE-Crops

• Pest resistant plants have lead to a decreased use of environmentally harmful chemicals • Ease of operation and time for farmers • Increased crop yields - Reduces the amount of land that must be used for agriculture - Increased food production for constantly growing world population - Higher income for seed producers and farmers • "GoldenRice2" -produces unusually high levels of beta- carotene (form of vitamin A) - Contains genes from daffodil, corn and bacteria - Can provide up to 50% of the daily requirement for vitamin A - Could be used as a staple crop in third world countries where 500,000 children go blind every year due to vitamin A deficiencies

Treating Disease with Transgenic Animals

• Pompe disease - An inability to make α-glucosidase (GAA) - Can't break down glycogen in lysosomes - Glycogen accumulates in muscles, resulting in muscle weakness - Treated by enzyme replacement therapy • Transgenic animals produce human GAA - Transgenic female rabbits produce human GAA enzyme in their milk

Measuring Allelic Frequencies in Populations

• Population genetics studies allelic frequencies in populations, not offspring of single matings • In some cases allelic frequency in a population can be measured directly - p = f(A) = (2nAA + nAa)/2N - q = f(a) = (2naa + nAa)/2N - p+q=1 • In some cases allelic frequency in a population can be calculated from genotypic frequencies - p=f(A)=f(AA)+0.5f(Aa) - q=f(a)=f(aa)+0.5f(Aa) • In other cases, the Hardy-Weinberg Law is used to estimate allelic frequencies within populations

C) Genetic Bottleneck

• Population undergoes drastic reduction • Example) Northern elephant seals in CA - In 1800, there were thousands - Between 1820-1880 = hunting - 1884 = as few as 20 seals survived on Isla de Guadelupe - Due to restrictions on hunting, there are now close to 30,000 • All very similar, since offspring from few survivors

Inbreeding

• Preferential mating between related individuals • Form of positive assortative mating • However, it affects ALL genes, not just those that determine the preferred trait • Causes departure from Hardy-Weinberg equilibrium frequencies (p2, 2pq, q2) • Increases the proportion of homozygotes, while decreasing the proportion of heterozygotes

Natural Selection Affects the Frequency of Genetic Disorders

• Rare lethal or deleterious recessive alleles survive because the vast majority of them are carried in the heterozygous condition • Other factors can cause differential distribution of alleles in the human population - Migration, genetic drift, mutations, selection

Two Father Babies?

• Researchers were able to take embryonic stem cell and human skin cells and induce them to become Primordial Germ Cells (PGCs) • PGCs can become either sperm or egg cells by controlling the SOX17 gene • So potentially a male skin cell can be induced to form a egg, which could then be fertilized by a sperm cell from another male • Also this process also appears to erase any epigenetic markers in the gametes

Variations on PCR

• Reverse Transcriptase PCR - First copy mRNA into cDNA (c = complementary) using reverse transcriptase - cDNA is then amplified using normal PCR procedure • Real Time PCR - Used to determine starting amount of nucleic acid - A fluorescent probe to sequence being amplified is used to measure amount of DNA as reaction proceeds - Can combine with reverse transcriptase to determine amount of mRNA in sample

Genetically Engineering Plants

• Scientists have engineered a plasmid vector to transfer foreign DNA • Place in A. tumefaciens with Ti plasmid (required for infection) • Bacteria infects plant • Foreign DNA integrates into plant chromosome • Can transfer characteristics like resistance to herbicides, plant viruses and insect pests

Genetically-Engineered (GE) Crops

• Scientists noted plants infected with mild viruses are resistant to infection by more virulent strains - Viral Resistant Squash (Freedom II) • Squash plants carrying genes for Watermelon Mosaic Virus 2 and Zucchini Yellow Mosaic Virus • Makes them resistant to viral infection • Using a gene for cold resistance from cold water fish, plants can tolerate temperatures that would kill normal seedlings

Forces That Affect Allelic Frequencies

• Several forces can affect genetic diversity and lead to evolution of a population • 1) Mutation • 2) Migration (Gene Flow) • 3) Genetic Drift • 4) Nonrandom Mating • 5) Natural Selection

Oligonucleotide Drugs

• Short sequences of synthetic DNA or RNA that can be used to treat disease • a) Anti sense RNAs or DNAs complementary to viral mRNAs can inhibit translation in virally-infected cells • b) siRNAs can be used to treat viral and genetic diseases, and cancer • c) Ribozymes can be used to target and destroy specific mRNAs

Affect of Mutation on Population

• Since mutation rates are low, effect of mutation per generation is very small • Mutation alone has a minimal impact on the genetic variability present in a population • Example) replacement of recessive d allele by dominant D allele by mutation alone - Rate of mutation = 1 x 10-5 - Need 70,000 generations (1.4 million years) to change frequency of d allele from 1.0 (100%) to 0.5 (50%)

Restriction Enzymes (Ends)

• Some enzyme generate sticky (cohesive) ends - Overhangs can hydrogen bond to hold pieces together • Other enzymes generate blunt ends • Can take pieces generated from same enzyme and join them using DNA Ligase

Cloning Vectors

• Stable, replicating DNA molecule into which you can insert a foreign DNA fragment • Three important characteristics: - 1) Origin of Replication - 2) Selectable Markers to allow vector to be selected or identified - 3) One or more unique restriction sites • Four common types for cloning in bacteria: plasmids, bacteriophage, cosmids, bacterial artificial chromosomes (BACs)

Genetic Engineering of Plants with Pesticides (Resistance)

• Tested resistance of transgenic plants vs. tobacco hornworms • High mortality rate in 2/3 of plants with fragments of Bt gene • None of the plants containing full-length Bt gene produced any toxin - Plant cells were better able to translate fragments than entire gene - Fragments were sufficient to confer insect resistance • Interbred to yield plants resistant to both kanamycin and insects • Similar methods have also been used to introduce Bt gene into cotton, tomatoes, corn and others

DNA Microarrays

• Testing for a wide range of genetic disorders is possible using DNA chips (microarrays), which can hold thousands of genes • DNA microarray - A series of short nucleotide sequences placed on a solid support (such as glass) that have several different uses • Uses of Micro array Technology - *Detecting mutant genes* • Individuals who will develop late-onset genetic disorders such as polycystic kidney disease (PCKD) and Huntington disease • Individuals at risk for disorders such as diabetes - *Detecting differences in the pattern of gene expression in normal and cancerous cells* • Each field of the microarray contains a unique sequence of single-stranded DNA • Test DNA and normal DNA are converted to single strands, tagged with fluorescent dyes, and hybridized to the chip • Each result has a different color - Normal alone is green; mutant alone is red - Both together are yellow; a blank field is black

Using the Hardy-Weinberg Law in Human Genetics

• The Hardy-Weinberg Law can be used to: - Estimate frequencies of autosomal dominant and recessive alleles in a population - Detect when allele frequencies are shifting in a population (evolutionary change) - Measure the frequency of heterozygous carriers of deleterious recessive alleles in a population

The Relationship between Sickle-Cell Anemia and Malaria

• The allele for sickle-cell anemia is present in very high frequencies in certain populations - Many recessive homozygotes die in childhood • The sickle-cell allele confers resistance to the parasite Plasmodium, which causes malaria - Selection favors survival and differential reproduction of heterozygotes • Areas with highest allelic frequency of sickle cell allele (HbS) are also areas where malaria is endemic • 7% of black individuals in US are carriers for sickle cell - Up to 20% in Africa - No selective pressure to maintain allele in US • US has not had malaria in over 100 years • The rarity of heterozygotes in Caucasians corresponds with the rarity of malaria in northern climates, where immunity (and therefore, heterozygosity) has no selective advantage

Lactose Intolerance

• The enzyme lactase converts lactose (milk sugar) into glucose and galactose - Lactase production slows or stops after childhood • Lactose intolerant have low levels of lactase - Intestinal bacteria ferment lactose, generating acids and gas - Lactose intolerant develop gas, cramps, diarrhea and nausea upon consuming dairy • Some populations have a gene for adult lactose metabolism (LA) - Lactose absorbers (LA) is autosomal dominant - Human species originally was lactose intolerant as adults, like other mammals - The cultural practice of keeping dairy herds was a selective factor that provided an advantage for the LA genotype - Appeared in Europe around 4,000 years ago

Natural Selection (Penicillin-resistant bacteria)

• The evolution of penicillin-resistant bacteria illustrates the relationship between natural selection and evolution - The first widespread use of penicillin occurred during World War II - Penicillin killed almost all infection-causing bacteria - Penicillin did not affect bacteria possessing a rare allele that destroyed penicillin on contact - Bacteria carrying the rare allele survived, reproduced and passed the resistance gene to other species - Now many strains of pathogenic bacteria are resistant to penicillin

Allelic Frequency

• The frequency with which alleles of a particular gene are present in a population • The frequency of alleles in a population may change from generation to generation - Changes in allele frequency can cause change in phenotype frequency; long-term change in allele frequency is evolutionary change

Heterozygote Advantage

• The high frequency of genetic disorders in some populations is the result of selection that often confers increased fitness on heterozygotes - A single sickle-cell allele confers resistance to malaria - A single Tay-Sachs allele confers resistance to tuberculosis

Gene Therapy

• Thousands of individuals have been treated in many clinical trials • First licensed gene therapy treatment in 2016 in Europe for ADA-deficiency (SCID)! • Trying to treat: - Heart Disease • Using vascular endothelial growth factor (VEGF) gene to grow new blood vessels in those with blocked arteries - Genetic Disorders - Cancer - Certain infectious Diseases, including AIDS • Most common vectors are viruses

Xenotransplantation

• Transplanting organs or tissues from a different species • Biggest problem for organ transplants is insufficient supply of organs - Large number of patients die while waiting for transplant • Historically focus has been on chimpanzees, baboons and pigs - A few attempts in 1990's • Several problems: - 1) Vigorous immune rejection, even with immunosuppression - 2) Xenozoonoses - pathogens from donor causing disease in host - 3) Animal retroviruses might recombine with human retroviruses to produce NEW pathogens • Animals have antigens that are not present in the recipient - Especially Galactosyl-1,3-α-galactose (G13G) - G13G is present on all microbes, so humans have seen it before and have antibodies against it • Alternative: Cloning genetically-engineered knockout pigs that lack the enzyme that makes G13G - Why pigs? • Pigs have similar anatomy and physiology to humans, similar sized organs, large litters - Potential Problem: Endogenous pig retroviruses • Though a Boston company claims to have developed retroviral-free pigs

Homozygous Individuals

• Two copies of an allele may be the same in state - Alleles alike in structure and function, but do not have a common origin • Alternatively, two copies of an allele may be the same in descent - Copies are descended from a single allele that was present in an ancestor

DNA Forensics & Identical Twins

• Two unrelated people share a probability of DNA match at 1 in 1 billion • Full Siblings = 1 in 10,000 • Identical twins share same profile • Currently in court, if you can't conclusively prove which twin is responsible, both must be set free • A recent method has been developed to help differentiate between these twins by studying differences in DNA methylation patterns that occur through life experiences (epigenetics)

Polymerase Chain Reaction

• Used to amplify the quantity of DNA • Developed by Kary Mullis in 1983 • Can amplify a billion-fold in a few hours • Mixture of DNA, primers flanking the region to be amplified, Mg2+, buffer, all 4 deoxyribonucleotides, Taq DNA polymerase • Taq DNA polymerase from Thermus aquaticus, which lives in boiling hot springs - Stable at high temperatures necessary for procedure • 30-40 cycles run in automated thermal cycler • Each cycle involves: - 1) *Denaturation* - heat sample to 95°C to separate DNA strands - 2) *Annealing* - to around 55-60°C to allow primers to anneal to template DNA - 3) *Elongation* - heat to 72°C to allow Taq polymerase to add to 3'-OH of primer and copy template strands

Gel Electrophoresis

• Used to separate restriction fragments • Gel cast with *agarose* - Purified polysaccharide from seaweed • DNA samples are placed in wells • Electric field applied • DNA migrates to positive electrode due to negatively charged phosphates • Run lane of standard markers to determine size • Smaller fragments migrate farther through gel • Larger fragments migrate less far • Separate samples by size • Stain with ethidium bromide - Glow orange under UV light

Knock-in Mice

• Variation on the knockout procedure where you insert a particular DNA sequence into the inactivated gene • Example) Inserting the human disease-causing allele into the same locus in mice - Generate a precise model for studying the human disease - Example) inserting human prion protein allele into the mouse prion gene to study transmissible spongioform encephalopathies (TSEs)

Restriction Fragment Length Polymorphisms (RFLP)

• Variations in the patterns of fragments produced by restriction enzymes • Get different banding patterns on gel for different individuals using same enzyme

Relationship between Allelic Frequency and Genotype Frequency

• What are the chances of two heterozygotes mating and having a child with a recessive trait? - If 1 in 10,000 members of the population have the disorder, then 1 in 50 is a heterozygote - Chance of two mating is 1/50 x 1/50 = 1/2,500 - Chance of a given child being affected is 1⁄4 - Chance of mating and having an affected child is 1/2,500 x 1⁄4 = 1/10,000

3) Genetic Drift

• When a population is indefinitely large, gametes carry genes that are perfectly representative of parental gene pool • When a population is small, gametes carry a sample of alleles from parental gene pool - By chance, composition of sample will often deviate from parental gene pool (sampling error) • Causes change in allelic frequencies - Smaller sample size, greater deviation • Genetic drift - Random fluctuations of allele frequencies from generation to generation that take place in small, isolated populations such as island populations or socioreligious groups due to sampling error • Magnitude of change depends upon the effective population size (Ne) • Equivalent number of breeding adults • Several factors determine - One factor = Sex ratio • When males and females are equal, Ne = sum • Greater disparity in sexes, lower Ne - Increases genetic drift - 1/2 of genes are coming from small population - Other factors include variation between individuals in reproductive success, fluctuations in population size, the age structure of the population and whether mating is random

The $1,000 genome

• When the Human Genome Project completed in the 2003 it had taken 13 years and $3 billion dollars to obtain a complete sequencing of a human genome (the complete sequence of DNA needed to create a human) • A decade ago it still cost over a million dollars • In 2015 the $1,000 threshold was crossed • With the basement not yet in sight • By the end of this decade the cost should drop to that of a routine medical test • There is now an ocean of data • Bioinformatics needs to develop the tools to analyze that data

Recessive Allelic Frequencies

• With recessive alleles, there is no direct relationship between phenotype and genotype - Heterozygotes and dominant homozygotes have the same phenotype - Therefore, the frequency of recessive alleles in a population cannot be measured directly • In 1908, Godfrey Hardy and Wilhelm Weinberg independently developed a mathematical formula to determine frequency of alleles when one or more alleles are recessive - Under certain specified conditions

RFLP to Map Huntington's Disease

• a) Different banding patterns for alleles A, B & C using HindIII and probe for chrom. 8 • b) partial pedigree from large family. Note individuals with C allele have Huntington's disease. - C allele is closely linked to Huntington gene

Horizontal Gene Transfer

• compared genomes of 40 animals • Found evidence that as many as 145 genes in humans may have arose from horizontal gene transfer (non parent-to- offspring) - From bacteria, Archaea, microbes, fungi and plants to animals - Process is well established among microbes • Shows that evolution may not be a straight forward phylogeny

Creating a cDNA Library

• mRNA must first be separated from other types of cellular RNAs • Oligo (dT) bound to beads in column attaches to polyA tail of mRNAs • All other RNAs pass through • mRNAs washed of column and collected • Use reverse transcriptase and primer to poly-A tail to make mRNA-DNA hybrid molecule • Treat with RNAase to destroy RNA strand - Results in short pieces of RNA to be used as primers • DNA polymerase makes double stranded DNA molecule • DNA ligase seal snicks • Insert cDNA into vector • Insert vector in bacteria

Calculating Allelic Frequency in a New Generation

• p=f(A)=f(AA)+0.5f(Aa) - f(A) = p2 + 0.5(2pq) - f(A) = 0.36 + 0.5(0.48) = 0.6 = 60% • q=f(a)=f(aa)+0.5f(Aa) - f(a) = q2 + 0.5(2pq) - f(a) = 0.16 + 0.5(0.48) = 0.4 = 40%

Recombinant DNA Technology

•Molecular techniques for locating, altering, and studying DNA segments •Goal is to often combine DNA from two distinct sources •Cohen & Boyer (1973) = inserted piece of DNA from one DNA molecule into another plasmid, creating new recombinant plasmid •Techniques now used for genetic engineering, biochemistry, microbiology, developmental biology, neurobiology, evolution and ecology