Set 1: Mendelian Genetics Advantages to Mendels use of garden peas: o Easy to cross-fertilize o Large numbers of offspring o Short growing

g season o Clear-cut alternative forms of particular traits alternative phenotypes o Establishment of pure-breeding lines (offspring carry parental traits that remain constant from generation to generation) o Carefully controlled breeding use of reciprocal crosses and self-fertilization Theories prior to Mendel: o One parent contributes most to an offsprings inherited features disproved through reciprocal crosses o Parental traits become mixed and forever changed in the offspring (disproved through reappearance of recessive traits) P F1 F2 (refers only to inbreeding, no outcrosses) Alleles: discrete units of inheritance; alternative forms of a gene different DNA sequences Polymorphic gene may have several alleles that normally occur in a population Monomorphic gene has only one allele that is normally present in a population Mendels Law of Segregation: the two alleles for each trait separate (segregate) during gamete formation, then unite at random, one from each parent, at fertilization describes how alleles of one gene behave Mendels Law of Independent Assortment: during gamete formation, different pairs of allele s segregate independently of each other describes how alleles of different genes behave Dominant and recessive are terms used to describe the phenotypic effect of different alleles o At the DNA level, different alleles differ in nucleotide sequence, resulting in changes in the amino acid sequence or the amount of protein 3:1 phenotypic ratio simple dominant and recessive traits; cross between monohybrids Dihybrid crosses: matings between individuals that differ in two traits o When dihybrids are involved and the alleles act in a simple dominant and recessive manner, new phenotypic combinations not present in previous generations appear; 9:3:3:1 phenotypic ratio Set 2: Extensions of Mendelism Incomplete dominance F1 hybrid resembles neither purebred parent (often an intermediate phenotype) Codominance alternative traits are both visible in the F1 hybrid Genotypic and phenotypic ratio for both is 1:2:1 (F1 cross); designate alleles as both uppercase Multiple alleles o e.g. the gene controlling the cell surface sugars responsible for the ABO blood grouping system is a multiple allele system in which IA and IB are codominant to each other and both dominant to the I allele o IA attaches a sugar different from that of allele IB and allele i does not attach a sugar at all o IAIB heterozygotes have both types of sugars on their cell surface

Variation on complete dominance are consistent with Mendels law of segregation the type of dominance relationship exhibited by different alleles does not affect the way they are transmitted; rather, it is a reflection of the way in which the proteins they encode act in the cell A gene may have more than two alleles o Reciprocal crosses can be conducted between pure-breeding lines representing all phenotypes to establish the dominance relationships between all possible pairs of alleles o This reveals a dominance series (alleles are listed in order from dominant to recessive) o e.g. fur colour in rabbits agouti is dominant (>99% in nature c+ is a monomorphic gene; albinos killed by predation in the wild) New alleles arise through mutation o Chance alterations in the genetic material arise spontaneously in nature Allele frequency the percentage of the total number of copies of a gene in a population represented by a particular allele o Wild-type allele greater than 1%; mutant allele less than 1% o Monomorphic: gene with only one wild-type allele; polymorphic: gene with more than one wild-type allele One gene may contribute to several visible characteristics o Pleiotropy multiple phenotypic effects caused by a single gene, e.g. sickle cell o A variation on pleiotropy some allele may cause lethality Some alleles may result not only in a visible phenotype, but also affect viability Assignment of dominant/recessive pertains only to the phenotype being regarded e.g. in mice, allele for yellow coat is dominant for coat colour but recessive for lethality; F2 phenotypic ratio is always 2:1 instead of 3:1 (homozygous AYAY die) e.g. all Manx cats are heterozygous for the Manx allele (homozygous fetuses die in utero) Manx allele is dominant for taillessness and recessive for lethality Multifactorial inheritance a phenotype arises from the action of two or more genes (polygenic), or from interactions between genes and the environment o Most common traits are determined by more than one gene o Novel phenotypes can emerge from the combined action of the alleles of two genes, e.g. comb shape in chickens individual trait determined by more than one gene o Phenotypic ratio of 9:3:3:1 indicative of two genes responsible for one trait, acting independently in a simple dominant and recessive manner (same ratio as for a dihybrid cross, except only one trait is affected) Complementary gene action two or more genes can work in tandem, in the same biochemical pathway to produce a particular trait o Heterogeneous trait a mutation at any one of a number of genes can give rise to the same phenotype o e.g. in Drosophila, a mutation in two different genes results in the same phenotype (black body colour) the wild-type phenotype (wild-type body colour) can be rescued by complementation (breeding two black flies, each with a mutation in a different gene, results in a fly with wild-type body colour) o Complementary gene action (e.g. AAbb x aaBB AaBb) - phenotypic ratio of 9:7 o For heterogeneous traits to determine if one or multiple genes are involved in producing a particular phenotype, use complementation testing

Complementation testing can also be used to determine if two individuals have mutations in the same gene Epistasis: a gene interaction in which the effects of an allele at one gene hide the effects of alleles at another gene epistatic = control over another gene o Recessive epistasis phenotypic ratio of 9:3:4 e.g. h Bombay allele in blood typing hh is epistatic to gene I H/h controls production of lipid H to which A and B sugars are attached hh is always type O, no matter what I/i is Two parents with blood type O can have a child with type A blood (ex. Mother is IA_hh, father is ii H_) o Dominant epistasis phenotypic ratio of 12:3:1 e.g. summer squash colour allele C is epistatic to gene G Deciding between different hypotheses using specific breeding tests o Cross of true-breeding parents gives new phenotype in F1 progeny indicates more than one gene is involved OR incomplete dominance of alleles in one gene o Intercross F1 examine ratios o To reveal genotype of an F2 offspring, cross with individual recessive for the unknown gene of the offspring The same genotype does not always result in the same phenotype influence of environment, modifier genes, and chance o Penetrance: % of population with a particular genotype that demonstrate the expected trait o Expressivity the degree/intensity with which a particular genotype is expressed in a phenotype within a population Genes can also determine differences between sexes o Sex-linked traits due to genes located on the X or Y chromosome o Sex-limited traits affect a structure or process that is found in one sex but not the other (not on the sex chromosomes both sexes have the gene but it is only expressed in one sex), e.g. bright plumage in male birds, milk production, horns/antlers o Sex-influenced traits show up in both sexes, but expression may differ between the two sexes, e.g. pattern baldness influenced by sex hormones; expressed in heterozygous men but not in heterozygous women, baldness results in both homozygous men and women, but much earlier in men (dominant trait in men, recessive in women) Influence of the environment factors such as temperature, light, and altitude can affect the phenotypic expression of a genotype o e.g. Himalayan coat pattern allele with temperature-sensitive expression Enzyme that converts colourless precursor to melanin is nonfunctional in warmer temperatures, resulting in light fur, and functional in cooler temperatures, resulting in dark fur o Conditional lethality a particular type of allele lethal only under certain conditions (e.g. malignant hyperthermia genetic disease triggered by certain anesthetics) Permissive allele does not manifest itself Restrictive allele manifests itself death

Chance occurrences in the lives of individuals can influence the expression of some alleles, e.g. exposure to carcinogens, radiation

Set 3: Pedigree analysis Many human traits run in families; most do not show a simple Mendelian pattern of inheritance because most are influenced by more than one gene (multifactorial) Most confirmed single-gene traits in people are relatively rare, involving abnormalities that are either disabling or life-threatening; e.g. sickle-cell anemia (single nucleotide substitution results in an incorrect amino acid, resulting in a change in the conformation of the beta-globin protein when deoxygenated), Tay-Sachs disease, phenylketonuria (PKU), albinism, Huntington disease (dominant), cystic fibrosis Dominant trait appears frequently in each generation Recessive trait appears sporadically, depending on mate being homo- or heterozygous for the recessive allele Autosomal trait conferred by gene residing on a chromosome not involved in sex determination Set 4: Chromosomes Chromosomal theory of inheritance: o Hereditary information is on genes, which are located on chromosomes o Egg and sperm contribute equally to genetic endowment of offspring through their nuclei Centromere DNA sequence, on top of which sits a complex of proteins (kinetochore) Sex determination o Humans presence of Y chromosome determines maleness (e.g. XO is female, but sterile) o Drosophila sex is determined by the ratio of X chromosomes to autosomes o Sex is not determined by X and Y chromosomes in all species, e.g. in some moths, fertile males are XO, females are XX o In other species, sex is determined by environmental factors, e.g. in some reptiles (e.g. alligators), sex is determined by temperature of incubation Diploid (2n), haploid (n); n = number of chromosomes in a normal gamete Karyotype always uses metaphase chromosomes Metacentric centromere in middle of chromosome; acrocentric centromere near one end Trisomy 21 tolerated because chromosome 21 is small (relatively few genes); other trisomies (aside from XXX are not tolerated in humans) Set 5: Mitosis Cell cycle: repeating pattern of cell growth, mitosis, and cell division o G1 interphase, gap before duplication; unipartites o S DNA synthesis and chromosome duplication; bipartites o G2 interphase, gap before mitosis; bipartites (generally shorter than G1 and S) Mitosis: nuclear division in cells; produces daughter cells that are genetically identical Centrosome organizes spindle; microtubules of the mitotic spindle form the aster Kinetochore site at which chromosome is attached to microtubules Prophase

o Degradation of nuclear membrane o Microtubules invade the nuclear area o Sister chromatids attach to microtubules from opposite centrosomes at the kinetochore Metaphase o Chromosomes align on the metaphase plate, sister chromatids facing opposite sides Anaphase o Sister chromatids separate at centromere o Separated sister chromatids (chromosomes) move to opposite poles disjunction Telophase o Nuclear membrane and nucleoli reform o Spindle fibres disappear o Chromosomes uncoil into chromatin Cytokinesis division of the cytoplasm o Cytoplasm divides, splitting the parent cell into two daughter cells with identical nuclei

Set 6: Meiosis Somatic cells o Mitotically dividing and G-arrested cells o Make up vast majority of individuals tissues o Diploid in humans (2n) Germ cells o Pockets of specialized cells (diploid) o Produce gametes (egg/sperm) o Incorporated into ovaries and testes o Undergo meiosis o Gametes are haploid (n) in humans Centromere remains intact during meiosis I 4 stages prophase I is the longest phase with 5 substages: o Leptonema (looks like mitotic prophase) Chromosomes duplicate Chromosomes thicken and become visible Centrosomes begin to move to each pole and produce spindle fibers o Zygonema Condensed chromosomes seek out homologous partner Synaptonemal complex form (elaborate protein structure) Homologous chromosomes zipper together in an intimate association (synapsis) chromosomes are maximally condensed prior to synapsis Homologous chromosomes form tetrads (bivalents joined) o Pachynema Crossing over genetic exchange between nonsister chromatids of a homologous pair Recombination nodules appear along the synaptonemal complex Nodules facilitate the exchange of DNA at various points

Crossing over results in a mixing of genetic material between paternal and maternal homologous chromosomes (non-sister chromatids of homologous chromosomes) Crossover occurs during every meiosis Sites vary from meiosis to meiosis Average is 3 or 4 exchanges per chromosome >9 million different possibilities in gametes for humans o Diplonema Synaptonemal complex dissolves Tetrads (4 chromatids) are visible as they begin to move apart, but remain connected at chiasmata Crossover points appear as chiasmata, which hold non-sister chromatids together o Diakinesis Chromatids thicken and shorten The nuclear membrane breaks down and spindle begins to attach to kinetochore Metaphase I o Tetrads line up along metaphase plate o Each chromosomes of a homologous pair attaches to fibers from opposite poles Anaphase I o Chiasmata are removed o Homologous chromosomes move to opposite poles Telophase o Haploid number of chromosomes, sister chromatids attached at centromere o Nuclear membranes form around the chromosomes that have moved to the poles o Cytokinesis follows Prophase II o Chromosomes condense o Centrioles move to poles o Sister chromatids attach to spindle fibers from opposite poles Metaphase II o Chromosomes align at metaphase plate Anaphase II o Centromeres divide and sister chromatids move to opposite poles Telophase o Chromosomes begin to uncoil o Nuclear envelope re-forms Cytokinesis o Cytoplasm divides, forming four new haploid cells Gametogenesis involves mitosis and meiosis o Oogenesis egg formation in humans Diploid germ cells multiply by mitosis to produce primary oocytes Primary oocytes undergo meiosis I to produce one secondary oocyte and one small polar body

Secondary oocyte undergoes meiosis II to produce one ovum and another polar body Polar bodies disintegrate, leaving one large functional gamete Stored oocytes in the ovaries arrest at prophase I Released mature haploid oocyte completes meiosis as far as metaphase II At fertilization, it quickly completes meiosis II o Ovum and sperm then fuse to form the diploid zygote o Zygote then begins mitosis to develop into the embryo o Thought that the long interval at arrested meiosis in women (>40 years) contributes to segregational errors, e.g. trisomies Circumstantial evidence for chromosomal theory of inheritance so far: o Demonstrated by early biologists that the phenotype of sexual identity is associated with the inheritance of particular chromosomes suggests specific traits are carried by specific chromosomes o Events of mitosis and meiosis ensure a constant number of chromosomes in the somatic cells of a species no other molecule is so conserved in each cell, suggests chromosomes are the agent of inheritance Need evidence that: o The inheritance of genes corresponds to the inheritance of chromosomes o Transmission of particular chromosomes coincides with the transmission of specific traits (other than just sex) e.g. experiments of T.H. Morgan (first to show a sex-linked gene) in Drosophila gene determining eye colour resides on the X chromosome o Found a mutant white-eyed male amongst thousands of wild-type red-eyed flies o Decided to trace its inheritance Crisscross inheritance males get trait from mother, females get trait from father (typical for sexlinked genes) X-linked traits o Results of reciprocal crosses are not identical to what Mendel had observed o An observed pattern of criss-cross inheritance o Males are said to be hemizygous for genes on the X chromosomes, i.e. their diploid cells have only half the number of alleles carried by the female on her two X chromosomes Back to chromosomal theory of inheritance: o The inheritance of genes corresponds to the inheritance of chromosomes o The transmission of particular chromosomes coincides with the transmission of specific traits (other than just sex) Inheritance of the gene for the trait eye colour corresponds to the inheritance of a particular chromosomes (X chromosomes, not just looking at sex phenotype) Tracking X and Y chromosomes allows tracking of the gene for eye colour through simple breeding experiments Further proof came from extended experiments o Reciprocal crosses between homozygous white-eyed females and hemizygous red-eyed males, every now and then, produced white-eyed females and red-eyed males rare events of disjunction (XXY females and XO males)

o o o

Confirmed hypothesis by microscopically examining the chromosomes of the aberrant males and females Transmission of white allele for eye colour followed the predicted behavior of X chromosomes during rare meiotic mistakes Compelling evidence that specific genes reside on specific chromosomes

Set 7: Changes in chromosome number Aneuploidy o Results from the loss or gain of one or more chromosomes o Aneuploids individuals whose chromosome number is not an exact multiple of the haploid number for the species o Monosomic lacking one chromosome from the diploid number (2n 1) o Trisomic having one chromosome in addition to the diploid set (2n + 1) o Tetrasomic having four copies of a particular chromosome (2n + 2) o Autosomal aneuploidy is generally deleterious to the organism Monosomy is generally lethal in humans Trisomy is not always lethal in humans Trisomies of chromosomes 1 or 2 (large) are almost always spontaneously aborted excessive amounts of protein produced from these genes result in gross, lethal abnormalities in cells Trisomy 21 (small) is well tolerated, but abnormalities still result Trisomy 13 and 18 and the only other trisomies recorded to survive in utero o Humans must have one X for viability gene dosage (number of copies of a gene present in a cell); need dosage of one for most genes on X chromosome o If all genes were expressed on both X chromosomes in females, we would have a gene dosage of two (results in double the amount of proteins) detrimental o X-inactivation expression of X-linked genes on all but one X chromosome is repressed (some genes near telomere and centromere of short arm escape repression); results in no increase in protein levels for most X-linked genes in cells Occurs in many mammalian species 16 day gestation (~800 cells) One X chromosome is randomly inactivated maternal in some cells, paternal in others Exists as a highly condensed body within the cell Barr body Tendency to clonally keep the same X off all descendants of the cell have the same X turned off 50/50 expression mix of fathers X genes and mothers X genes females are genetic mosaics (usually dont see in humans) Commonly seen in certain animals, e.g. calico/tortoiseshell (patches of orange and black) cats/rabbits/guinea pigs are always female o Sex chromosome aneuploidy XXX normal, fertile female

XO Tuner syndrome; abnormal, infertile female (true XOs are not viable spontaneous abortion, Turner females are actually mosaics arises from error in early mitosis, not meiosis) X gets inactivated, except for some genes (e.g. those responsible for female sexual development explains Klinefelter males) Turner females lack the genes responsible for normal female development XXY Klinefelter syndrome; abnormal, infertile male XYY normal, fertile male OY lethal o Meiosis in aneuploidy individuals theoretically 50% should be normal gametes Reality is that trisomy 21 males are always infertile, females have very low fertility o Aneuploidy mosaics can result from mitotic nondisjunction (results in one trisomic daughter cell and one monosomic daughter cell) or chromosome loss (results in one monosomic cell and one normal diploid cell) occurs early in embryo development o Humans Turner females are mosaics carrying XX and XO cells (true XOs not viable) o Drosophila when an XX female loses one of her X chromosomes during the first mitotic division after fertilization, a gynandromorph results, composed of equal parts male and female; lateral division between left and right sides of body Euploidy o Euploid species contain only complete sets of chromosomes o Most species of eukaryotes are diploid o Polyploids euploids that carry three or more complete sets of chromosomes, e.g. triploids, tetraploids, etc. o Monoploids only one complete set of chromosomes (usually infertile) o Monoploidy and polyploidy are rare in the higher animals o e.g. insects ants, bees, wasps Sterile males (workers) are produced by parthenogenesis, whereby an unfertilized gamete from the female develops into an embryo o Salamanders, frogs, and some reptiles commonly exhibit polyploidy; all salmon are tetraploid o Euploidy is often induced in plants o Triploidy results from the union of monoploid and diploid gametes (from a diploid and tetraploid cross) Triploids are viable, but almost always sterile extra chromosomes cant pair at meiosis; sexual reproduction results in unbalanced gametes o Tetraploidy can arise if during mitosis the chromosomes in a diploid tissue fail to segregate after replication; union of diploid gametes can produce tetraploid organisms Usually sterile because synapsis is not uniform gametes are aneuploid o In plants, larger chromosomes numbers often lead to increased size (presumably because there is an increased amount of gene product present); seedless varieties are polyploid

Set 8: Linkage and Recombination

Some genes on the same chromosome assort together more often than not In dihybrid crosses, departures from a 1:1:1:1 genotypic ratio of F1 gametes indicates that the two genes are on the same chromosome not completely independent of each other; can see this in F2 offspring o They are in close proximity to one another and therefor tend to assort together genetically linked o Some genes are more closely linked than others Parental class phenotype of P generation (not parents of F2) When genes are linked, parental combinations outnumber recombinant types Drosophila model for genetics o ~75% of known human disease genes have a similar gene match in Drosophila genome o ~50% of fly protein sequences have a similar protein match in mammals o Used in research on neurodegenerative diseases, e.g. Parkinsons, Huntington, Alzheimers; research on processes of aging, diabetes, drug abuse Only concerned with phenotype when classing Recombination between non-sister chromatids during meiosis (pachynema) gives rise to recombinant phenotypes The number of individuals in the recombinant phenotype classes varies with how closely the genes are linked The number in the recombinant class will always be less than the number in the parental class if the genes are linked Linkage is never 100% if you observe enough offspring, you will always see recombinant phenotypes, even if 2 genes are directly adjacent to each other Chi squared test o Used to evaluate hypotheses that two genes assort independently or are genetically linked o Indicates how often an experimentally observed deviation from a particular hypothesis will occur solely by chance o Requires large sample sizes o When geneticists want to determine if two genes are linked, they test whether the data are consistent with a null hypothesis no linkage o Use chi-square value and number of degrees of freedom to determine a p value the probability that a deviation from the predicted numbers at least as large as that observed in the experiment occurred by chance; the convention is that a 0.05 p value is the boundary between accepting and rejecting the null hypothesis Recombination frequencies of 50% between two genes indicates that the genes are either not on the same chromosome, or are at a great distance from each other on the same chromosome o In this case, one way to determine if the genes are on the same chromosome is to show linkage with other genes that lie between them

Set 9: Map distances Limitations of two point crosses: o In crosses involving genes lying close together, it may be difficult to determine the correct order

The actual distances on the map do not always add up for genes that are far apart a single two point cross is not accurate for determining the distance between genes that are far away from each other Would have to do a series of crosses using the genes that lie between, OR Three point crosses o A faster, more accurate method for mapping genes, through the simultaneous analysis of three marker genes o Allows correction for double crossovers o Parental class will be the two with the highest frequencies; all others are recombinants o Next two highest frequencies will be single crossover recombinants between the two most distant adjacent genes Interference o The number of double crossovers may be less than expected sometimes a crossover in one region of the chromosome will reduce the likelihood of a crossover in an adjacent part of the chromosome o Not uniform, and may vary for different regions of the chromosome o A quantitative measure of the amount of interference in a particular chromosomal region is first obtained by calculating the coefficient of coincidence o Coefficient of coincidence = frequency observed/frequency expected o Interference = 1 coefficient of coincidence o

Set 10: DNA The discovery that DNA was a chromosomal constituent did not establish that it had anything to do with genes many scientists at the time pointed to proteins as the likely genetic material Use of bacteria to implicate DNA as the substance of genes o Bacteria carry their genetic material as a single circular chromosome, without it being enclosed by a nuclear membrane (prokaryotes) o Griffith performed studies with Streptococcus pneumoniae two forms: smooth (S), which is wild type, and rough (R) Smooth form is virulent, R form is not Heat killed type S bacteria does not kill mice when injected; R form does not kill Heat killed type S bacteria mixed with live R form inject into mice, mice die Something in the heat killed S was transforming the live R bacterial into virulent form transforming principle o Transformation The ability of a substance to change the genetic characteristics of an organism By 1929 two other labs had repeated the results of Griffith Achieved transformation by simply growing R-form bacteria in the presence of components from dead S-forms no animal required o What component is responsible for transformation? Avery & co.; finding the transforming principle Prepared various purified and semi-purified components from the mixture and tested to see if they could transform R form bacteria; the only one that could was determined to be DNA

Further tested this preparation by subjecting it to various chemicals Protease, RNase, DNase o More evidence that genes are made of DNA experiments of Hershey and Chase, infecting bacterial cells with bacteriophages (Waring blender experiment) T2 bacteriophages are very small, and are composed of approximately equal weights of protein and DNA; depend on the host cell machinery for replication Electron microscope revealed that when phage infect bacteria, they leave the viral shell (ghost) on the surface of the cell It appeared that the substance for phage replication was injected into the cell Chemical constituents of DNA phosphate, deoxyribose sugar, nitrogenous bases o Cytosine and thymine (and uracil) are pyramidines (one ring) o Guanine and adenine are purines (two rings) o Nucleotides are linked in a directional chain through phosphodiester bonds DNA is a double helix o First proposed by Watson and Crick, based on both X-ray diffraction data and studies of chemical composition of DNA, incl. the finding that the ratio of both A:T and G:C is 1:1 Antiparallel strands held together by hydrogen bonding between the base pairs One complete turn ever 3.4 nm (0.34 nm between adjacent base pairs) A and T pair with two hydrogen bonds; G and C with three Sugars and phosphates form backbone Right handed turn o The DNA helix may assume alternative forms The majority of naturally occurring DNA molecules have the classic WatsonCrick structure B-form DNA; spiral to the right Some nucleotide sequences (alternating purine:pyrimidine base-pair repeats) cause DNA to assume a Z-form helix spirals to the left and the backbone takes on a zigzag shape The biological role of Z DNA has yet to be defined, but it appears to be present in certain highly active genes Proteins that specifically bind Z DNA have recently been identified The A form is dehydrated Some DNA molecules are circular rather than linear chromosomes of prokaryotic bacteria, mitochondria, chloroplasts, and those of some viruses Some viruses carry single-stranded DNA which serves as the template for synthesis of a second strand once inside the host cell Some viruses use RNA as their primary genetic material In the case of retroviruses, like HIV, an intermediate DNA is made from the RNA template using viral reverse transcriptase DNA stores information in the sequence of its bases

Set 11: DNA replication Watson-Crick model of DNA replication o Strands separate o Complementary bases align opposite templates

Enzymes link sugar-phosphates of aligned nucleotides into a continuous new strand This model proposed a mechanism whereby DNA replication is semiconservative in daughter molecules, one strand is conserved from the parental molecule and the other is newly synthesized Experimental proof of semiconservative replication o Meselson and Stahl studies with controlled isotopic composition of nucleotides incorporated into daughter DNA strands o Ruled out conservative replication after first generation, ruled out dispersive after second The molecular mechanism of DNA replication o Complex process; occurs at a precise moment in the cell cycle (S phase in eukaryotes) o Two steps: initiation and elongation o E. coli DNA replication as a model: Circular chromosome Initiation Origin of DNA replication, OriC AT rich (only two hydrogen bonds between A and T) DnaA protein binds to the four 9-bp repeats in oriC (one of which is AT rich, other region signals DnaA) Additional molecules of DnaA protein bind cooperatively, forming a complex with oriC wrapped on the surface DnaB protein (DNA helicase) and DnaC protein join the initiation complex and produce a replication bubble DNA helicase catalyzes the unwinding of the parental double helix (moves in both directions two helicases, one moving on each end of replication bubble; synthesis occurs in both directions); SSB proteins (single-stranded binding proteins) keep strands separated as helicase unwinds them Elongation DNA polymerase (DNA-dependent DNA polymerase uses DNA as a template to make more DNA, vs. RNA-dependent DNA polymerases, only in retroviruses) must have a free 3-OH to extend upon requires a primer to begin synthesis DNA polymerase can only add nucleotides to the 3 OH end of the new strands synthesis is always in the 5 to 3 direction, so the orientation of the strands is very important An RNA primer is needed to provide a free 3OH for DNA polymerase; RNA primer is made by DNA primase DNA polymerase III (in E. coli) extends DNA using RNA primers DNA synthesis is discontinuous in lagging strand Okazaki fragments (fragments of DNA and RNA primers) During elongation, the RNA primers are removed by polymerase I DNA polymerase I (not III) fills in the gap (still 5 to 3)

o o

DNA ligase seals the 3 OH and 5 PO4 nicks by catalyzing the formation of phosphodiester bonds This completes the formation of the lagging strand E. coli DNA polymerase Adds nucleotides to the end of a DNA strand Must have a single-strand template (DNA must be unwound) Can only add nucleotides in the 5 to 3 direction Needs a free 3-OH to polymerize Adds nucleotides on basis of complementarity to template Two types of E. coli DNA polymerase are involved in DNA replication I and III; similar forms are found in other species, including eukaryotes o III is the major enzyme responsible for synthesis of new strands o I is responsible for gap filling after removal of the RNA primer o Others are needed for repair Three activities of polymerase I o 5 to 3 polymerase activity o 3 to 5 exonuclease activity proofreading ability o 5 to 3 exonuclease activity primer degradation The problems with circular chromosomes: Without a swivel or axis of rotation, the unwinding process would produce positive supercoils in front of the replication forks o As replication forks move in both directions, a -like structure is produced in the DNA, causing supercoiling of chromosome o Topoisomerases make transient nicks to relieve torsion Newly replicated chromosomes are intertwined o Topoisomerases nick both DNA strands and separate the two daughter molecules Eukaryotes Large linear chromosomes Problem with having large chromosomes: must replicate all DNA in S phase, would take too long if each chromosome only had one origin of replication (one replication bubble) Eukaryotes initiate DNA replication at several points along the chromosome Problem with having linear chromosomes Cellular lifespan is linked to DNA replication Since chromosomes are linear, problem with DNA replication at the ends of the chromosomes (requirement of DNA polymerase for free 3OH end to add to) Telomeres are present at the ends of each chromosome they contain repeat units of (in humans) TTAGGG x 250-1500 No 3OH for covalent extension after RNA primer at the end of the chromosome is removed every round of replication shortens the ends

of DNA; eventually lose genetic information cells cannot replicate anymore, either go into senescence or die Repeats on telomeres absorb the loss so that critical information (genes) are not immediately affected Eventually information is lost with continuous cell division most mature somatic cells do not express telomerase Telomeres shorten slightly at each cell division; senescence after <50 generations in culture telomerase is NOT active in most somatic cells (only active in stem cells, reproductive cells, and embryonic cells) People with longer telomeres tend to live longer, healthier lives, whereas those with shorter telomeres suffer from more age-related diseases, e.g. diabetes, Alzheimers, heart disease Reinstigating telomerase production in mice without active telomerase spurred almost total recovery But re-activating telomerase in somatic cells greatly increases probability of cancer

Set 12: The Genetic Code Gene expression: the flow of genetic information from DNA via RNA to protein Nucleotides code amino acids through groupings of triplets Each nucleotide triplet is known as a codon o AUG initiator/start codon o UAA, UAG, UGA terminator/stop codons A genes nucleotide sequence is collinear with the encoded polypeptide o Linear relationship between the nucleotide sequence in a gene (or mRNA transcript) and the order of amino acids in the polypeptide chain specified by the gene Reading frame o Crick and Brenner published a report of their studies of a bacteriophage T4 gene o Conclusion: a codon is composed of three nucleotides and the designated starting point for each gene establishes the reading frame for these triplets o Reading frame: the partitioning of groups of three nucleotides such that the sequential interpretation of each succeeding triplet generates the correct order of amino acids in the resulting polypeptide o Start triplet must be in frame with the stop triplet o Intragenic suppression: the restoration of gene function by one mutation cancelling the other in the same gene o Frameshift mutations: changes that alter the groupings of nucleotides into codons If a gene sustains 3 or multiples of 3 nucleotide mutations of the same sign (insertions or deletions), the protein can still function as long as the mutation did not affect the majority of amino acids signified that code is read in threes Single nucleotide insertions or deletions will completely shift the reading frame and alter the protein from that point on frameshift o Mutations between genes do not interfere with translation of the two genes not transcribed, let along translated

o Open reading frame refers to genes (coding regions, not noncoding regions) o Reads 5 to 3 Cracking the code o Researchers using radioactively labeled amino acids discover that protein synthesis takes place in the cytoplasm, even though genes are located in the nucleus o Conclusion: an intermediate molecule must exist that transports DNA sequence information to the cytoplasm, later shown to be messenger RNA o Experiments involving in vitro translation systems (cell-free extracts) Cellular extracts that contain ribosomes (composed of two subunits one small, one large), tRNAs and amino acids Added synthetic mRNA of known sequence to see which codons corresponded to which amino acids Correlation of polarities in DNA, mRNA and polypeptide o RNA-like strand = coding strand/sense strand/+ strand o Non-coding strand = non-coding strand/non-sense strand/- strand o Moving from 5 to 3 end of an mRNA, each successive codon is sequentially interpreted into an amino acid, starting with the N-terminus and ending with the C-terminus Nonsense codons three different triplets, UAA, UAG, and UGA, do not correspond to any of the amino acids; when these codons appear in frame, translation stops, and the polypeptide chain is terminated; also known as stop codons But recently, it has been discovered that a 21st and 22nd coded-for amino acid exists o Selenocysteine coded for by UGA; L-pyrrolysine coded for by UAG o Interpretation as stop or amino acid appears to depend on the surrounding context of the codons The genetic code is almost universal, some exceptions *AUG is not a start codon if there is no inframe stop codon Open reading frame inframe start, inframe stop

Set 13: Transcription The process by which the polymerization of ribonucleotides guided by complementary base pairing produces an RNA transcript of a gene Nucleotides are added in the 5 to 3 direction Uracil is incorporated in place of thymine in RNA In prokaryotes: o RNA polymerase (DNA-dependent RNA polymerase) enzyme that catalyzes transcription o (In bacteria) made of two components: sigma factor and RNA polymerase core enzyme; together, called the RNA polymerase holoenzyme Sigma factor recognizes promoter region, binds to promoter region Once bound, sigma factor is released; core enzyme carries out synthesis Promoters DNA sequences near the beginnings of genes that signal RNA polymerase where to begin transcription o AT rich

Terminators sequences in the RNA products that tell RNA polymerase where to stop (encoded by DNA that polymerase can recognize and bind to) o Terminator signal is encoded in the gene o In bacteria, at least two methods for transcription termination: Rho-dependent some terminators; recognized by a protein; disengages RNA polymerase Rho-independent no protein involved; sequence-mediated; two complementary regions Sequences that can bind with itself (complementary with itself) stem loop/hairpin structure, which destabilizes the polymerase Second complementary region is flanked by strings of As or Ts weak hydrogen bonding, releases transcript During transcription, no single stranded binding proteins makes a single-stranded molecule of RNA that is released, DNA pairing snaps back to keep the molecule intact, forcing out messenger RNA molecule (nascent strand) Prokaryotes genes are what you see is what you get; mRNA and proteins can be directly deduced from the DNA sequence In eukaryotes (not WYSIWYG): RNA processing after transcription produces a mature mRNA o Addition of a methylated cap at the 5 end A special capping enzyme adds a guanidine triphosphate in reverse orientation to the 5 end after polymerization of the transcripts first few nucleotides (not a phosphodiester bond) this G is NOT encoded by the gene Methyl transferases then add methyl groups to the backward G and to one or more of the succeeding nucleotides in the RNA critical for efficient translation Cap protects mRNA when it enters the cytoplasm In prokaryotes: 5 end of transcript has a triphosphate, rather than a methylated cap o Addition of 100-200 adenosines to the 3 end, known as the poly-A tail (NOT encoded by the gene post-transcriptional modification) First, ribonuclease cleaves the primary transcript to form a new 3 end (sequence AAUAAA is encoded 11 to 30 nucleotides upstream of the position where the tail is added) part of the terminator region in eukaryotes Then, poly-A polymerase adds As onto this new 3 end Thought to stabilize mRNA (prevent degradation) and aid efficiency of translation Capping and tailing are also related to translation initiation enzymes recognize the cap and tail, set up complex necessary for ribosome to initiate translation (only in eukaryotes) cap and tail are a signal for the ribosome o RNA splicing removal of introns Exons sequences found in both a genes DNA and in the mature mRNA; exons are amino acid coding sequences for the gene product, contain expressed sequences Introns sequences found in a genes DNA but NOT in the mature mRNA removed from the primary transcript

Splicing is usually carried out by a complex known as the spliceosome, although some RNA transcripts are self-splicing Not all eukaryotic genes contain introns Why are introns present? o Allows for alternate splicing in some cases, splicing may occur between the splice donor site of one intron and the acceptor site of a different intron downstream; produces different mature mRNA molecules that may encode related proteins with different, though partially overlapping amino acid sequences o Species comparisons re alternative splicing across phyla Generally more splicing in more complex organisms, e.g. in mammals Differences in the level of alternative splicing suggests that alternative splicing may contribute greatly to the mammalian higher level of phenotypic complexity Accumulation of introns confers an evolutionary advantage as it allows increasing the number of alternative splicing forms Number of genes/genome size says nothing about complexity of organism (e.g. human has same number of genes as C. elegans roundworm) o Allows for trans-splicing (rare) a form of alternative splicing in which an exon from one transcript can be joined to an exon from a different transcript (exons from two genes joined together in nucleus)

Set 14: Translation *by definition, stop codon is not part of the ORF (includes start codon, ends just before stop codon) doesnt get read and converted into an amino acid Translation: the process in which the genetic code carried by mRNA directs the synthesis of proteins from amino acids Requires: mRNA, tRNA with attached amino acid, ribosomes tRNAs o Short, single stranded RNA molecules 74-95 nucleotides long coded by DNA (not all genes code for proteins!) o Lots of base pairing within single strand o One end (3OH end) carries amino acid o Other end of tRNA has an anticodon, which matches a codon on the mRNA o Carry some modified bases produced by chemical alterations of the A, G, C, and U nucleotides o Each tRNa carries one particular amino acid o Aminoacyl-tRNA synthetases catalyze the attachment of a tRNA to its cognate amino acid o Base pairing between an mRNA codon and a tRNA anticodon determines where an amino acid becomes incorporated in a growing polypeptide not a function of the amino acid, but rather the tRNA Codons and anticodons are always read/referred to in the 5 to 3 direction Genetic code is degenerate o Wobble: some tRNAs recognize more than one codon for the amino acid they carry; base pairing is not as strict (e.g. U can bind to A or G)

Ribosomes o Sites of protein synthesis o Complex structures composed of protein and RNA o Prokaryotic ribosomes have a large subunit and a small subunit, each made up of many proteins and rRNAs (final products of their own genes never translated to proteins); 16S rRNA, part of small subunit, brings ribosome to the mRNA o Eukaryotic ribosome large subunit and small subunit, equivalent part that directs ribosome to mRNA is the 18S rRNA o Ribosome size differs between prokaryotes and eukaryotes In prokaryotes (E. coli) o Initiation The E. coli Shine-Dalgarno (unique to prokaryotes reads AGGAGG on mRNA) sequence is specifically recognized by complementary sequences in the 16S rRNA of the small subunit (ribosome-binding site includes SD box and AUG start codon) Not all prokaryotes have the same ribosome binding site recognition sequence as E. coli, but many have sequences that are very similar in that they are purine rich The presence of a nearby downstream AUG codon signals the initiation of translation by the addition of tRNA carrying formylmethionine This tRNA carries the same anti-codon as tRNA carrying unmodified methionine; however, the rest of the tRNA is unique and is only used for initiation; AUG is not the only start codon some bacteria occasionally use GUG (valine) The large ribosomal subunit then binds such that the tRNAfMet is placed in the P (peptidyl) site of the ribosome; this completes initiation of translation o Elongation phase Elongation factors (EFs) escort the next tRNA into the A site of the ribosome Peptidyl transferase catalyzes the formation of a peptide bond between the carboxyl (C) terminus of formylmethionine and the amino (N) terminus of the second amino acid Causes the release of the amino acid (fMet) from its tRNA in P site Ribosome shifts/pulls mRNA through Free tRNA moves to E site A site now available for incoming tRNA:amino acid Free tRNA in E site is released Process repeats When a nonsense (stop) codon is encountered (for which there is no tRNA), a release factor, which recognizes the stop codon, moves into the A site The polypeptide is released from the C-terminal tRNA The mRNA, tRNA, and ribosomal subunits all dissociate from each other o mRNA has 5 and 3 untranslated regions (UTRs) Eukaryotes o Initiation is slightly different no Shine-Dalgarno sequence

Small ribosome subunit recognizes and binds to the 5 methylated cap; it then scans along the mRNA until it finds the initiation of translation sequence (which in lower eukaryotes is only the AUG, but in mammals, it also includes the surrounding sequences) o Initiator tRNA carries Met, not fMet o Rest of translation is similar to prokaryotes o Polyribosome: a complex of several ribosomes translating from the same mRNA Translation compartments o Prokaryotes no cellular organelles Replication, transcription, and translation all occur in the cytoplasm (tightly coupled) As soon as enough of the transcript is made translation can begin (transcription and translation can be simultaneous) mRNA is very short-lived (seconds) o Eukaryotes membrane-bound organelles Compartmentalization Replication and transcription in nucleus; translation in cytoplasm (ER) uncoupled, complete transcription before translation begins mRNA has to travel out of nucleus to cytoplasm before being translated longlived (minutes to hours) Posttranslational processing modifications that occur to the protein after translation o Cleavage may remove an amino acid or split a polyprotein into several smaller polypeptides, e.g. removal of methionine on N-terminus o The addition of chemical constituents may modify a polypeptide after translation How mutations affect the products of gene expression o Types of mutation in a genes coding sequence: Silent mutation same amino acid due to degeneracy of genetic code/wobble but still new allele (change in DNA sequence) Missense mutation changed codon, new amino acid; may or may not affect proteins function e.g. sickle cell anemia single missense mutation in the beta-globin polypeptide chain (hemoglobin is composed of two beta-globin and two alpha-globin) Nonsense mutation codon changed into a stop codon, prematurely truncates translation of polypeptide; may or may not affect protein function Frameshift mutation added/deleted nucleotide(s); probably going to affect polypeptides function (affects all amino acids downstream of the mutation) o Sites of mutation outside the coding sequence that can disrupt gene expression Mutation in promoter RNA polymerase may not recognize promoter, obliterates transcription Mutations in introns may affect splicesomes ability, affecting splicing activity affects translation (some introns may not be cleaved out and get translated) Terminator mutation could message sequence for cleaving at poly-A tail, may not have translation if there is no poly-A tail o

Set 15: Gene regulation in prokaryotes Can change expression of genes in various ways o Increase/decrease transcription of DNA Modify promoter sequences RNA polymerase doesnt recognize it as well, or recognizes it more strongly Change the half-life of mRNA degrade quickly decrease level of protein o Control level of translation Modify ribosome binding site, e.g. tweak Shine-Dalgarno sequence o Posttranslational modification activate/deactivate protein The role of RNA polymerase in transcription recognition of the promoter o Sigma factor recognizes two particular consensus sequences within the much larger promoter sequences: -10 and -35 boxes (generally about 10 and 35 bases upstream of +1 region, where transcription begins); sigma factor binds to both simultaneously -10 is also known as the TATA box (TATAAT in prokaryotes) -35 starts with TTG (conserved part) +1 of transcription is random; every gene starts in a slightly different place Not all genes are being transcribed all the time Studies of E. coli metabolism o Used to determine fundamental principles of gene regulation in prokaryotes: The binding of regulatory proteins to DNA targets controls transcription These regulatory proteins either inhibit or enhance transcription o Lactose utilization in E. coli negative regulation Monod and Jacob studied E. coli lactose-utilization mutants Need permease to transport lactose into inner membrane of E. coli Beta-galactosidase break down lactose into glucose and galactose A small amount of lactose gets rearranged into allolactose, which is what controls the system The presence of lactose (allolactose) induces expression of the genes required for lactose utilization Addition of lactose to growth medium induces a 1000-fold increase in the production of lac permease and -galactosidase Discovered through studies of lac- mutants (cells unable to utilize lactose) lac Z encodes -galactosidase lac Y encodes permease lac A encodes a transacetylase that adds an acetyl group to lactose (not required for lactose breakdown mutating it doesnt prevent cell from bringing lactose in and breaking it down) The lac genes are transcribed together When lacZ expression is repressed or induced, the same effect is observed for lacY and lacA Led to the proposal that the three genes are transcribed as part of the same polycistronic mRNA (one mRNA with two or more transcribed genes on it) The operon theory of gene regulation was published

Operon a unit of DNA composed of specific genes (polycistronic), plus a promoter and operator, which act in unison to regulate the response of the structural genes to environmental changes Three operators in lac operon Each gene has its own SD sequence, start codon and stop codon (and UTRs) Evidence for a lac repressor protein Mutations in another gene, lacI, result in mutants that synthesize galactosidase and lac permease even in the absence of lactose These are examples of constitutive mutants ones that result in protein synthesis, irrespective of environmental conditions Hypothesized that lacI encodes a negative regulator (repressor) that binds near the promoter of the lactose utilization genes at the operator site Lac repressor forms an active tetramer and binds to two 21 bp conserved sequences in DNA of lac operon Polymerase cannot bind to promoter In the presence of lactose, some gets converted to allolactose, which changes the shape of the repressor, taking it off the operator Concentration dependent always a small amount of transcription of these genes (otherwise lactose wouldnt be able to enter cell at all, without permease always some permease in the cell) Operator mutants OC or O- nucleotide sequence is changed, repressor cannot recognize and bind to operator, lac enzymes are synthesized constitutively Proteins act in trans, but DNA sites act only in cis Trans-acting proteins (repressors) and cis-acting sites Use of plasmids to make partial diploid cells (meroploids or merodiploids) to study the lac operon First look at promoter, then repressor Inducible when lactose is absent, genes are turned off; when lactose is present, three proteins are produced and lactose is broken down if on all the time (constitutive), non-inducible *If cell cant make permease (mutant Y), cant ever bring lactose in to induce operon no genes expressed cant get lactose into cell to make allolactose

Set 16: Gene regulation in eukaryotes Prokaryotes can organize their genes into operons; eukaryotes generally do not do this o Once one gene is translated, ribosome goes back to cap never reaches next genes because of the requirement for the binding of the cap as the signal for the ribosome), vs. in prokaryotes, each gene has SD sequence before its start codon Generally, transcription in eukaryotes is very similar to that in prokaryotes Control of transcription is much more complex in eukaryotes

How cis-acting and trans-acting factors affect transcription o Cis-acting elements Enhancer element (may be far before promoter) retains function even when reversed or moved far from gene whose transcription it influences Promoter is close to a genes initiation site o Trans-acting gene products interact with cis-acting elements In eukaryotes, three RNA polymerases (vs. one in E. coli) transcribe different sets of genes o Protein-encoding genes (structural genes) are transcribed by RNA polymerase II o Cis-acting regulatory regions recognized by polymerase II: Core promoter always very close to the genes coding region Includes an initiation site where transcription begins (+1 of transcription) TATA box consisting of roughly seven nucleotides (5-TATAAAA-3 on nontemplate strand) located at about -30 position (different position and consensus sequence from prokaryotes) Other cis promoter elements Enhancer regulatory site that can be located far away (even >10000 bp) from the core promoter, or can be very close; some can be very large Basal factors are required for binding to the promoter (core promoter region) to maintain a basal level of transcription TBP TATA box-binding protein, essential to the initiation of transcription from all class II genes with a TATA box TAFs TBP-associated factors *anything that is involved in transcription and is a protein is a transcription factor Transcriptional activators transcription factors that bind to specific enhancer sequences and can increase transcription 100-fold (or more) above the basal level; have two important structural domains: the DNA-binding domain and the transcription-activation domain Most eukaryotic activators must form dimers to function o Homomers multimeric proteins composed of identical subunits; two subunits: homodimers o Heteromers multimeric proteins composed of nonidentical subunits; two subunits: heterodimers Many transcription factors possess common motifs o One of the families of activator proteins have a zinc-finger motif all proteins with zinc finger motif forms a dimer and is an activator protein, involved in the enhancement of a gene e.g. steroids (e.g. hormones such as estrogens, testosterone, and glucocorticoids) work at the level of co-activation (hormones are co-activators) of transcription factors; they bind to specific transcription factors (activators); by doing so they impart a reversible allosteric effect, causing the transcription factor to undergo a conformational change to allow it to bind its enhancer element; transcription rate of the target gene is greatly increased

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The DNA sequence of the glucocorticoid response element (enhancer element) is palindromic; the glucocorticoid receptor (the activator protein), like all steroid hormone receptors, is a zinc-finger transcription factor For a steroid hormone to turn gene transcription on, its receptor (the activator protein) must bind to the hormone, bind to a second copy of itself to form a homodimer, be in the nucleus (moving from the cytosol if necessary), bind to its enhancer element each of these functions depend upon a particular region of the protein (e.g. the zinc fingers for binding DNA); mutations in any one region may upset the function of that region without necessarily interfering with other functions of the receptor Transcriptional repressors diminish transcriptional activity; may bind to enhancer element (competitive) to prevent enhancement or may bind to other specific DNA-encoded sequences (silencers) to diminish transcription Basal level of expression DNA of eukaryotes is packaged into chromatin by the wrapping of DNA around histone proteins to form units of nucleosomes This structural arrangement of the DNA keeps it turned off so transcription is very low or non-existent in individual genes In prokaryotes, transcription is actively turned off by repressor proteins binding to the DNA; in eukaryotes, transcription is passively minimized due to the normal structure of chromatin high or low levels of transcription depends on the interactive balance between remodeling of the chromatin structure and the subsequence interactions involving the various transcription factors Methylation patterns of DNA and histone proteins affect transcription When genes are tightly wrapped not accessible Remodeling enzymes can change this Acetyl groups added to histone tails reduce electrical attraction of negative DNA to otherwise positive lysine in tails makes it easier to unwrap the DNA from the histone and expose the gene In vitro studies show basal levels of transcription (no activators) from most eukaryotic genes is quite high Why are transcription levels observed in vivo so much lower? Highly methylated areas of DNA can keep nearby genes off Gene silencing epigenetics In higher eukaryotes (e.g. mammals) silencing is associated with methylation of CpG dinucleotides in highly condensed heterochromatin, e.g. silenced X chromosome (Barr body) Two levels of biochemical modification histones and DNA; both of these modification levels called epigenetics (changing gene expression on top of actual genetic sequence second genome) Genomic imprinting and epigenetic effects Methylation of CpG dinucleotides in DNA frequently inhibits transcription Methylation patterns can be inherited from mother or father

Diet can also change methylation patterns and these changes can be inherited by other cells or offspring End up with different phenotypes, despite having identical sequences (genotypes) e.g. lgf2 gene (produces insulin-like growth factor) In germ cells of females, both copies are methylated and repressed, but not in males Offspring get one of each; only paternal gene is expressed

Set 17: Restriction enzymes/cloning Restriction enzymes o Endonucleases that exist naturally in prokaryotes o In the natural host, their function is to recognize foreign DNA sequences and cut it into pieces for further degradation by host exonucleases o Restricts what type of DNA can exist in the host o e.g. E. coli will accept the entry of DNA from other E. coli of the same strain, but will destroy DNA from some phage o Protect own DNA using methylation Type II REN (restriction endonucleases) o Recognize inverted palindromic sequences in DNA o Each REN has a unique recognition sequence o When they cut within these sequences, they leave either cohesive ends or blunt ends o Sequence of recognition read from 5 to 3 (REN doesnt recognize if it is in reverse) Gel electrophoresis distinguishes DNA fragments according to size o Electrophoresis the movement of charged molecules in an electric field; DNA in solution is weakly acidic (negative charge); the larger the DNA molecule, the more slowly it moves through the gel matrix Restriction maps o Show the order of different size fragments within a clone o Consist of linear (or circular) diagrams of the sites along a DNA segment at which one or another restriction enzyme cleaves the molecule Molecular cloning (DNA fragments DNA cloning) o The process of using living cells to make many exact replicas of fragment(s) of foreign DNA normal shape of bacterial chromosome is circular, linear will be recognized as foreign; so linear fragment must be put in a circular holder a vector o Inserting linear, cut fragments of DNA into a bacterial cell will result in its degradation o 1. Purify DNA fragment(s) and insert the fragment into a specialized chromosome-like carrier termed a vector (e.g. a plasmid); 2. Transfer the vector with its insert (together a construct, or recombinant plasmid) into cells; copies will be made through replication these are therefore known as DNA clones o Types of vectors Plasmids (usually) circular double-stranded DNA

Each has an origin of replication, several unique sites for restriction enzymes, and a gene referred to as a selectable marker (engineered to have these elements) Typically 2-4 kb in size, with an ability to carry inserts up to 15 kb in length (limitations due to size) Problems: o Not all plasmids will have an insert (self-ligation) o Not all cells will take up a plasmid (with or without insert) o How to distinguish cells without plasmids make sure there is a selectable marker, grow on an antibiotic o How to distinguish cells without inserts e.g. Bluescript, using lacZ gene as a differential marker (vs. selective marker e.g. antibiotic) Yeast artificial chromosomes (YACs) o Linear vectors built to mimic a yeast chromosome (including telomeres and centromeres) o Yeast DNA allows YACs to segregate as if they were natural chromosomes in the yeast host o Can carry inserts up to 1 million base pairs

Genomic library o A collection of DNA clones that theoretically contains copies of every DNA fragment in the whole genome inserted into a suitable vector and placed into storage cDNA (complementary DNA) library o A library that stores DNA sequences that have been copied from mRNA transcripts o These sequences do not contain the intron sequences present in the DNA o They contain the exon sequences that actually code for the protein product o Genomic: Contains all sequences Contains same information no matter what cell type from the organism is used to construct the library Used for prokaryotes mostly (no introns) or sequencing projects o cDNA: Contains only exon sequences Information is conditional upon cell type Used for eukaryotes mostly (to study expressed sequences)

Set 18: Population genetics Phenotype frequency proportion of individuals in a population that are of a particular phenotype Genotype frequency proportion of individuals in a population that are of a particular genotype Allele frequency proportion of all copies of a gene in a population that are of a given allele type Hardy-Weinberg law o Defines the relationships between genotype and allele frequencies within a generation and from one generation to the next o Depends on five assumptions:

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The population includes a very large (virtually infinite) number of individuals that all have equal access to mating The individuals mate at random in the sense that each individuals genotype at the locus (location on the genome genes, but also includes DNA sequence (alleles change in DNA sequence, doesnt have to be a gene) outside of genes, e.g. enhancer) of interest does not influence his or her choice of a mate No new mutations appear in the gene pool (the alleles in question do not change) No migration into or out of the population There are no genotype-dependent differences in the ability to survive to reproductive age and transmit genes to the next generation (no natural selection) If all these assumptions hold true, can accurately calculate allele frequency, genotype frequency (and phenotype frequency) for a particular gene Hypothetical populations that satisfy all five of these assumptions are said to be at HardyWeinberg equilibrium (not changing for that gene/alleles) No actual population is at Hardy-Weinberg equilibrium; however, the equations derived on the basis of these assumptions are still powerful tools for providing estimates of genotype and allele frequencies in real populations In the real world no population is in equilibrium for all genes if it were, it would stop evolving Populations often face barriers to mating (1) e.g. geographical barriers Genotypes do affect viability (5) Migration does occur (4) Individuals do not always randomly choose mates with respect to certain genotypes (2) Mutations do happen (3) H-W does NOT look at all genes simultaneously only gene(s) of interest, in isolation of effects of others Evolution is the change in the inherited characteristics of populations over generations change in allele frequencies Populations that are in Hardy-Weinberg equilibrium therefore will show: Constant allele frequencies p and q that do not change over time Genotypics proportions that are described by p2 (RR), 2pq (Rr), and q2 (rr) = (p + q)2 (binomial equation) A population is said to be in Hardy-Weinberg equilibrium if p2 + 2pq + q2 = 1 If the observed genotype proportions are different than what we expect, then one of the H-W assumptions has been violated and the population is not in genetic equilibrium i.e. it is changing for that locus Calculations using the H-W law show that Allele frequencies for a particular gene do not change after one generation in a hypothetical population at H-W equilibrium they remain stable The genotypic frequencies, p2, 2pq, and q2 will be directly determined by the allele frequencies the frequency of heterozygous carriers can be estimated based on observed frequencies of diseased (mutant) and healthy individuals Computing genotype, allele and phenotype frequencies (diploid)

It is not practical to genetically test all the genotypes in such large populations, so a representative smaller sample is used for calculations Computing genotype frequencies add up the number of individuals possessing the genotype and divide by the number of individuals in the sample (N) Calculating allele frequencies using the Hardy-Weignberg law allows us to see if changes are occurring for the genes (alleles in question) If a population is not in H-W equilibrium, it should reach H-W equilibrium after one generation if assumptions hold DO NOT assume a population is in H-W equilibrium Genetic drift has an unpredictable effect on allele frequencies o Genetic drift unpredictable, chance fluctuations in allele frequency that have no effect on survival o The smaller the population size, the greater effects of drift o In small populations, chance dictates that some gametes unite more often than others e.g. coin flip, sampling error, Tristan de Cuna Changes to H-W equilibrium: evolution and natural selection o Most of the genetic variation observed in large natural sexually-reproducing populations arises from recombination due to sexual reproduction allele shuffling during meiosis o Not spontaneous, random mutations or genetic drift or migration or assertive mating these factors tend to make minor contributions to allelic variation in very large populations o Natural selection does not produce variation, but can select for certain alleles o Genetic natural selection interactions between genetically determined phenotypes and environmental conditions that cause differential reproduction of certain genotypes; results in genetically based adaptive traits appearing with a greater frequency (changes in allele frequencies) after many generations (evolution) o Natural selection acts on differences in fitness to alter allele frequencies Fitness an individuals relative ability to survive and transmit its genes to the next generation; two basic components: viability and reproductive success Natural selection the process that progressively eliminates individuals whose fitness is low and chooses individuals of high fitness to survive and become the parents of the next generation Fitness (w) measured by number of offspring; 0 = no offspring (or dies); 1 = survives and has 1 offspring; 2 = survives and has 2 offspring, etc. Can calculate average fitness for a population, genotype, or phenotype Average fitness > 1 growing population Average fitness < 1 declining population Average fitness = 1 stable population Relative fitness (RF) ranges from 0 to 1 Used to calculate the relative fitness for a given genotype If all genotypes have a relative fitness of 1, then selection is not occurring for that genotype Why is selection unable to reduce the frequency of a recessive lethal allele to zero?

Because when the level of the allele in the population is low, the incidence of homozygotes will be rare Heterozygous advantage sometimes heterozygotes have a higher fitness than either homozygote, e.g. heterozygotes for sickle-cell syndrome are resistant to malaria Time of onset can influence the frequency of disease alleles diseases that cause death after the completion of reproduction will sustain little or no negative selection, e.g. Huntingtons disease (dominant); alleles associated with some cancers e.g. some mutant brca-1 and brca-2 alleles

Set 19: Genes and mutation Mutation: heritable changes in base sequences that modify the information content of DNA To identify mutations, we first need to identify what the normal or wildtype allele is o Generally it is the allele(s) that dominates in a wild population o Others consider any allele present in more than 1% of the population to be a wildtype allele; mutant is less than 1% o In this class, we use the 1% cut-off (better accounts for polymorphic genes) o Wildtype alleles can be recessive or dominant o Dominant and recessive are really a reflection of what goes on at the molecular level; a dominant allele usually masks the presence (effect) of the recessive allele o More than one wildtype allele can exist for a gene polymorphic gene, e.g. seed coat colour in Indian corn o Different alleles result from changes in DNA sequence (doesnt matter if it is in a gene or outside a gene Classes of DNA mutations (anything that changes the DNA sequence) o Twelve different base substitutions can occur in DNA Transition mutation switch from a purine to a purine, or switch from a pyrimidine to a pyrimidine Transversion purine for pyrimidine, or pyrimidine for purine o Deletion can be single base pairs or multiple; losing DNA information o Insertion adding a base pair, or multiple (whole piece of DNA) o Inversion a piece of DNA switched around; e.g. double stranded break in the DNA, inverted when repaired o Translocations part of one chromosome has been replaced with a region from another chromosome and vice versa changing places (reciprocal translocation) In general, spontaneous mutations occur at a very low rate How to estimate rates of mutation for a particular gene look at mutations in haploids (sustained mutations are more likely manifested) o Can use inbred homozygous individuals for a particular trait; breed large numbers of offspring and count how many show an alternative phenotype for the trait, e.g. mouse coat colour o Problems: most mutations wouldnt happen inside genes, most happening in intragenic regions wont see by looking at a phenotype; wouldnt see recessive mutations;

wouldnt see mutations that did happen but did not manifest/cause a difference in protein (silent mutation) o Mutations occur that do not affect phenotype (most mutations are not in genes) the only way to detect these is through molecular methods, e.g. DNA sequencing o In multicellular organisms, only mutations in the germ cells will be passed along to future offspring, mutations in somatic cells are only passed to daughter cell within that individual Evolution: adaptation vs. mutation o Evolution is the change in the inherited characteristics of populations over generations o Mutations are chance occurrences modifying the genome o Luria and Delbruck jackpot/fluctuation test Used E. coli and bacteriophage T1 to test hypotheses regarding the mechanisms responsible for the appearance of a new phenotype Set up small vials of bacterial culture and let them grow for a specified amount of time, then added equal portions of each culture to plates containing media along with phage Testing the hypothesis that resistance is the result of active adaptation to the selective pressure (phage) Based on Lamarcks theory of evolution by acquired traits The selective pressure causes/drives the mutation Based on this hypothesis, wouldnt be mutation until the phage is added; all plated cultures should adapt similarly, resulting in similar numbers of resistant colonies Darwinian hypothesis: resistance is a result of random mutations, exclusive of the selective pressure Based on Darwins theory of evolution of natural variation and natural selection If so, then there will be differences in the numbers of resistant colonies obtained due to random mutation Numbers of resistant colonies fluctuated (jackpot colony with a lot of mutants) Conclusion: Resistance occurs as a result of random, spontaneous mutations that happen at various time points before exposure to the selective agent selective pressure is not causing the mutation Mutations occurring early in the growth of the liquid culture resulted in many resistant colonies when plated; those occurring late resulted in few resistance colonies (fluctuations in numbers obtained) Spontaneous mutations occur naturally For evolution, luck is a strategy Verification replica plating o Resistant cells were present before the selective agent, as evidenced by their position on the original plate o Mutations occur prior to selective pressure and are stable for many generations o No new positions or loss of resistant colonies

Mutations o Mistakes in DNA occur at a low frequency all the time o Natural processes of deanimation, UVA light, errors by DNA polymerase o Mutagens induce mutations Any physical or chemical agent that raises the frequency of mutations above the spontaneous rate, e.g. X rays, base analogs, intercalators o Processes which can change the information stored in DNA: Depurination purine lost from strand, replaced with another base during replication, may result in a substitution Deamination with nitrous acid (carcinogen) takes amino groups off e.g. adenine gets converted with hypoxanthine (base analog), which polymerase tends to pair with cytosine instead of thymine replication: pairs with guanine (T to C, A to G) Cytosine deamination results in uracil, which pairs with adenine replication: pairs with thymine (G to A, C to T) Both transition substitutions Oxidation e.g. guanine converted into GO, which pairs with adenine replication: pairs with thymine (G to T transversion) X-rays break the DNA backbone (phosphodiester bonds) deletion (cell attempts to fix by just ligating together remaining strands), or translocation, or inversion Thymine dimers produced by UV light Adjacent thymines in a strand base pair to each other (UV causes carbon bonds to form across) When two thymines are adjacent to one another in a DNA strand, the absorption of UV radiation can cause the formation of a covalent bond between them a thymine dimer Such a dimer introduces a kink into the double helix, which prevents replication of the duplex by DNA polymerase (polymerase either stops at dimer and replication is aborted, or throws something else in mutation) Torsion can break the strand if there are enough thymine dimers o Light-dependent repair of DNA in bacteria Photolyase binds to the thymine dimer in DNA Photolyase is activated by the absorption of blue light (close to UV light) Cleaves cross links o Eukaryotes do not have photolyase repair T-T dimers in a different way Nucleotide excision repair removes lesions of damaged DNA in bacteria and humans In E. Coli: (exinuclease activated excises) Polypeptide trimer recognizes and binds to damaged DNA (recognizes kink) Energy from ATP is used to bend DNA Cleaves DNA 5 and 3 to dimer

Releases the excised oligomer Gap filled in by DNA polymerase I, using complementary strand as template DNA ligase seals the nick left by polymerase Higher rise for mutations as polymerase mistakes during repair Adjacent thymines are hotspots for mutations Apoptosis too much damage to repair Epidermal cells die all at once peeling Some mutations derail apoptosis so some damaged cells dont die (could lead to cancer, etc.) Xeroderma pigmentosum (autosomal recessive) Mutation resulting in inactive excinuclease (excision repair removes damaged DNA) Excision repair cannot occur No treatment available Fixing other basepairing problems: Base excision repair (vs. nucleotide excision repair, which removes whole stretch of nucleotides) removes damaged DNA in bacteria and humans Glycosylases recognize the mismatch and remove the base Endonucleases recognize the gap and cleaves out the sugar-phosphate DNA polymerase and ligase fix Mistakes during DNA replication can also alter genetic information Replication errors are extremely rare The rate of replication errors is kept low due to correction or proofreading activity of DNA polymerase I and III, which reduces the error rate DNA polymerase mismatch repair DNA polymerase has a 3-5 exonuclease activity that can recognize and remove mispaired bases during synthesis, backs up The 5 to 3 nucleotide addition function then adds the correct nucleotide Only works during cant go back after replication Methyl-directed mismatch repair Bacterial recognition and repair system that can fix mutations after DNA replication Relies on tagging parental strands with methyl groups (bacteria dont use methyl groups to turn genes off) Eukaryotic cells also have a mismatch repair system, but the tag is not yet known (not methyl groups) Parental strands are marked with methyl groups in E. coli, this methylation is on A in GATC sequences Proteins recognize mismatch in replicated DNA and bind to it Recruits another enzyme that scans along and finds the nearby methylated GATC strand

o o

Only parental strand is methylated (daughter strand has not yet been methylated) this system can recognize whether which base is wrong (which strand is the newly synthesized strand) by looking at which strand the methyl group is on Can only cleave daughter strand because methylation protects parental strand from cleavage Corrected daughter strand methylated Mutations that cannot be corrected Unequal crossing over and transposon movement can change the information content of DNA one chromosome ends up with a duplication while the other ends up with a deletion These types of changes are not susceptible to excision or mismatch repair nothing biochemically wrong with the DNA *some mutations never get detected and repaired ex. insertions and deletions gained/lost sequences, but nothing wrong with the rest of the DNA Mutations can be detected in somatic cells e.g. persons with one blue eye and one brown eye somatic mutation occurring in early ocular development in one eye only e.g. some types of cancer somatic mutation in certain genes (e.g. p53) controlling the cell cycle (uncontrolled growth) e.g. aging accumulation of oxidation reactions within individual cells (usually triggered to go through apoptosis, and are replaced with stem cells, although stem cells decrease with aging) Not passed through the germline

Set 20 Genetic Analysis (clone isolation/analysis, PCR and DNA sequencing) Genomic library: a collection of DNA clones that theoretically contains copies of every DNA fragment in the whole genome inserted into a suitable vector and placed into storage DNA probes o Short single-stranded stretches of DNA of known composition o Usually short; from 25 to several thousand nucleotides in length o To visualize them, the probes are labeled, usually with 32P or fluorescent dyes o Used in the identification of clones that contain complementary DNA sequences (e.g. make probe out of human gene to identify corresponding gene in mouse, which is expected to have a similar DNA sequence) Screening libraries by hybridization (colony hybridization) o Synthesize radioactive* cDNA using purified mRNA as template o Plate bacteria carrying recombinant DNA molecules on agar medium and incubate until small colonies form o Replica-plate colonies to nylon membrane o Lyse cells in situ, denature and bind DNA to the membrane o Add radioactive cDNA and incubate under annealing conditions o Rinse membrane thoroughly to remove nonannealed cDNA and expose to X-ray film

Pick colony containing DNA sequence of interest from agar medium and isolate recombinant DNA *Fluorescent labelling mainly used nowadays Several ways to make labeled probes o Can be chemically synthesized, and are termed oligonucleotides (usually 25-100 nucleotides in length) o Fluorescent dyes can be added as part of the synthesis procedure o Must know the DNA sequence of the probe o OR can label a previously cloned fragment removal from the vector by restriction endonuclease digestion, followed by chemical labelling; usually 100-500 base pairs in length; dont need to know the sequence of this probe Requirements of limitations of hybridization probes o The region of complementarity between probe and target sequence must be long enough to allow sufficient hydrogen bonds to provide a cohesive force o In general, two single DNA strands that are longer than 50-100 bp will hybridize so long as the extent of their complementarity is more than 80% Southern blot analysis o Used not to isolate gene sequences, but to find out if organisms have the gene of interest, how many copies it has o Used to show the location of a gene within a larger fragment of DNA o Involves the transfer of DNA fragments from an agarose gel to nitrocellulose filter paper o The relative position of DNA fragments is preserved in the transfer o Hybridization between a specific probe and DNA on the filter can be carried out Polymerase chain reaction (PCR) o Artificial DNA Xeroxing o With the use of two specific oligonucleotides (primers), any fragment of DNA can be amplified from one to one billion copies in as little as two hours o Has uses in DNA cloning, DNA sequencing, making DNA probes, identification of organisms without Southern blotting o Design two primers that are complementary to each end of the sequence of interest (forward and reverse primer) o Mix the primers, template DNA, thermostable DNA polymerase (e.g. Taq), and the four nucleotide bases in a microtube o Begin thermocycling procedure denature, anneal, extend o Each cycle of PCR results in duplication of the strands exponential o PCR: Cycle One Denature DNA a PCR reaction starts with a denaturing step. Samples are heated to 94-96C for one to several minutes to denature (separate into single strands) the target DNA Anneal primers the temperature is lowered for one to several minutes. This allows the left and right primers to anneal (base pair) to their complementary sequences. The primers are designed to bracket the DNA region to be amplified Extend primers the temperature is raised for one to several minutes. This allows Taq polymerase to attach at each priming site and extend a new DNA strand

PCR: Cycle Two (each strand from Cycle One becomes a template strand) Denature DNA the temperature is raised, denaturing the target DNA as in cycle one Anneal primers The temperature cools, allowing more primers to anneal Extend primers The temperature is again raised. Taq polymerase binds to each priming site and synthesizes a new DNA strand o Gets long strands (longer than target fragment) in cycle one eventually get only short strands synthesized (more of them for template), short strands (targets) eventually outnumber the long strands o Uses: Cloning Identification of genetic elements in an organism Diagnosis of infection Paleomolecular biology amplify sequences from highly degraded DNA Forensic science DNA sequencing o Provides the highest resolution of a cloned DNA fragment a complete description of its genetic information at the level of the nucleotide o Sanger dideoxy sequencing Based on lack of 3OH group to terminate chain elongation Chain-terminating sequencing technique Always needs a primer (oligonucleotide) to the flanking template sequence to provide 3OH; primer designed to known (cloned) sequence from vector Primer radioactively labelled Take cloned template DNA (rendered single-stranded), add primer, add DNA polymerase and all the normal deoxynucleotides Divide solution into four aliquots and add low concentration of dideoxynucleotide to each Polymerase will, at random, add either the normal deoxynucleotide and synthesis continues or add a dideoxynucleotide, stopping synthesis (no 3OH on dideoxynucleotide) Analyze lengths of resulting strands (resolve lengths by gel electrophoresis) Incorporation of the dideoxynucleotide is RANDOM end up with a mixture of strand lengths, each one ending where the dideoxy has been incorporated Polyacrylamide gels can resolve strands that differ in length by just a single nucleotide Read from smallest fragment to longest (bottom to top) 5 to 3 New way (no radioactivity) fluorescently label dideoxynucleotides, different colour (fluorescent dye) for each; only need a single tube Couple to computer laser reads fluorophores Scenario 1: 2 parents, both carriers of sickle cell mutant allele (single substitution mutation within beta-globin gene A-T to T-A, single transversion), wish to know the genotype of their newest baby genetic testing using molecule tools

PCR amplification of 500 bp fragment of interest (that contains the mutation) in betaglobin gene, followed by REN digestion with MstII if wild-type allele, MstII would recognize, bind, and cut the 500 bp fragment into two binds; mutant allele restriction endonuclease site has been obliterated, end up with intact 500 bp fragment (REN cannot recognize and cut) o Gel electrophoresis to look at fragment length Scenario 2: two parents, both carriers of CF mutant allele, wish to avoid having a child with CF o Preimplantation genetic testing o Eggs are retrieved from the ovary, fertilized with sperm o At 6-10 cell stage, one egg is removed from each viable embryo o In each of the isolated cells, site of common mutation in CF gene is amplified with PCR o Divide PCR product into two portions; denature o Apply one dot of each sample onto nitrocellulose filter o Hybridize one dot for normal CF allele (wildtype probe); hybridize other with ASO (mutant probe) for mutant allele

Set 21 Cancer genetics Cancer is a genetic disorder involving mutations in cells Cancer is not inherited, but certain inherited mutations can dispose one to cancer inheritance of cancer only happens from cell to cell within a clone of cells, not through generational lines Begins with loss of cell cycle control tumor Tumor (transformed) cells undergo further changes that allow them to invade and disrupt other tissues cancer Types of cancer o Carcinoma epithelia origin, e.g. skin o Sarcoma connective tissue, e.g. bone, cartilage o Leukemia blood-forming tissue, e.g. marrow o Lymphoma cells of the immune system, e.g. B-cells (specific kind of leukemia) Cancer arises when controls over cell division no longer function properly two groups of genes o Tumor-suppressor genes (act recessively, but phenotype is dominant due to high penetrance; mutations inactivate) mutant tumor-suppressor alleles release a brake on cell division, and can decrease the accuracy of cell division in homozygotes Involved in cell cycle control Facilitate suppression of tumors through their very strict control on the cell cycle p53 (TP53) and pRB p53 regulates G1 to S checkpoint Normally, when DNA is damaged in G1 phase, entry into S phase is delayed to allow repair before DNA replication proceeds In mammals, S phase delay is initiated by activation of the p53 pathway Cyclin-dependent kinase (CDK) if CDK is not inactivated, then the cell commits to S phase p53 is a protein transcription factor that induces expression of p21 p21 inactivates CDK, stops movement into S phase, cell cycle arrest

If the damage cannot be repaired (within a certain amount of time p53 accumulates, time/concentration dependent), then apoptosis is initiated by p53 protein End result is prevention of cell division (checkpoint apoptosis if damage is not repaired p53 acts as a tumor suppressor *again, does not detect mutations that dont have some sort of physical or biochemical damage to the DNA Homozygosity for mutations in the p53 gene have the ability to disrupt this checkpoint (acts recessively) Consequently, the damaged DNA gets replicated and strand breaks occur that cause serious chromosomal abnormalities Cells with mutations in p53 may allow a cell to rapidly acquire further gene mutations Approximately 50% of all cancers are associated with mutations in p53 e.g. colon, breast, liver, lung, ovarian, pancreatic Located on chromosome 17 in humans, along with BRCA1 gene (also a tumor suppressor gene) It takes more than just disruptions in p53 to cause cancer, but this is a common first strike leading down the road to cancer pRB regulates G1 to S checkpoint Protein product pRB (transcription factor) controls cell cycle In absence of functional pRB (brake), S phase cannot be prevented uncontrolled cell growth Normally p53 will become activated to induce apoptosis and prevent this cell from replicating pRB interacts with another transcription factor, which, on its own away from pRB, is involved in the transcription of genes that are needed to move into the cell cycle Early in G1, pRB binds this transcription factor (E2), bound E2F proteins are unable to stimulate transcription of their target genes pRB is taken off by a complex The brake is always on, sitting on the transcription factor, has to be actively removed to allow transcription of genes needed in S phase, committing the cell to S phase Two-hit mutational model for some cancers supported by studies of retinoblastoma Tumor of the eye Can be hereditary or sporadic (somatic) Both involve mutation in tumor suppressor gene pRB Hereditary o One mutant pRB copy is inherited from a parent = first hit o Mutation present in germ line passed on

If good pRB becomes mutated (second hit), then retinoblastoma begins in that eye (somatic) o Recessive allele, but end effect is dominant o Autosomal dominant because there exists a strong likelihood that another mutation in the normal allele will eventually occur incomplete penetrance, variable expression (every cell in body has one mutant, some may get it in one eye but not necessarily the other/both eyes) Sporadic o Requires two independent mutations of pRB to begin oncogenesis o Somatic mutation, not passed on BRCA1 and BRCA2 are tumor suppressor genes o Products are part of a surveillance system needed to repairing DNA breaks o If mutations occur in these genes, then damaged DNA may get replicated o Heterozygotes are at higher risk for acquiring mutation in remaining copy o Promotes mutations in other genes leading to cancer dominant Oncogenes mutant alleles that act dominantly to stimulate cell proliferation; mutations activate Studying tumor-causing retroviruses (oncoviruses) has led to the discovery of oncogenes Proto-oncogene normal allele involved in stimulating cell proliferation Retroviruses integrate into host chromosome Proto-oncogene is now under control of a viral promoter oncogene Viral machinery replicates this oncogene and it gets packaged into progeny virus particles, which then insert it into other cells/hosts could become cancerous Some DNA viruses carry oncogenes as well (does not normally integrate into host chromosome), e.g. human papilloma virus strains 16 and 18 (genital warts) 90% of cervical cancer in women is associated with strains of HPV; carrying oncogenes that inactivate the p53 and pRB Is the first step in tumorigenesis (other mutations must follow to develop cancer) Every now and then, DNA virus accidently recombined into the chromosomes in the nucleus by recombination enzymes viruses are no longer made, but the products that interfere with pRB and p53 are produced by the cell, forcing the cells to undergo rapid division; this time, the cells arent killed (no more virus to kill the cells) Accidental integration of viral DNA o Disruption of cell cycle, but no virus cells arent killed, continue to replicate

o Increases probability of mutations cancer Note: only oncogenic strains, like 16 and 18, destroy p53 and pRB proteins; non-oncogenic strains cannot bind p53, and bind pRB, but do not destroy it when they integrate, they do not continue to disrupt cell cycle Normally, cells with damaged DNA are caught by p53 repair or apoptosis (tumor suppressed) Mutations in p53 (2 hits) cell cant detect damage and DNA get replicated o But normally, most such cells will only undergo a limited number of replication rounds as telomers shorten each time, then die o Mutations in genes controlling telomerase expression allows telomers to be repaired and cells become immortalized (could have happened as a result of mutations of p53 gene) Mutations in genes that induce cell proliferation (proto-oncogenes oncogenes) o Cause cell to reproduce rapidly o Excessive proliferation enhances the potential for mutation o Mutations are more likely to occur in a large clone of rapidly growing cells o Every round of DNA replication enhances the potential for new mutation to occur to lead to cancerous state Typically, multiple mutations must occur to change a normal cell to a cancerous e.g. analysis of cells from human colon cancers show that these cells contain mutations in at least 5-10 genes Cancer develops over time the incidence of cancer rises with age; supports idea that cancer is generally caused by a series of mutations Cancer is thought to arise by successive mutations in a clone of proliferating cells Phenotypes for cancer include many types of cellular abnormalities o Loss of contact inhibitions (loss of cell to cell communication) normally, cells stop growing when there is no more space o Autocrine stimulation o Disruption of local tissue and invasion of distant tissues Tumor cells remain localized Angiogenesis formation of new blood vessels; nutrient sources for the tumor Metastasis mutant cells invade another tissue and continue dividing there