Genetics Unit 3

Haplotypes and Linkage Disequilibrium

*Haplotype*: regions of the genome where all of the polymorphisms are linked. We have different haplotypes - Each SNP is associated with other SNPs on the same chromosome. - The set of SNPs observed on a single chromosome, or part of a chromosome is called a *haplotype.* - SNPs within haplotype are inherited together.

Implications of Violating HWE

*Positive assortative mating* - selecting to mate with individuals with similar traits (Ex - height) - leads to more homozygosity *Negative assortative mating*- selecting to mate with individuals with different traits - increase in heterozygosity. Generally good for immunity - Example of nonrandom mating = inbreeding. Affects all genes. ---> Causes increase in homozygotes.

Selection Mechanisms that Result in Evolution

- "Nothing in biology makes sense except in light of evolution". - Genetic variation → selection → evolution.

Genes that Control the Cell Cycle

- *CDKs*: enzymes that add phosphates to other proteins. - Cyclin specifies which proteins the CDK will phosphorylate. - *G1-S transition*: Retinoblastoma protein. Mutated RB → G1-S transition unregulated. - Cyclin D over expressed in 50% of breast cancers. RB binds E2F and keeps it inactive. Increasing concentrations of Cyclins D and E lead to phosphorylation of RB, which activates RB, and releases E2F. E2F can then stimulate the genes required for DNA rep

Epigenetic Effects of Chemicals

- *Endocrine disruptors*: chemicals can bind to hormonal receptors, modifying the response of the normal hormone pathway - *Vinclozolin* mimics testosterone, and binds to androgen receptors. - Increased DNA methylation in Vinclozolin treated males.

Cause and Effect of Drift

- *Founder effect*: small number of individuals colonize a new habitat - *Genetic bottleneck*: large population size goes through drastic reduction in population size - Produces a change in allele frequencies. - Allelic fixation. - Increased genetic divergence with time.

What is genomics?

- *Genomics* is the study of genomes. You are looking at data at the genome level - Sequence: looking at A's G's T's C's and U's - Function: Look at the function of individual genes - what is the role of everything - Variation: SNPs and changes in coding regions

Homeotic Genes in Drosphila

- *Homeotic* genes become active and determine the identity of each segment. - On after the morphogens have acted - which segments get legs, antennas, etc. - Activated by segmentation genes. - Mutations in these genes cause some spectacular phenotypes, e.g. Antennapedia mutations! (generally not severe enough to cause death - NOT lethal)

Evolutionary Conservation of Homeobox Genes

- *Hox genes*, includes homologs of homeotic genes. - In humans due to duplication, there are Four clusters of Hox genes each one containing about 10 genes. - Mammalian Hox genes encode transcription factors that help determine identity of body regions along anterior-posterior axis. Homologs between species are expressed in the same order. Gene A - Anterior; Gene Z - Posterior. - the Hox genes in humans are not all organized next to each other as in Drosophila. They are scattered throughout the genome

Gene Expression and Development

- *Morphogen*: A protein/mRNA relative amount determines fate of cells: Expression gradient. - Increased expression of a morphogen puts a cell under a particular plan. A different amount puts a cell under a different plan. - "French Flag" model. - Shh good example.

Anterior-Posterior Axis

- *bicoid*, first transcribed in the ovary during oogenesis. - The bicoid mRNA is incorporated into the cytoplasm of the egg. - bicoid becomes anchored to the anterior end.

Application: using RNAi for treatment of human disease

- Disorders of cholesterol metabolism. - ApoB protein key part of lipoproteins. - Some people possess genetic mutations that cause elevated levels of ApoB → increased chance of coronary artery disease. - In 2006 it was demonstrated that RNAi could reduce the levels of ApoB. - Not easy! Had to find a way to get the synthesized siRNA into the primate cell. - Encapsulated ApoB-siRNAs in lipids → increased the time the siRNAs spent in the cells. - Injected macaques with ApoB-siRNAs as well as control group. Results: direct relationship between siRNA dosage and serum cholesterol levels

Gap Genes

- Divide the embryo up into broad regions. - Mutations in gap genes eliminate whole groups of adjacent segments. - E.g. Krüppel gene, mutations cause the absence of neighboring segments.

Cell division regulated by different groups of genes

- Cell division regulated by signals that stimulate and inhibit division. - Stimulatory gene can be made hyperactive. Such mutations are normally dominant: *Oncogenes.* - Cell division may be stimulated when inhibitory genes are made inactive: recessive effects → *tumor suppressor genes.*

Epigenetic marks and human behavior

- Childhood abuse increases chance of adult depression, anxiety, and suicide. - Relationship between abuse and methylation of glucocorticoid receptor genes. - Low socioeconomic environment as children alters expression of over 100 immune genes in adults. - Effect on adult immunity.

Example of DNA methylation in Honey Bees

- Colony all female, usually all related - Queens and workers differ in many respects, but all are sisters. - Diet: Royal jelly --> silences *Dnmt3* (DNA (cytosine-5)-methyltransferase 3) : this leads to repression of the repressor and queen genes are expressed - Workers injected with siRNA that targets Dnmt3 developed into queens

Genome-wide Epigenetic Marks

- Compared methylation sites between undifferentiated human stem cells, and fibroblasts. - Lots of differences. - Compared methylation patterns of cancer versus non cancerous cells. Targets for gene therapy? Hyper/Hypo methylation in cancer genes? - X-ChiP, allows us to look at modified histones.

Behavioral Epigenetics

- DNA methylation differences and acetylation of histones → alter expression of glucocorticoid receptors: stress. (cortisol binds to these - stress response) - Experimental reduction of deactylase inhibitor removed this effect. Two mice groups: one was low licking and grooming brood and the other was a high licking and grooming brood. The low licking brood had reduced GR expression (high methylated gene) which comes with high cortisol levels, high anxiety, and low licking and grooming in further generations. In contrast, the high lick group had high levels of GR expression (low methylation patterns)

Class 2 transposable elements

- DNA transposons. - Use both cut and paste and copy and paste mechanisms. - in cut and paste, no copy made. It is cut out and placed somewhere else in the genome

Applications of NGS

- De novo genome assembly. (new species sequenced) - Genome re-sequencing. - RNA-seq. (allows us to look at gene expression) - SNP discovery (finding polymorphisms in the genome and looking at effects) - Epigenetic profiling (figuring out which Cs are methylated) - Ancient DNA (sequencing organisms which are extinct)

DNA-repair genes and cancer

- Defects in genes that encode part of DNA-repair systems have been associated with number of cancers. - 13% of stomach cancers: mismatch repair. - Heterozygote: normal allele sufficient for normal mismatch repair. BUT, likely normal allele will be mutated in a few cells. - No mismatch repair → higher rates of mutation. Homozygote recessives usually do NOT make it far past fertilization

Determining Changes in Allele Frequency due to Selection (General Selection Model)

Look in Notes, kind of complicated p2WAA /ϖ ..... 2pqWAa/ϖ .... q2Waa/ϖ Next Generation Calculations A = 0.11 + ½ 0.44 = 0.33 a = 0.44 + ½ 0.44 = 0.66*

How we Define Selection

Selection (and evolution) occurs when there is: - Variation in a phenotype. - Consistent relationship between phenotype and fitness. - Additive genetic variance for phenotype: *additive effects* refer to the relationship between parents and offspring (the match) - *Directional*: one side is favored, a shift in the graph to either side - *Stabilizing*: middle is favored (bell shape curve) - *Disruptive*: individuals in the center are at disadvantage

Transposable Elements

Sequences of DNA that move around to different positions in the genome - mobile DNA sequences. - Can insert at many different locations → transposition. - Often cause mutations; insert within genes disrupting transcription or by promoting rearrangements. - Transposable elements recognize specific sequences. - All have *short flanking direct repeats* on both sides of the element. Not part of the repeat and do not travel with it. - At the end of most transposable elements are *terminal inverted repeats*- 9 to 40bps in length that are inverted complements of one another. ARE part of the transposable element

Calculating Allele Frequencies

- Alleles have continuity across generations: - Alleles (e.g. A and a) represented by p and q p = *2n (AA) + n (Aa) / 2N* q = *2n (aa) + n (Aa) / 2N* p + q = 1

Bioinformatics

- Analysis and cataloging these data = huge task! - Bioinformatics = the science of analyzing large datasets. use databases to annotate genes - codons, peptides, function, etc.

Genes that promote Vascularization and cancer

- Angiogenesis important to tumor progression. - Stimulated by growth factors: overexpressed in tumors. - In development of many cancers, tumors give rise to secondary tumors elsewhere. - Metastasis cause of death in 90% of human cases. - Microarrays identified genes overexpressed in metastatic cells. With Microarrays, we can compare metastatic cells and tumor cells. If we can stop metastasis, prognosis is much better

Determination of Dorsal Ventral Axis

- At least 12 different genes determine this axis. - *dorsal*- expressed in the ventral end of the embryo - After nuclei have moved to the periphery of the embryo, dorsal protein becomes redistributed.

BLAST

- Basic Local Alignment Search Tool. - Compare against annotated databases of model organisms. - *E-values*: number of hits expected due to chance, smaller e-value more likely true hit.

Out of HWE: Mutation

- Big question is what does that mutation do? is it good, bad, equal? Mutations generally reach an equilibrium, assuming there is no selection and they are equally fit.

Clonal Evolution of Tumors

- Cancer begins with a cell that undergoes mutation causing it to divide rapidly. - Clone of cells. - Increased rate of mutation: increased proliferation. Mutations lead to cancer, so upon mitosis, division leads to more cells (more cells = increased chances of mutation due to division rate)

The Role of the Environment in Cancer

- Cancer rates vary across the world. - Genetics or environment? Allelic differences between populations may increase/decrease cancer rates. Environment also chances cancer rates - Migrant populations typically take on cancer rates of new country. - Tobacco use, diet, exercise, alcohol, UV radiation. - Chemicals: Benzene, PCBs

Loss-of-function mutations

complete or partial absence of normal protein function. Frequently recessive. E.g., mutation that causes cystic fibrosis. (nonsense and maybe missense)

Epigenetics

defined as variation NOT in the nucleotide sequence that gets transmitted to multiple generations - Epigenetics first defined in 1942 by Conrad Waddington. - Phenotypes and processes transmitted to cells but not the result of genome sequence. - Heritable changes.

Underdominance

selection in which the heterozygote has lower fitness than that of either homozygote - Extremely rare

polymerase chain reaction (PCR)

steps: 1. Denature - strand separation (cook to 94 degrees centigrade) 2. Anneal - primer attachment; from gene of interest 3. Extension/replication - Taq polymerase is using primers to elongate - Need to know sequence to make a primer - multiple products can occur - forward and reverse primers may have complementarity. Also, gene families may have similar sequences so you may accidentally amplify multiple genes (multiple amplicons)

genetic anticipation

the phenomenon in which the severity of symptoms in genetic disorders increases from generation to generation and the age of onset decreases from generation to generation. It is caused by the expansion of trinucleotide repeats within or near a gene and was first observed in myotonic dystrophy

Making Sense out of 250 million sequences

- Need to align sequences to form *contigs*: consensus sequences from numerous reads - Can now annotate using BLAST or other databases. Lets you know which genes are similar between organisms

Other Genes in the Dorsal-Ventral Axis

- *cactus* binds to *dorsal* trapping it in cytoplasm. This leads to degradation of dorsal in dorsal side - Presence of *toll* leads to phosphorylation of *cactus* --> degradation. *Toll* is expressed in ventral side - When *cactus* is degraded, *dorsal* is released and can move into nucleus. - Together *cactus* and *toll* regulate the distribution of dorsal. - High nuclear concentrations of *dorsal* activates *twist*, causes ventral tissues to develop. - Low nuclear concentrations of dorsal activate *decapentaplegic* = dorsal tissues. Twist and decapentaplegic are NOT morphogens because they are not a concentration. They are purely on or off. Cactus and Toll are morphogens

Nanos and Development of the A-P Axis

- *nanos* acts at the posterior end of the axis. - mRNA becomes localized at the posterior end of the egg. - After fertilization nanos is transcribed into protein, which diffuses towards the anterior end. *Hunchback* is activated by bicoid in anterior end and *Caudal* is activated by Nanos in the posterior end

Tumor-Suppressor Genes

- 10% of cancer causing genes are thought to be tumor-suppressor genes. - Heterozygote predisposition. - Haploinsufficiency - heterozygotes may not be enough to have WT function If individual cancer cases have different mutations, likelihood of curing it is low (Ex -> breast cancer). Some cancers, like prostate cancer, may be cured because they have similar dysregulations

Sequencing the Entire Genome

- 1995 the first free-living organism Haemophilus was sequenced: 1.8 million bps. - In 1996 yeast. - In 2000 Drosophila - In 2000 first draft of the human genome.

Artificial Selection can Still Cause Evolutionary Change

- 20 Microsatellites run in 241 bighorn sheep from around the Canadian Rockies. - Quantitative genetic methods determined that ram weight and horn size were highly heritable. - Selection for rams with big horns: Positive (good for mating and dominance in polygyny) and negative (big rams are hunted by humans - Between 1971 and 2002 horn size decreased by about a quarter.

Loci with Multiple Alleles

- 3 alleles: A1, A2, A3 (p, q, and r) p = 2n(A1A1) + n(A1 A2) + n(A1 A3) / 2N - The frequencies for all alleles at a locus should ALWAYS sum to 1.

Epigenetics in cognition

- Abnormalities in DNA methylation associated with disorders of development, and IQ. - Training mice to avoid adverse stimulus reduced methylation of Bdnf (growth factor stimulates neuron connection). - Demethylated Bdnf is more active = memory. - Injecting mice that inhibits demethylation, less Bdnf expression, less memory. - Connection between deacetylation of histones and Alzheimer disease. learning decreases methylation

Signal-transduction pathways and cancer: e.g. Ras

- Activated when a growth factor binds to a receptor on the cell membrane. - Ras becomes activated when binding adaptor molecules. - Genes that encode Ras proteins are often oncogenes. - 75% of pancreatic and 50% of thyroid tumors have mutations in ras genes. Impacts many other genes

Site directed Mutagenesis: Transgenic Animals

- Add sequences of interest to the genome of an organism that normally lacks sequence. - Transgenic mice often used in study of human genes. - Oocytes of mammals large enough that DNA can be injected directly into them. Leads to all cells past birth having a gene How the Process goes down: - Mice are mated and fertilized eggs are removed from the female mouse. Foreign DNA is injected into one of the pronuclei. Embryos are implanted in a *pseudopregnant female* (a female that is not pregnant but was mated with a sterile male mouse; allows oocyte to take to uterus and develop). Offspring are tested for the presence of the introduced transgene. Mice carrying the gene are are bred to produce a strain of mice homozygous for the foreign gene. Now observe and can cross recombinants and non-recombinants. - Success rate is low (10%-30% of eggs survive and most do not have copy of altered gene of interest). - Advantage of working with mice! -- fast generation time and large liters - Insertion of foreign DNA early in development so most, if not all copies of chromosomes should have it. (need to work in early stages of zygote) - Including germ cells.

Pair-rule Genes

- Affect the development of pairs of segments. - E.g. mutations in the even-skipped gene cause the deletion of even-numbered segments. - Mutations in the fushi tarazu gene cause absence of odd-numbered segments.

How a Fish Lost Its Eyes

- All vertebrates have a set of genes that encode for eye development. (conserved amongst vertebrates) - The Mexican tetra. - About 24 hours after fertilization eye development aborts --> blind Tetra - Expression of *sonic hedgehog (shh)* and *tiggy-winkle hedgehog (twhh)* increased in blind tetras → apoptosis. The cave version of the tetra is a closely related blind species. It went blind because developing eyes was a waste of resources

Allele Frequencies used to Describe Gene Pool

- Allele Frequencies used to Describe Gene Pool - Genetic variation is the basis of evolution. Ability to pass on genetically fit allele is evolutionarily important - Although genetic change occurs within the individual, evolution operates on the population Rate of Evolution is NOT constant. Varies with environmental conditions

Magnitude of Genetic Drift

- Drift can be quantified by *variance* in allele frequency: Sp2 = (p x q)/2N E.g. p = 0.5, N = 50: Sp2 = 0.5 x 0.5 / 2 x 50 = 0.0025. if p = 0.5, N = 10: Sp2 = 0.5 x 0.5 / 2 x 10 = 0.0125. - Smaller populations more likely to be influenced by genetic drift. -- smaller number of individuals you can select for to make the next generation. May lead to *fixation* over time: loss of heterozygotes; alleles are fixed - Experiments with Drosophila and two alleles: bw75 and bw. 107 populations: 8 males and 8 females. Equal allele frequencies. 19 generations. - This experiment shows that as a result of genetic drift, allelic frequencies in the different populations diverged and often became fixed for one allele or the other

Paramutation in Mice

- E.g. Kit locus; tyrosine kinase receptor, pigment. - Kitt allele is an engineered allele (lacZ). - Kit+ Kit+ mice = WT, Kit+ Kitt = white pigment. - Kit* allele passed on to future generations. - miRNAs that degrade Kit mRNA. miRNAs passed on. Sometimes Kit-t modifies Kit-t and sometimes it doesn't. About 50/50 - so *penetrance* of alleles is different. Kit-t not 100% whereas B' in corn is 100% effective

Spontaneous Chemical Changes

- E.g., depurination, the loss of a purine base from a nucleotide. - Occurs when the covalent bond connecting the purine to the 1'-carbon atom breaks.

Understanding Drosophila Developmental Genetics

- Each cell has the same genome. - Changes in expression due to inducing or suppressing expression by morphogens results in the unique development of each fly segment. - Interruption/mutation results in death, or modified phenotypes (usually appendage additions)

Out of HWE: Migration

- Effect of migration: 1) it increases gene flow, and 2) increases genetic variation within populations. - decreases genetic distinction between populations - With each generation of migration, frequencies of two populations will become similar. *Gene Flow*: How different populations are swapping alleles. This erodes the uniqueness of different populations and lead to two populations becoming genetically similiar

What Controls timing of Hox Gene Expression?

- Epigenetic changes are very important in controlling organism development. - Hox genes highly methylated. - Methylation reduced in areas where expression is needed. - miRNAs may be important in determining hox gene expression. miRNAs are less specific - due to duplications of Hox (A,B,C,D), there will be polymorphisms between the genes. Similar, but not perfect match; therefore, miRNAs are the best option

Egg polarity Genes

- Establish the two main axes of development. - *Egg-polarity genes are transcribed into mRNAs in the course of egg formation in the maternal parent.* - These mRNAs become incorporated into the cytoplasm of the egg. - After fertilization = translation. Morphogens.

Map-based Sequencing

- First create high detailed genetic and physical maps (known locations of markers). - Large pieces of chromosomes cut up by partial digest (shotgun approach). Genes are too big so you need to chop it up with restriction enzymes - Cloned into *BACs, YACs, or cosmids.* BAC - bacterial artificial chromosomes YAC - Yeast Artificial Chromosome - entirely synthetic chromosome put into genome, short generation time = fast sequencing. More efficient because you can sequence larger pieces --> build contigs that limit spontaneous mutation mistakes

Mutagenic Effects of Retrotransposition

- For example color in grapes. - Black grapes produce by anthocyanin pigments. - Gret1 10,422 bp retrotransposon disrupts anthocyanin = green grapes. Retrotransposon inserts near promoter. Modifies RNA pol's ability to bind, lowering transcription - In black grapes second mutation = red grapes. transcription levels increase and red grapes are produced - Can cause diseases.

Functional Genomics

- Function genomics characterize what sequences do. - *Transcriptome* - the part of the genome that is transcribed (can find protein coding regions) - Start/stop codons. - Use a closely related model organism (comparative approach). - Can use related models to infer *homology* - gene A in species 1 is the same as gene B in species 2

Utilising SNPs for GWAS (Genome wide association studies)

- GWAS = approach to determine link between phenotype and genotype. - 17,000 people from the UK were genotyped for 500,000 SNPs. - Detected strong associations between 24 SNPs and incidences of Crohn's disease, rheumatoid arthritis, bipolar disorder, hypertension and two types of diabetes. Direct relationship between alleles and chances at getting sick. NOT mendelian inheritance. May or may not develop disease. Polygenic effects usually cause diseases and its hard to tell

Epigenetic Changes and Cancer

- Genes encoding proteins that are regulators of epigenetic change often associated with cancers. - 90% of cases of follicular lymphoma exhibit mutations in MLL2: histone methyltransferase enzyme. - Comparisons of cancer and non-cancer cells from same individual confirms methylation changes (hypomethylation). - Some regions hypermethylation seems to be involved.

Application of Genomics

- Genome duplications and gene family duplications. - *Gene deserts*: regions of low numbers of genes; unknown as to WHY - Transposable element evolution. - Protein diversity. - Tracking changes in homologous genes. - 99% of genes in common between human and mouse. 50% of genes in common between human and Drosophila. 18% of genes in common between human and Arabidopsis. --> this shows there is a lot of conserved sequences and similarity

Oncogenes

- Genomes within all normal cells have proto-oncogenes. - When these genes become mutated → oncogenes. - Viruses may serve as a vector in the development of cancers. - Mutation. - Recombination. - About 90% of all cancer genes = oncogenes Most oncogenes are transcription factors or activators of growth pathways

Illumina Sequencing

- Illumina currently most favored method. - Sequencing like Sanger, but automated. - Tradeoff between getting longer reads with Sanger, and shorter reads with NGS. - shorter bp reads (100-110 bps) difficult to work with because it is difficult to piece these small parts together

Changes in Chromosome Number (cancer)

- Some cancers associated with chromosomal abnormalities. - Deletions can result in the loss of tumor suppressor genes. Inversions and translocations can lead to cancer via disruption, or fused protein. Ex --> philadelphia chromosome - 9 and 22 translocation (BCR on 22 and 9 has c-ABL) - Fusion proteins seen in chronic myelogenous leukemia. - Transfer of a potential cancer causing gene to a new location. - Burkitt lymphoma cancer of B cells: Reciprocal translocation between 8 and 14 --> C-MYC now controlled by regulatory protein → Igs.

Genomic Imprinting

- Imprinting results in "Switching off" of one copy of a, or a group, of gene(s). - Several genetic diseases are caused by deletions of the non-imprinted copy. - Prada-Willi and Angelman syndromes: In humans, chromosome 15 is a hotspot for genomic imprinting. Disease caused by non mutated copies. Extreme methylation silences the region of the chromosome - Why does imprinting occur? Maybe one way of regulating amount of mRNA (dosage regulation) for a particular gene EX - *igf2 growth factor gene in mice*: this gene is connected with size. Maternally imprinted allele. Dad wants offspring to be large, regardless of sex. Mom would have to use hella resources for this so she wants smaller offspring. More beneficial to be large and have mom suffer - evolutionary advantage

The Human Genome Project

- In 1980 first proposal to sequence the human genome. Estimated time 15 years, budget of $3 billion. - Started in 1990 and finished in 2002 ---> *still parts of the genome which are hard to sequence* - Ex. centromeres and other heterochromatin areas - 20 research groups from around the world. - International Human Genome Sequencing Consortium.

Out of HWE and Inbreeding

- Inbreeding coefficient (F) is the probability that two alleles are identical by descent. - When inbreeding occurs proportion of heterozygotes decreases. - Each generation of inbreeding decreases heterozygotes. As F goes down, the level of variability in the population gets higher

Out of HWE: Selection

- Individuals with adaptive traits produce more offspring than others: Higher fitness. - *Fitness* = relative reproductive success of a genotype. Ranges from 0 to 1 (W). A1A1 =10, A1A2 = 5, A2A2 = 2. *(Number of offspring produced by each genotype/no. of offspring produced by most successful genotype)* W11 = 10/10 =1; W12 = 5/10 = 0.5, W22 = 2/10 = 0.2

Developmental and Cell Determination

- Initially, each cell in embryo is totipotent: has the potential to develop into any cell type. - Many cells in *plants and fungi remain totipotent*, but animal cells become committed to a certain function. This is why you can cut a piece of plant and grow it to an entire new plant - Commitment begins early in a cells life and is not reversible (determination). - Different expression between cells.

Insertion of Gene of interest (to multiply gene of interest)

- Insert gene of interest within bacterial plasmid and let it multiply. - Vector is a stable replicating DNA molecule that foreign fragments can be attached. Need three things in the plasmid: 1. must contain an ori 2. should carry selectable markers 3. needs a single cleavage site for each of one or more restriction enzymes used

Next Generation Sequencing Technology

- Introduced in 2005. - Millions of fragments sequenced simultaneously. - Initial costs very high, about $2000 currently for one lane on a machine, plus library construction costs. - But can get up to 240,000,000 sequences back!

Viruses Associated with Cancer

- Link between viruses and development of cancer. - 95% of all women with cervical cancer are infected with HPV. - Retrovirus: Cause cancer by mutating and/or rearranging host genes. - Affecting expression of host genes.

The Study of Development Reveals Patterns of Evolution

- Many biologists are turning to *ontogeny* (evo-devo study) for a better understanding of the processes of evolution. - Revealing that the same genes often shape developmental pathways in distantly related organisms. - E.g., development of eyes in Drosophila, mice, and humans. all very similar to eachother --> mutated Pax6 gene leads to blindness and improper eye development in all three species

MicroRNAs and Cancer

- Many tumor cells suppression of miRNAs. - May contribute by allowing oncogenes to be expressed at high levels. - Let-7 normally controls ras, ↓ expression of let-7 in people who smoke. - Transcription factor c-MYC often over expressed in cancer cells. - Binds to promoters of miRNAs and decreases their transcription

HPV and Cervical Cancer

- Many types of HPV: In the US 70% of cases of cervical cancer are caused by HPV-16 and HPV-18. - Inactivate RB and p53 à loss of control of cell cycle. - About 75% of sexually active women have HPV, but development into cervical cancer has declined. - Pap test: early detection = treatment. - Very high infection and death for women in developing countries. No access to test, and treatment.

Epigenetic Marks: Methylation

- Methylated cytosine. - Methylated changes are preserved and can be passed on. Role of Methyltransferase - NOT known why/how the specific new DNA sequence is methylated

Histone Modification (epigenetics)

- Methylation, acetylation, phosphorylation. - Modification of histones carried out by *polycomb (PcG) proteins.* - PRC2 adds 2 methyl groups to lysine 27 of H3. - Mutations of PcG can produce bizarre phenotypes

Mutations in Homeobox Genes: Ultrabithorax

- Mutations in *ubx* result in changes in wing development. - Expression of ubx in T3 changes structure of *halteres* (residual wings flys have in the thorax): normally confined to T2. - Other mutations in ubx result in "in between" phenotypes. Can get partial wing development or full blown extra pair - Two major clusters of homeotic genes in Drosophila Located on chr3. - The *Antennapedia complex*, affects the development of the fly's head and anterior thorax. - The *bithorax complex*, affects development of the posterior thoracic and abdominal segments The arrangement of the genes on the chromosome corresponds to the sequence in which the genes are expressed along the anterior-posterior axis of the body - Geneticists have altered expression by moving order of genes on the chromosome - NOT a happy accident --> Timing is important

Site-directed mutagenesis: CRISPR-Cas9

- Naturally occurring in bacteria: CRISPR RNAs have sections of foreign DNA within them → "memory RNA" → Cas destroys foreign DNA. Cas9. (kind of like an immune system for bacteria) - *CRISPR portion*:20 nt region of guide RNA pairs with DNA and Cas cleaves. - DNA to be cleaved must have protospace-adjacent motif (NGG). PAM - recognized by caspase for attachment. CRISPR RNAs come phages (viruses) attempting to attack a bacteria. - Seed Sequence: cRNA which recognizes DNA sequence to attack and cut - Cut leads to ds break. DNA doesn't like this. Either 1) deletion occurs which makes gene of interest non functional (KNOCK OUT) or 2) insert the correct sequence to give correct phenotype (homologous recombination) - *increased chance it will slot into right place by homologous recombination*

How to get plasmid in cell and check to make sure its doing what it should

- Need to place plasmid in cell. - Typically via *transformation.* - Can use fragment of lacZ and a gene that encodes resistance to ampicillin. - Can then sequence gene of interest. - these bacteria are usually E coli with a non functional Lac operon - ampicillin resistance is part of the plasmid and the partial LacZ+ is switched off because the gene of interest is inserted here in the plasmid. - Bacteria with an original (nonrecombinant) plasmid produce β-galactosidase, which cleaves X-gal and makes the colonies blue. - Bacteria with a recombinant plasmid do not synthesize β-galactosidase. Their colonies remain white. Rate of success for foreign DNA insertion into the plasmid is very low.

Out of HWE: Small Populations and Genetic Drift

- No population is infinitely large. - Just by chance the individuals that contribute to the next generation may alter in allele frequencies for whole population. - Role of chance in altering alleles The role of Chance

Transposable Elements in Maize

- Noticed that two genes (Ds and Ac) move together to different chromosomes. - Both genes are DNA transposons that possess terminal inverted repeats. - Ds has lost its transposase and so requires Ac to move. - Ds elements are Ac elements with one or more deletions. - Inactivation of transposase. Each kernel is a uniquely fertilized ovule. - Pigment encoding locus has two alleles C = pigment, c = no pigment. - Timing of insertion or secondary transposition is everything

Microarrays (gene chips)

- Nucleic acid hybridization. - Allow us to ask questions about gene expression. A microarray consists of DNA probes fixed to a solid support. Each spot has a different DNA probe. RNA is extracted from cells and reverse transcription in the presence of labeled nucleotide produces cDNA molecules with a fluorescence tag. The tagged cDNA will pair with any complementary probe. After hybridization, the color of the dot indicates the relative amount of mRNA in the samples. - mRNA is more unstable than DNA. Harder to work with. This is why we convert to DNA right away - Used to compare relative amounts of transcription between different types of cells (ex cancerous and noncancerous cells)

The Epigenome

- Overall pattern of chromatin modification possessed by each individual. - More complex than the genome! - Can find methylated cytosines with enzymes. (one reason why meth-C's are easy to study) Two restriction enzymes Hpdll and Mspl: - no difference between the cut sites (nucleotides). However, Hpdll only cuts at non methylated sites. Different banding patterns on gel may show you where methylated regions are.

Epigenetic Change and Its Effects

- Paramutation in corn. - r1 locus. Rr allele = purple kernels, Rst = spotted kernels. - RrRst = spotted kernels. - Subsequent Rr plants still produced spotted kernels. - Interaction of alleles that leads to heritable change in expression of one allele.

Impacts of Transposable Elements

- Parasite/host relationship. - Retrotransposons act very like retroviruses. - Maize genome up 85% of genome. - Humans about 40% - What can the host genome do to stop it? Mehtylation may increase after SB and other transposons are inserted. Is the cell responding? who knows This may be a reason why Alu only inserts into introns

Applications of Molecular Techniques: Biotechnology

- Pharmaceuticals. --> drug development: drugs react differently in different people (negative affects in certain areas) - Resistance to infection in crops. - Increased yield in livestock. - Medical testing and rarely, correcting human genetic defects (gene therapy).

Types of Vectors

- Plasmids are great at taking up short fragments of DNA. (~5000 bps) - For whole genes, or genome sequencing need something else. - *Cosmids* contain lambda phage cos sequence. Can handle about 45-50 KB of sequence. - *Fosmids*, similar to cosmids based on F- plasmid.

Implications of the Hardy-Weinberg Law

- Population cannot evolve if it meets HW assumptions. - HWE only applies for that generation: environment may change leading to a certain genotype having increased fitness

Limitations to HWE

- Population must be "large". - Random mating, means each genotype mates relative to its frequency. Ex --> If high frequency of p allele, than most of next generation should have p. - Allele frequencies not affected by migration, selection, or mutation. - These laws apply to a locus under consideration - NOT for other loci - Population maybe in HWE for one locus, and not for others.

Predicting the Effects of Inbreeding

- Recessive allele (a) frequency (q) of 0.01. - Random mating (F=0); frequency of disease: *q2 + F(pq)*= 0.012+ 0 x(0.99 x 0.01) = 0.0001: 1 in 10000. F= 0.25; q2 + F(pq) = (0.01)2 + 0.25 x (0.99 x 0.01) = 0.0026: 1 in 2600. ***significantly increases frequency of genetic disease - *Inbreeding depression*: when this starts to have an effect on the phenotype. Gets worse with more inbreeding - Negative consequences of inbreeding has been recognized for some time: cultural taboo. - William Schull and James Neel found that for each 10% increase in F mean IQ dropped 6 points. - Inbreeding studies in lab organisms confirm harmful. Ex --> as inbreeding goes up in corn, corn yield goes down

Class 1 transposable elements

- Retrotransposons - Transpose through RNA intermediate. - Copy and paste mechanism: individual transposon inserts and is used as template. This means transposon amount can increase over time - Examples include LINE and SINEs. Long interspersed Nuclear Elements and Short interspersed nuclear elements. Ex --> Alu 100s bp makes up 18% of our genome.

X-Linked Loci Frequencies

- Same principles, but need to account for differences between males and females. - E.g. two alleles XA and Xa (p and q). - For *p = 2n XAXA + n XAXa + n XAY/ 2n females + n males.* q = 2n XaXa + n XAXa + n XaY/ 2n females + n males.

Segment-Polarity Genes

- Segment-polarity genes, guide the development of individual segments. - E.g. gooseberry mutation cause the posterior half of each segment to be replaced by anterior half of adjacent segment.

Cancer as a Genetic Disease

- Some cancers are consistently associated with chromosomal abnormalities: translocation between chr22 and chr9 and chronic myeloid leukemia. - Some cancers run in families. This means there has to be a genetic bases - highly heritable component (additive or general) - But, if cancer is a genetic disease why don't all cells in the body become cancerous? - different genes expressed in different cells. Lots of genes that control cell cycle get disrupted Heritability - belongs to a POPULATION used in the study. Predisposition - individual has increased chance --> ex you have a mutated allele

End Result of Map based sequencing: The Human Genome

- Summer of 2000 a rough draft of the human genome announced. Completed in spring 2003. (again, still NOT completely finished - high heterochromatin and repetitive DNA. Hard to determine sequence at these regions) - 99.999% accurate. - Cost of sequencing a complete genome is constantly dropping. - Race for the $1,000 genome! - Availability of multiple human genomes means we can ask and answer many questions. Why do we care about cheap access? -> easier and cheaper means we can study the variability of DNA and its phentypic effects within a species. Compare genomes to study diseases. Look for shared mutations ---> important to science

Sleeping Beauty: A Homemade Transposable Element

- Synthetic transposable element. potential for gene therapy - Components have their origin from multiple organisms. - Fusing previously inactive parts of two different transposable elements. - Inserted into both somatic and germ cells. Examples of Using SB to Induce Mutations - Although sleeping beauty is 11 years old, few studies have successfully identified a previously unknown gene as being important in human health. - Insertion of SB into Slc16a10(solute carrier protein) in mice increased Type 1 diabetes --- might be useful to recognize this sequence and give people odds of getting T1DB

Interactions Between Gap and Pair-Rule Genes

- The gradient of hunchback critical for regulation of other gap genes. - *Krüppel* repressed by hunchback and giant (where bicoid is present but low concentrations) - Set boundaries between genes. - Pair-rule gene expression determined by gap genes. In the pic: eve stripe 2 is determined by the absence of giant and Kruppel. - Segmentation is complex - #/concentration of morphogens leads to development

Potential and limitations of CRISPR

- The specificity of the sgRNA and the need for PAM. --> allows us to *multiplex* (use CAS for a different gene at 1 time) - Change the Cas9 to make single-stranded breaks...why? --> ss breaks creates sticky ends which are more likely to lead to recombination - Limitations: Off-target cleavage. sgRNA does not have to match 100%. - When to insert? End up with mosaics. --> best time to insert is very early on after fertilization; is 50/50 enough to give proper phenotype (haploinsufficiency) - Already been conducted on human embryos. --> gene therapy, hard part is inserting the gene - Certainly the potential to add problems: cancer.

Problems: How do you find gene of interest?

- To analyze a gene must first locate and isolate. - "shotgun cloning": fun method - multiple copies of genomic DNA are digested by a restriction enzyme for a limited time so that only some of the restriction sites in each molecule are cut. Different DNA molecules are cut in different places, providing a set of overlapping fragments. Each fragment is then joined to a cloning vector and transferred to a bacterial cell, producing a set of clones containing overlapping genomic fragments, some of which may include segments of the gene of interest - problem at first is that you don't know the sequence of the gene, so you can't design primers. BUT, once recombined in a bacterial plasmid, you know the sequences of the plasmid so you can amplify the gene that way - sequencing fragments from the plasmid

Isolating a gene of interest - digestion

- Type II restriction enzymes recognize specific sequences and cut the DNA at defined sites. - When cutting enzymes either leave "sticky" or "blunt" ends. - These can be used to isolate gene of interest. *Sticky ends*: staggered cuts (Ex hindIII); easier to use and more common *Blunt Ends*: cut both strands at same place (ex. PvuIII)

Forward and Reverse Genetics

- Typically begin with a phenotype and proceed to a gene that encodes the phenotype = *forward genetics.* (usually not model organism) - Alternatively, begin with a genotype and proceed to the phenotype via altering the sequence (cause mutations) = *reverse genetics.* Ex --> Knock out mice, silenced genes, etc. Need to know genes to manipulate (usually model organism) - Both approaches are used in analyses of gene function.

DNA Sequencing: Sanger Method

- Uses *dideoxyribonucleotide triphosphate (ddNTPs)* - Lack a 3'OH group. - This allows them to be incorporated into the sequenced strand and stopping additional nucleotides from being added - basically allow replication to occur with ddNTPs and then extrapolate sequence from electrophoresis results

Interested in Variation: Single-Nucleotide Polymorphisms

- We are all identical for about 99.99% of positions in the genome. - As the human genome is so large 3.2 billion base pairs, you and I are different at about 3 million base pairs. - MOST of this variation = SNPs. - About 1 SNP ever 1000 bps. - NOT constant, varies with region of the genome

X-Inactivation

- Which X is inactivated within a cell is controlled by the *X-inactivation center.* - Inactivation then spreads to the remainder of the inactivated X. - Both Xs have Xist, so why isn't the other one silenced? - Several long noncoding RNAs (lncRNAs) involved. - *Tsix* is transcribed on active X: antisense to Xist. Suppresses Xist on active X. - *Jpx* stimulates transcription of Xist on inactive X. Not clear how start up occurs. There is some expression of genes on the silenced X. NOT totally silenced

Radiation

- X-rays, gamma rays and cosmic rays are all capable of penetrating tissues. - Do so by dislodging electrons, causing free-radicals. - UV light is absorbed by DNA, can cause pyrimidine dimers. - Can block replication, causing cell death. Dimers may stop DNA pol from replicating

Suppressor Mutations

- mutation occurs at a site that is distinct from the site of the original mutation, i.e., a double- mutant. Reverts the organism back to normal - Can be intragenic or intergenic.

insertions and deletions (indels)

- mutations in which extra base pair(s) are inserted into a new place in the DNA or a section (one or more base pairs) of DNA is lost, or deleted

Silencing Genes with RNAi

- siRNAs mechanism to switch off expression. - Needs to be recognized and cleaved by Dicer. - Designed siRNA MUST be unique. - Once synthesized, the siRNA can be cloned into a plasmid, inserted into E. coli. - OR, synthesize double-stranded mRNA that Dicer will recognize.

linkage disequilibrium (LD)

- the nonrandom association between genetic variants. AKA the nonrandom association of alleles at different loci - LD is tightly connected to recombination. - Why do we care about LD? Gives us info about diseases High LD (LD = 1) equals the SNPs are always linked/inherited together. LD=0 means they completely unlinked Further apart two markers are on the genome, the more likely association is broken up. Recombination is NOT constant --> if you're looking at diseased individuals and you find high LD, you cannot tell which polymorphism is causing the disease. But if you have low LD, you may isolate which polymorphism causes disease

Mismatch Repair Enzymes

1) Detection: Damaged region of DNA is recognized. 2) Excision: DNA-repair endonucleases nick the backbone near to the damaged region. 3) Polymerization: DNA polymerase adds new bases to the 3'OH group by using other strand as template. 4) Ligation: DNA ligase seals nicks in backbone. - multiple different mismatch repair enzymes in the genome - Numerous diseases are directly linked to ineffective repair enzymes

Testing for Deviations from Hardy-Weinberg

1) calculate allele frequencies. 2) expected genotypic frequencies assuming HWE. 3) compare using chi-square. Chi-squared: (135-129.8)2/129.8 + (44-54.5)2/54.5 + (11-5.7)2/5.7 = 7.16. D.O.F. = number of expected genotypic classes (3) - the number of associated alleles (2) = 1.

Transposition

1) staggered breaks are made in target DNA. 2) the transposable element is joined to single-stranded ends of the target DNA. 3) DNA is replicated. - Transposase encoded by element makes staggered breaks.

Two factors that affect selection effects

1. how abundant the allele under question is 2. how strong the selection pressure is. - selection against rare recessive allele is inefficient

Missense Mutation

A point mutation in which a codon that specifies an amino acid is mutated into a codon that specifies a different amino acid.

Silent Mutation

A point mutation in which a codon that specifies an amino acid is mutated into a new codon that specifies the same amino acid. (may still affect translation - kinetics) - Transitions are most likely to be silent. Translations are most likely to give missense or nonsense

Nonsense Mutation

A point mutation in which a condon that specifies an amino acid is mutated into a stop (nonsense) codon.

Overdominance

AKA heterozygote advantage - being heterozygote is advantageous compared to being either heterozygote - Example of overdominance is frequency of sickle-cell allele in sub-Saharan Africa.

Gain-of-function mutations

An entirely new protein is produced that may have unrelated function. Frequently dominant.

Human Epigenome Project

An international collaborative project which aims to identify, catalogue, and interpret the epigenome and links it to diseases/phenotypes

Hardy-Weinberg Law

Assumption: if population is large, randomly mating, and not affected by mutation, migration, selection then: Prediction: 1) the allelic frequencies do not change, and 2), p2 + 2pq + q2 = 1. When genotypes are in expected proportions, the population is in HWE.

Southern blotting

Basically, you have digested DNA and you put it through gel electrophoresis. Use buffer to separate dsDNA and transfer to nylon membrane. Then use radioactive probe to tag complementary fragments of DNA on the membrane - Probe - radioactive DNA strand which is complementary to DNA gene of interest *Limitations* - you need to know what your working on to be able to make probe for DNA complementarity

Epigenetics: Bisulfite Sequencing

Bisulfite converts non mehtylated C to U. Can look at where nucleotides are altered pre and post treatment. Can NOW look at variations between individuals - can look at multiple different regions of the genome

Mutation Rates

Calculations of mutation rates are affected by 3 factors 1) frequency with which a change in DNA takes place. 2) The probability of repair. If repair systems are effective, mutation will be low. 3) Probability that a mutation will be recognized and recorded. (needs to be a phenotypic effect to record a mutation) - Are mutation rates constant within the genome? No, Introns/noncoding regions liekly to have mutations (no selection pressure = mutation). Genes where selection for variation occurs. Ex --> immunoglobin - What happens next? Depends, No idea. Completely silent = flatline mutation rate (not selected for or against). Better = increase in frequency. Worse = decrease in frequency

What is Cancer?

Cancer the presence of cells that do not respond to normal controls of division - One in every 5 people in the U.S. will die from cancer: Billions of $ per year. - Not a homogenous disease: different cancers can be caused by varying mutations All cancers are NOT equally abundant. Some have a higher mortality rate then others

What Causes These Mutations?

Caused by both internal factors and external factors. - Random error in DNA pol and/or RNA pol. - Exposure to mutagenic compounds. - E.g., *tautomeric shift*: Where the positions of protons within the bases changes. New "form" or tautomer is produced. Still the same base, but allows mis-priming... Allows for incorrect pairing during replication/transcription Rare form of C can pair with A. Rare form of G can pair with T.

Transversions

Change of a chemical group - Purine to pyrimidine or vice versa - a lot more options but not as common

Transposase

Enzyme encoded by many types of transposable elements that is required for their transposition. The enzyme makes single-strand breaks at each end of the transposable element and on either side of the target sequence where the element inserts.

Paramutation

Epigenetic change in which one allele of a genotype alters the expression of another allele; the altered expression persists for several generations, even after the altering allele is no longer present. 1) Newly established expression pattern is transmitted to future generations, even though original allele is absent. 2) altered allele is now able to convert other alleles to new phenotype. 3) no associated DNA sequence difference in altered alleles. *Epialleles*: alleles where DNA is the same, but different forms give rise to different phenotype

Automated Sanger Sequencing

Instead of four different tubes with different ddNTPs, each of the four ddNTPs is tagged with a different fluorescent dye, and the Sanger sequencing reaction is carried out. (blue - C, black - G, Red - T, green - A) gel electrophoresis is used and the flourescent dye on the DNA is detected by a laser beam. Each fragment appears as a peak on the computer printou; the color of the peak indicates which base is present - The sequence information is read directly into the computer, which converts it into the complementary sequence

Linkage maps vs Physical Maps in Genomics

Maps are constructed by crossing individuals of known genotype and seeing the frequency of recombinants. *Linkage Maps* look at amount of recombination. Recombination does not tell you how close and far genes are. It more or less estimates order and distance (limited accuracy) *Physical Maps* - Based on the direct analysis of DNA, place genes in relation to distance in base pairs. The very basic orientation is roughly placed by linkage maps

Are mutations always bad or good?

Mutations are both the sustainer of life and the cause of suffering. Long term effect of a mutation will depend on many factors. - Somatic mutations are only passed on by mitosis. Therefore it cannot be passed to the next generation. Ex of bad --> cancer - Germ-line mutations occur in cells that give rise to gametes. About half the next generation gets it (all cells within this organism have the mutation)

Drosophila serves as a Model For Genetic Control of Development

Pattern formation consists of the developmental processes that lead to the shape and structure of complex multicellular organisms. - 2 reasons drosophila are dope: *1) short generation time (9 days)allows you to quickly see genetic effects and 2) Segmentation: can see what is expressed in each segment, what becomes what* Initial Development: sperm and egg fuse to create a single-celled diploid zygote. Multiple nuclear divisions create a single multinucleate cell, the syncytium. The nuclei migrate to the periphery of the embryo and divide several more times, creating the syncytial blastoderm. The cell membrane grows around each nucleus, producing a layer of cells that surrounds the embryo. The resulting structure is the cellular blastoderm. Nuclei at one end of the blastoderm develop into pole cells, which becomes the primordial germ cells. The anterior-posterior and dorsal-ventral axes of the embryo are established

Working at a molecular level - how to sequence and find genes

Research on a gene of interest. How do we find it? How do we separate it? How do we "grow" it? Success rate of gene transferal is very low!

Segmentation Genes

Segmentation genes control the differentiation of the embryo. - 3 main classes: 1) Gap Genes: Ex - Kruppel 2) Pair-rule Genes: Ex - fushi tarazu 3) Segment polarity genes: Ex - gooseberry

Base Substitutions (Mutations)

Switching out the nucleotide at 1 position

Neutral mutation

a mutation that does not change the function of the protein (silent or maybe missense)

Eastern Blotting

looks at post-translational modification of peptides

Microsatellites or Tandem Repeats

often in nonprotein coding regions of the genome but are found in coding regions - Often the number of copies correlates with the severity of the disease. - Can either affect protein, or repeat is located in trans. Ex. in protein coding regions - Huntington's disease Example: *Myotonic dystrophy* - mental defects, muscle weakness, cataracts, death after birth - Linked to two genes: Myotonin-protein kinase and ZNF9. - Both contain repeat sequences (tri and tetra). - Genetic anticipation. - Tetra repeats really **** people up because it alters the reading frame of the DNA.

Western Blotting

probe is an antibody which binds to certain proteins of interest

Transition mutation

purine to purine or pyrimidine to pyrimidine mutation - far more abundant than transversions although less options available

Northern Blotting

same concept as Southern blotting but uses RNA not DNA (probe is a mature mRNA) - looks at transcriptome - part of the genome that is transcribed