genetics 1-6

genetics 1-6 - Genetics: From Genes to Genomes Genetics:...

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Unformatted text preview: Genetics: From Genes to Genomes Genetics: Hartwell ● Hood ● Goldberg ● Reynolds ● Silver ● Veres Hartwell Hood Goldberg Second Edition Genetics Genetics The Study of Biological Information • DNA molecules encode the biological information fundamental • • • • • • Chapter Outline ***print outline 1 to all life forms (not necessarily alive) Proteins are the primary unit of biological function Regulatory networks specify the behavior of genes All living forms are closely related Genomes are modular, allowing rapid evolution Genetic techniques permit dissection of biological complexity Focus of this course in on human genetics • Process of evolution has taken 4 billion years to generate organisms seen today Information in DNA generates diversity • Four bases – G (guanine), A (adenine), T (thymine), and C (cytosine) are the nucleotide building blocks of DNA • DNA is a double stranded helix composed of A­T and G­C complementary bases • Order of nucleotide sequences determine which proteins are synthesized , as well as when and where the synthesis actually occurs. • Hydrogen bonds between bases (relatively weak) • • Although DNA is 3­D, information within the molecule is 1­D Genetic Information is Digital and “digital” • The sequence of bases in DNA can be read by DNA sequenceers, stored in computers and synthesizes DNA synthesis—can be read about 10^6 bases/day • Huge DNA database potentially possible Genes are sequences of DNA that encode proteins DNA resides in within cells packaged as units called chromosomes • The entire collection of chromosomes in each cell of an organism is called a genome • Humans have 24 different chromosomes (why not 23?) • 1­22 are autosomes, 23 are sexosomes • The human genome has about 3 x 10^9 base pairs and 40,000 to 60,000 genes Biological function emerges primarily from proteins Proteins are polymers of amino acids • • • • • The diversity of protein structure generates extraordinary diversity Proteins interact with DNA and other proteins • Proteins have three dimensional structures Information in DNA dictates the sequence of its amino acids There are 20 different amino acids The order of amino acids determines the type of protein and its structure Biological systems function as complex interactive networks of proteins and DNA that interact with one another RNA was probably the first information processing molecule, but RNA is unstable All living things are closely related • • Had the ability to store, replicate, mutate, express, and fold in 3 • • • • dimensions DNA took over the linear formation and replication functions Proteins took over the 3-dimensional folding functions All organisms alive now descended from the first organisms that adopted this molecular specialization RNA is composed of four bases: guanine (G), adenine (A), cytosine ©, and uracil (U) All living organisms use the same arbitrary codes for RNA, DNA, and protein (amino acid sequences) Many genes have similar functions in very different organisms Convergent Evolution organisms that are not directly related—examines analogous characteristics • Streamlines bodies of penguins and dolphins • The independent development of similar structures or capabilities in • Analogous structures perform the same function but are structurally different • Human legs vs. insect legs (both for locomotion but humans use muscles and tendons while insects use hydrolytic pumps) • Homologous structures: similar structure anatomically, but performs different functions • Human arms vs. front flipper of whale (both look like “arm” structures, we use to hold items and they use to swim) Divergent Evolution Process by which an ancestral characteristic becomes adapted to new roles —examines homologous characteristics Ex. Human arms and wings of bats • Common ancestor to both which had 4 limbs. In humans the one pair evolved into hands used for grasping and in bats they evolved into wings for flight Relatedness among organisms is important for the study of human genes Studies of genetics in model organisms help us understand how genes work in humans • Some model organisms include bacteria, yeast, roundworms, fruit flies, and mice • Model organisms may have simpler biological networks and can be manipulated experimentally • Modular construction of genomes has allowed rapid evolution of complexity • Gene families arise from primordial genes through duplication and rearrangements • Duplication and divergence of new genes can generate genes with new functions • We’ll examine traits that are controlled by multiple genes and single genes (for example… Widow’s Peak Hairline Ear Lobes Tongue Rolling So, what do these traits mean? Mid­digital Hair Mid­digital Hair The process of duplication and divergence Duplication and divergence has made rapid evolution possible Genetic techniques permit the dissection of complexity • Nothing really… • Genes can be identified and inactivated one at a time using genetic techniques Dissection of genomes gene­by­gene unravels the complexity of biological systems The challenge for modern biology lies in the • understanding how the multitude of networks of genes and higher level systems interact to produce complex systems Genome sequencing projects are a step in understanding the complexity of genomes New technological tools facilitate the dissection of New genomes and integration of information genomes Dna chips detect the expression of thousands of genes in a response to environmental changes • Genetics is a field of science that will have an enormous impact Focus on human genetics on society • Our understanding of biological complexity using genetic approaches is proceeding at a very rapid pace • Recent technological advances have shifted the focus of genetics from analysis of single genes and proteins to entire networks – the systems approach • • • Gene therapy • Diagnostics Discovery of genes with variations that cause or predispose one to disease will continue at a rapid pace Genetics Predictive and Preventative Medicine • Therapeutic drugs to block or reverse effects of mutant genes • Detections of disease and treatment before its actual onset with may increase life span significantly Social issues and genetics Should an individual’s genetic profiles be freely available to insurance companies, employers, government? • Should our government regulate the use of genetic and genomic information to reflect society’s social values? • 2008 • Genetic Information Nondiscrimination Act was passed by government in • Is it okay to permanently alter genes in humans for medical or social reasons? Ch. 2 Notes Mendel’s Breakthrough Patterns, Particles, and Principles of Heredity Outline of Mendelian Genetics • The historical puzzle of inheritance and how Mendel’s experimental approach helped solve it • Mendel’s approach to genetic analysis including his experiments and related analytic tools • A comprehensive example of Mendelian inheritance in humans; disease transmission of single gene traits will also be examined Gregor Mendel (1822-1844) Themes of Mendel’s work • Variation is widespread in nature • Observable variation is essential for following genes (other wise no visible way of noting differences among progeny • Variation is inherited according to genetic LAWS and not solely by chance • Mendel's laws apply to ALL sexually reproducing organisms • The historical puzzle of inheritance • • • • • Artificial selection has been an important practice since before recorded history Domestication of animals (ancient civilizations) Selective breeding of plants (aesthetic and commercial) 19th century – precise techniques for controlled matings in plants and animals to produce desirable traits in many of the offspring Breeders could not explain why traits would sometimes disappear and then reappear in subsequent generations State of genetics in early 1800’s • WHAT IS INHHERITED? • HOW IS IT INHERITED? • WHAT IS THE ROLE OF CHANCE IN HEREDITY? Mendel’s workplace Historical theories of inheritance • One parent contributes most of the feature (homunculus, 1694) • Blending inheritance – parental traits become mixed and forever changed in the offspring Keys to Mendel’s experiments • The garden pea was an ideal organism • vigorous growth • self fertilization • easy to cross fertilize • produced large number of offspring each generation • Mendel analyzed traits with discrete alternative forms • purple vs. white flowers • yellow vs. green peas • round vs. wrinkled seeds • long vs. short stem length • Mendel established pure breeding lines to conduct experiments Monohybrid crosses reveal units of inheritance and Law of Segregation Traits have dominant and recessive forms • Disappearance of traits in F1 generation and reappearance in the F2 generation disproves the hypothesis that traits blend • Trait must have two forms that can each breed true • One form must be hidden when plants with each trait are interbred • Trait that appears in F1 is dominant • Trait that is hidden in F2 is recessive Alternative forms of traits are alleles • Each trait carries two copies of a unti of inheritance, one inherited from the mother and the other from the father • Alternative forms of traits are called alleles • Some traits are controlled by multiple genes, while others are controlled by single genes • Another example... Law of Segregation • Two alleles for each trait separate (segregate) during gamete formation, and then unite at random, one from each parent, at fertilization The Punnet Square Rules of Probability • Product rule: probability of two independent events occurring together is the product of their individual probabilities o What’s the probability that event 1 and event 2 will occur? • ***Look up Sum Rule Probability and Mendel’s Results • Cross Yy x Yy pea plants • Chance of Y sperm uniting with a Y egg • ½ chance of sperm with Y allele • ½ chance of egg with Y allele • Chance of Y and Y uniting = ½ x ½ = ¼ • Chance of Yy offspring • ½ chance of sperm with y allele and egg with Y allele • ½ chance of sperm with Y allele and egg with y allele • Chance of Yy – ( ½ x ½ ) + ( ½ x ½ ) = 2/4 or 1/2 Further crosses confirm predicted ratios Genotypes and Phenotypes • Phenotype – observable characteristic of an organism • Genotype – pair of alleles present in an individual • Homozygous – 2 alleles of trait are the same (YY or yy) • Heterozygous – 2 alleles of trait are different (Yy) Genotypes versus phenotpyes Test cross reveals unkown genotpye Dihybrid crosses reveal the law of independent assortment • A dihybrid is an individual that is heterozygous at two genes • Mendel designed experiments to determine if two genes segregate independently of one another in dihybrids • First constructed true breeding line for both traits, crossed them to produce dihybrid offspring and examine the F2 for parental or recombinant types (new combinations not present in the parents” Results of Mendels dihybrid crosses • F2 generation contained both parental types and recombinant types • Alleles of genes assort independently, and can thus appear in any combination in the offspring Dihybrid cross shows parental and recombinant types Dihybrid cross produces a predictable ratio of phenotypes The law of independent assortment • During gamete formation different pairs of alleles segregate independently of each other Summary of Mendel's work • Inheritance is particulate – not blending • There are two copies of each trait in a germ cell • Gametes contain one copy of the trait • Alleles (different forms of the trait) segregate randomly • Alleles are dominant or recessive – thus the difference between genotype and phenotype • Different traits assort independently Laws of probability for multiple genes ****LOOK UP Rediscovery of Mendel • Mendel’s work was unappreciated and remained dormant for 34 years • Even Darwin’s theories were viewed with skepticism in the late 1800’s because he could not explain the mode of inheritance of variation • In 1900, 16 years after Mendel died, four scientists rediscovered and acknowledged Mendel’s work, giving birth to the science of genetics 1900 - Carl Correns, Hugo deVries, and Erich von Tschermak rediscover and confirm Mendel’s laws Mendelian inheritance in humans • Most traits in humans are due to the interaction of multiple genes and do not show a simple Mendelian pattern of inheritance • A few traits represent single gene interactions: sickle cell, cystic fibrosis, Tay-Sachs, and Huntington’s (Table 2.1) • Because we can not do breeding experiments on humans, we usually examine large groups of people from a particular region, ethnic group, or controlled breeding to gain more information about a disease In humans we must use pedigrees to study inheritance • Pedigrees are an orderly diagram of a families relevant genetic features extending though multiple generations • Pedigrees help us infer if a trait is from a single gene and if the trait is dominant or recessive Anatomy of a pedigree A vertical pattern of inheritance indicates a rare dominant trait A horizontal pattern of inheritance indicates a rare recessive trait Ch. 3 Notes Extensions to Mendel Complexities in relating genotype to phenotype Chapter Outline Chapter Outline (cont’d.) Single Gene Inheritance Models Summary of dominance relationships Incomplete dominance in snapdragons Co­dominant blood group alleles Co­dominant lentil coat patterns Do variations on dominance relations negate Mendel’s law of segregation? • • • • • Dominance relations affect phenotype and have no bearing on the segregation of alleles Alleles still segregate randomly Gene products control expression of phenotypes differently Mendel’s law of segregation still applies Interpretation of phenotype/genotype relation is more complex Genes may have multiple alleles that segregate in populations Good example of this includes blood type in humans IA, IB, i (or use A, B, O, respectively) are the three different alleles that contribute towards blood type A is dominant to O B is dominant to O A and B are codominant towards eachother A gene can have more than two alleles • • • • • • • • Type “O” is the UNIVERSAL DONOR Type “AB” is the UNIVERSAL RECIPIANT The Chromosome Theory of Inheritance • • • • Observations and experiments that placed the hereditary material in the nucleus on the chromosomes Mitosis ensures that every cell in an organism carries same set of chromosomes Meiosis distributes one member of each chromosome pair to gamete cells Gametogenesis, the process by which germ cells differentiate into gametes Outline of Chromosome Theory of Inheritance • Validation of the chromosome theory of inheritance A. Evidence that Genes Reside in the Nucleus • • Microscopist • Semen contains spermatozoa (sperm animals) • Hypothesized that sperm enter egg to achieve fertilization 1667 – Anton van Leeuwenhoek • 1854 – 1874: confirmation of fertilization through union of eggs and sperm • Recorded frog and sea urchin fertilization using microscopy and time­ B. Evidence that Genes Reside in Chromosomes • • • • lapse drawings and micrographs 1880’s – innovations in microscopy and staining techniques identified thread­like structures Provided a means to follow movement of chromosomes during cell division Mitosis – two daughter cells contained same number of chromosomes as parent cell (somatic cells) Meiosis – daughter cells contained half the number of chromosomes as the parents (sperm and eggs) • • Walter Sutton – studied great lubber grasshopper 1. One Chromosome Pair Determines and Individual’s Sex Parent cells contained 22 chromosomes plus an X and Y chromosome • Daughter cells contained 11 chromosomes and X or Y in equal numbers • • Cells with XX were females • Cells with XY were males After fertilization • • Provide basis for sex determination • One sex has matching pair • Other sex has one of each type of chromosome (heterogametic) Sex chromosome • Sex determination in humans • Children receive only an X chromosome from mother but X or Y • Gamete contains one­half the number of chromosomes as the zygote • Haploid (N) – cells that carry only a single chromosome set • Diploid (2N) – cells that carry two matching chromosome that appear 2. At Fertilization, Haploid Gametes Produce Diploid Zygotes from father as pairs • N – the number of chromosomes in a haploid cell • 2N – the number of chromosomes in a diploid cell 3. The number and shape of chromosomes vary from species to species Diploid vs. haploid cell in Diploid vs. haploid cell in Drosophila melanogaster Homologous chromosomes match in size, shape, and banding patterns • Anatomy of a chromosome Homologous chromosomes (homologs) contain the same set of genes Genes may carry different alleles • • Nonhomologous chromosomes carry completely unrelated sets of genes Karyotypes can be produced by cutting micrograph images of stained chromosomes and arranging them in matched pairs 4. There is variation between species in how chromosomes determine an individual’s sex II. Mitosis ensures that every cell in an organism carries the same chromosomes • • Interphase – period of cell cycle between divisions/ cells grow and replicate chromosomes • G1 – gap phase – birth of cell to onset of chromosome replication/ cell growth • S – synthesis phase – duplication of DNA • G2 – gap phase – end of chromosome replication to onset of mitosis • alternates between interphase (90%) and mitosis (10%) Cell cycle – repeating pattern of cell growth and division Chromosome replication during S phase of cell cycle • • G1, S, and G2 phase – cell growth, protein synthesis, chromosome Within nucleus A.Interphase replication • • Formation of microtubules radiating out into cytoplasm crucial for Outside of nucleus interphase processes • Centrosome – organizing center for microtubules located near nuclear envelope • Centrioles – pair of small darkly stained bodies at center of centrosome in animals (not found in plants) • • Inside nucleus • Chromosomes condense into structures suitable for replication • Nucleoli begin to break down and disappear • Outside nucleus 1. Prophase – chromosomes condense B. Mitosis – Sister Chromatids separate • Centrioles which replicated during interphase move apart and migrate to opposite ends of the nucleus • Interphase microtubules disappear and are replaced by microtubules that rapidly grow from and contract back to centrosomal organizing centers • • • • • 2. Pro­metaphase: The Spindle Forms Nuclear Envelope breaks down Microtubules invade nucleus Chromosomes attach to microtubules through kinetochore Mitotic spindle – composed of 3 types of microtubules • Kinetochore microtubules – centrosome to kinetochore • Polar microtubules – centrosome to middle of cell • Astral microtubules – centrosome to cell’s periphery • • Chromosomes move towards imaginary equator called metaphase plate 3. Metaphase – Chromosomes align at cell equator • 4. Anaphase Sister chromatids move to opposite spindle poles • Separation of sister chromatids allows each chromatid to be pulled towards spindle pole connected to by kinetochore microtubule, metacentric chromosomes have traditional “V” shape during this process • • • • • • Telophase: Identical sets of Chromosomes are enclosed in 2 nuclei Spindle fibers disperse Nuclear envelope forms around group of chromosomes at each pole One or more nucleoli reappear Chromosomes de­condense Mitosis complete • 6. Cytokinesis : Cytoplasm divides • Starts during anaphase and ends during telophase • Animal cells: contractile ring pinches cells into two halves • Plant cells: cell plate forms dividing cell into two halves C. Regulatory checkpoints ensure correct chromosome separation during Mitosis III. Meiosis produces haploid germ cells • Somatic cells – divide mitotically and make up vast majority of organism’s tissues Germ cells – specialized role in the production of gametes • Arise during embryonic development in animals and floral development in plants • • Undergo meiosis to produce haploid (N) gametes • Gametes unite with gamete from opposite sex to produce diploid (2N) offspring • B. Meiosis I, Homologous chromosomes pair, exchange parts, then segregate from eachother Chromosomes replicate once Nuclei divide twice Crossing over during prophase produces recombined chromosomes 1. Prophase I continued 2 & 3. Meiosis I – Metaphase I and Anaphase How crossing over produces recombined gametes How crossing over produces recombined gametes à genetic diversity *know number, where, and (un) or duplicated chromosome are 4. MEIOSIS I C. Meiosis II: Sister chromatids separated to produce haploid gametes 1 & 2. 3 & 4. Meiosis II – Anaphase II and Telophase II Prophase II and Metaphase II D. Summary of Significant Events of Meiosis • Meiosis ­ Cytokinesis One round of duplication, two rounds of nuclear division • Non­disjunction: when homologs of a chromosome pair do not segregate during meiosis I or II properly • Can lead to trisomy 21 (downs syndrome) • Also, donkey x horse example • offspring are sterile • chromosomes can’t pair up properly because donkey has 31 • Meiosis contributes to genetic diversity in two ways chrom. pairs and horse has 32 chrom pairs • Independent assortment of non­homologous chromosome creates different combinations of alleles among chromosomes Crossing­over between homologous chromosomes creates different combinations of alleles within each chromosome rule: 2^23 creates 8,388,608 different gamete types without even considering any crossing over events 2n • • • Gametogenesis involved mitosis and meiosis A. Oogenesis – egg formation in humans • • Diploid germ cells called oogonia multiply by mitosis to produce primary oocytes • Primary oocyte undergo meiosis I to produce one 2ndary oocyte and one small polar body (which arrests during development) • Secondary oocyte undergoes meiosis II to produce one ovum and one small polar body • Polar bodies disintegrate leaving one large functional gamete • • Symmetrical meiotic divisions produce 4 functional sperm • Begins in the male testis in germ cells called spermatogonia B. Spermatogenesis in humans Oogenesis in humans Gametogenesis • Mitosis produces diploid primary spermatocytes • Meiosis I produces 2 secondary spermatocytes per cell • Meiosis II produces 4 equivalent spermatids • Spermatids mature into functional sperm cells Spermatogenesis in humans **LOOK UP A. The chromosome theory correlates Mendel’s laws chromosome behavior during meiosis • • • • • • with • • • • • • B. Specific traits are transmitted with specific chromosomes Chromosome Behavior Each cell contains two copies of each chromosome Chromosome complements appear unchanged during transmission from parent to Chromosome complements appear unchanged during transmission from parent to offspring Homologous chromosomes pair and then separate to different gametes Maternal and paternal copies of chromosomes pairs separate without regard to the assortment of other homologous chromosome pairs At fertilization an egg’s set of chromosome untie with randomly encountered sperm’s chromosomes In all cells derived from a fertilized egg, ½ of chromosomes are of maternal origin and ½ of paternal Behavior of genes Each cell contains 2 copies of each gene Genes appear unchanged during transmission from parent of offspring Alternative alleles segregate to different gametes Alternative alleles of unrelated genes assort independently Alleles obtained from one parent unite randomly with those from another parent In all cells derived from a fertilized gamete, ½ of genes are from maternal origin and ½ from paternal • • If genes are on specific chromosomes, then traits determine by the 1. A test of the chromosome theory gene should be transmitted with the chromosome • T.H Morgan’s experiments demonstrating sex­linked inheritance of a gene determining eye color demonstrate the transmission of traits with chromosomes • 1910 – T.H. Morgan discovered a white – eyed male Drosophila Nomenclature for Drosophila genetics and alleles in populations • melanogaster among his stock • population • Denoted with a “+” (greater than 1 percent) Wild­type allele – allele that is found in high frequency in a ***look up monomorphic, polymorphic, allele frequency Mutant allele – allele found in low frequency; considered rare • no symbol • Polymorphic example: plant incompatibility—alleles determine acceptance or rejection of pollen; idea that self-pollination in some plants is avoided o Again multiple alleles are used in the pollen and ovules o Separate male and female parts on the plant However each plant can still only carry a maximum of 2 different allele type on one particular plant There are greater than 90 alleles for the incompatibility complex • The mouse agouti gene controls hair color: One wild-type allele, many mutant alleles (monomorphic) o Wild-type agouti allele (A) produces yellow and black pigment in hair o 14 different agouti alleles in lab mice but only A allele in wild mice o E.g. mutant alleles a and a^t a recessive to A • aa has black only a^t dominant to a but recessive to A • a^ta^ mouse has black on back and yellow on belly • • Recessive mutation – gene symbol is in lower case Dominant mutation – gene symbol is in upper case Dominant • Pleiotropy is the phenomenon of a single gene determining several distinct and seemingly unrelated characteristics o E.g. many aboriginal Maori men have respiratory problems and are sterile Defects due to mutations in a gene required for functions of cilia (failure to clear lungs) and flagella (immotile sperm) • With some pleiotropic genes… o Heterozygotes can have visible phenotype o Homozygotes can be inviable (e.g. AY allele of agouti gene in mice, see Fig. 3.9) • The A^y allele produces a dominant coat color phenotype in mice (pleiotrophic) o A^y allele of agouti gene causes yellow hair with no black o Cross agouti with yellow mice Progeny in 1:1 ratio of agouti to yellow Yellow mice must be for A and A^y A^y is dominant to A • The A^y allele is a recessive lethal allele o A^y is dominant to A for hair color, but is recessive to A for lethality o Cross yellow with yellow mice F1 mice are 2/3 yellow and 1/3 agouti 2:1 ratio is indicative of a recessive lethal allele Pure-breeding yellow mice (A^yA^y) cannot be obtained because they are not viable One gene may contribute to several characteristics Sickle Cell Anemia: Extensions to Single Gene Inheritance (slightly explained ^above) • Multiple alleles o Hemoglobin is composed of two polypeptide chains (alpha and beta globin chains) Beta globin gene is the normal wild-type allele called “A” One type of common mutant allele called “S” because it causes sickling of red blood cells • Pleiotropy o The mutant “S” allele affects more than one trait o SS individuals Red blood cells clog after releasing oxygen Low oxygen causes tissues to cramp Anemia also results However, these individuals are resistant to malaria o SA individuals o AA individuals • Recessive lethality o SS individuals develop heart failure because of stress on circulatory system o Lethality of “S” allele obvious here when homozygous o SA and AA individuals have a normal circulatory system • Different dominance relations o Codominance SS, SA, AA individuals • A and S are codominant for beta globin polypeptide production o Complete dominace and recessiveness SS, SA, AA individuals • A>S for red blood cell shape and concentration at sea level SS, SA, AA individuals • S>A for resistance to malaria o Incomplete dominance SS, SA, AA individuals • A and S alleles are incompletely dominant for red blood cell shape and concentration at high altitudes “Criss­cross inheritance” of the white gene demonstrates X­linkage X and Y linked traits in humans are identified by pedigree analysis dads à daughters moms à sons • • • Trait appears in more moles than females X­linked traits exhibit 5 characteristics seen in pedigrees • Mutation and trait never passes from father to son • Affected males does pass X­linked mutation to all daughters, who are heterozygous carriers • Trait often skips a generation • Trait only appears in successive generations if sister of an affected male is a carrier. If so, ½ of her sons will show trait Example of X­linked recessive trait in human pedigree – hemophilia Example of sex­linked dominant trait in human pedigree – hypophosphatemia Two genes can interact to determine one trait Extensions to Mendel for Multifactorial Inheritance • In all of these cases, F2 phenotypes from dihybrid crosses are in a variation of the 9:3:3:1 ration expected for independently assorting genes 1. Novel phenotypes can results from combined gene interactions (ex. Seed coat in lentils) • Dihybrid cross of lentils, tan with gray o All F1 seeds are brown o F2 progeny: 9/16 brown, 3/16 tan, 3/16 gray, 1/16 green Suggests two indepentenly assorting genes for seed coat color • The two-gene hypothesis explains why there is: o Only one green phenotype (pure-breeding aabb) o Two types of tans (pure-breeding AAbb; tan and green producing Aabb) o Two types of grays (pure-breeding aaBB; gray and green producing aaBb) o Four types of browns (pure-breeding AABB; brown and tan producing AABb; brown and gray producing AaBB; and AaBb dihybrids that give rise to all four colors) • Four color phenotypes arise from four genotypic classes (9:3:3:1 indicates dihybrid cross of this type) • Sorting out the dominance relations by select crosses of lentils 2. Complementary gene action (ex. Flower color) • Purple F1 progeny are produced by crosses by two pure-breeding white lines o A modified 9:3:3:1 ratio is observed o Instead of four phenotypic classes, only two emerge Why? The 9:3:3:1 ratio gets “lumped” Dyhybrid cross generates 9:7 ratio in F2 progeny 9/16 purple (A-B-) 7/16 white (A-bb, aaB-, aabb) • Possible biochemical explanation for complementary gene action for flower color in sweet peas o One pathway has two reaction catalyzed by different enzymes At least one dominant allele of both genes is required for purple pigment Homozygous recessive for either or both genes results in no pigment So both gene need to be turned “on” in order for purple to be expressed 3. Epistasis, where one gene influences another one (ex. Dog fur, Bombay phenotype in humans, squash color, chicken feather color) • The gene that does the masking is spistatic to the other gene • The gene that is masked is hypostatic to the other gene • Epistasis can be recessive or dominant o Recessive—epistasic gene must be homozygous recessive (ee) o Dominant—epistatic gene must have at least one dominant allele present (E-); two types of dominant epistasis seen • Recessive epistasis in Golden Labs o 9:3:4 ratio in F2 progeny of dihybrid crosses indicates recessive epistasis 9/16 black (B-E-) 3/16 brown (bbE-) 4/16 yellow (B-ee, bbee) Genotype ee masks the effect of all B genotypes • Recessive epistasis in humans with a rare blood type (Bombay phenotype) o Gene for substance H is epistatic to the ABO gene Without the H substance (H antigen), there is nothing for the A or B sugar to attach to o All type A, AB, B, and O people are Ho People with hh genotype with appear to be type O o 1st discovered in Mumbai; very rare allele 1/1 million worldwide but in India there are 1/10,000 Can only receive blood from Bombay phenotype individuals Linkage, Recombination, and the Mapping of Genes on Chromosomes Outline of Linkage, Recombination, and the Mapping of Genes on Chromosomes • • Genes linked together on the same chromosome usually assort together • Linked genes may become separated through recombination • • The frequency with which genes become separated reflects the physical distance between them Linkage and Meiotic recombination Mapping • • Rarely, recombination occurs during mitosis • In eukaryotes mitotic recombination produces genetic mosaics Mitotic Recombination Independent assortment – Genes on different chromosomes 1. In dihybrid crosses, departures from a 1:1:1:1 ration f F1 gametes indicate that the 2 genes are on the same chromosome 2. A bias in parental genotypes in the F2 generation defines linkage 3. The % parental and recombinant classes vary with the gene pair in question. For example, examine sex linkage… A. Some genes on the same chromosome assort together more often than not Linkage: Two genes on same chromosome Linkage: Two genes on same chromosome segregate together “Crossing over” and “linkage” lead to separation of linked genes 5. Chi square test pinpoints the probability that ratios are evidence of linkage • • Deviations from 1:1:1:1 rations can represent change events OR linkage • Ratios alone will never allow you to determine if observed data are significantly Transmission of gametes is based on chance events Chi square test measures “goodness of fit” between observed and expected (predicted ) results • Accounts for sample size, or the size of the experimental population different from predicted values • The larger you sample, the closer your observed values are expected to match the predicted values • Degrees of Freedom (DF) : # of independently varying parameters minus 1 • • Null hypothesis – observed values are not different from the expected Framing a hypothesis 6. Applying the chi square test values • For linkage studies – no linkage is null hypothesis • Expect a 1:1:1:1 ratio of gametes • Alternative Hypothesis – observed values are different from expected values • For linkage studies – genes are linked • expect significant deviation from 1:1:1:1 ratio 6. Applying the chi square test to a linkage study Chi Square – Experiment 1 & 2 Chi square table of critical values B. Recombination results when crossing­over during meiosis separates linked genes • 1909 – F. Janssens observed chiasmata, regions in which non­ sister chromatids of homologous chromosomes cross over each other • T.H. Morgan suggested these were sites of chromosome breakage and change resulting in genetic recombination 1. Reciprocal exchanges between homologous chromosomes are the physical basis of recombination • 1931 – Genetic recombination depends on the reciprocal exchange of parts between maternal and paternal chromosomes • • • • Harriet Creighton and Barbara McClintock studied corn Curtis Stern studied fruit flies Physical markers to keep track of specific chromosome parts Genetic markers were points of reference to determine if particular progeny were result of recombination 2. Chiasmata mark the sites of recombination Genetic recombination between car and Bar genes on the Drosophila X chromosome 3. Recombination frequencies for pairs of genes reflect distance between them • Chiasmata mark the sites of recombination A.H. Sturtevant – Percentage of recombination, or recombination frequency (RF) reflects the physical distance separating two genes • 1 RF = 1 map unit (or 1 centiMorgan) • Unlinked genes show a recombination of 50% Genes on different chromosomes (USE CHI­SQUARE test to verify this… frequency 4. Unlinked genes show a recombination frequency of 50% Summary of linkage and recombination • • • • • • • II. Mapping: Locating genes along a chromosome • • Genes close together on the same chromosome are linked and do not segregate independently Linked genes lead to a larger number of parental class than expected in double heterozygotes Mechanism of recombination is crossing over Chiasmata are the visible signs of crossing over Farther away genes are the greater the opportunity for chiasmata to form Recombination frequencies reflect physical distance between genes Recombination frequencies between 2 genes vary from 0% to 50% Difficult to determine gene order if two genes are close together • Actual distances between gene do NOT always add up • Pair­wise crosses are time and labor intensive • Mapping: Locating genes along a chromosome 3. Limitations of two point crosses • “signature” is 4 BIG classes and 4 small classes 1. comparisons help establish relative (order to eachother) 2. Genes are arranged in a length of a chromosome gene positions line along the B. Three Point Crosses: A faster more accurate method to map genes • 1. Analyzing the results of a three point cross Look at two genes at a time and compare to parental (NCO) 1. Analyzing the results of a three point cross 2. Interference: The number of double crossovers may be less than expected • Sometimes the number of observable double crossovers is less than expected if the two exchanges are independent • Occurrence of one crossover reduces likelihood that another crossover will occur in adjacent parts of the chromosome • Chromosomal interference – crossovers do NOT occur independently • Interference is not uniform among chromosomes or even within a chromosome Coefficient of coincidence = ratio between actual frequency of DCO and expected frequency of DCO • Interference = 1 – coefficient of coincidence • • If interference = 0, observed and expected frequencies are equal • If interference = 1, no double crossovers can occur Measuring interference 3. Double recombinants indicate order of three genes Summary of three point cross analysis • • • Parental class – most frequent • Double crossovers – least frequent • • • Cross a true breeding mutant with a true breeding wild­type Analyze F2 individuals (males if sex­linked) C. Do Genetic and Physical maps • • Determine order of genes based on parentals and recombinants Determine genetic distance between each pair of recombinants Calculate coefficient of coincidence and interference Order of genes is correctly predicted by using physical maps Limitations: Distance between genes is NOT always equal to physical maps • Double, triple, and more crossovers • Only 50% recombination frequency observable in a cross • Variation across chromosome in rate of recombination correspond? • D. Genes chained together by linkage relationships are known as linkage groups Model organisms for understanding the mechanism of recombination because all four haploid products of meiosis are contained in ascus • Ascospores within ascus germinate into haploid individuals • • Saccharaomyces cerevisiae – bakers yeast • Neurospora crassa – bread mold Mapping functions compensate for inaccuracies, but not often imprecise E. Tetrad analysis in fungi Saccharaomyces cerevisiae life cycle 1. Tetrads can be characterized by the number and recombinant spores they contain Neurospora crassa life cycle of parental 2. When PD=NPD, two genes are unlinked 2. When PD=NPD, two genes are unlinked (cont’d.) 3. Linkage is demonstrated by PDs outnumbering NPDs How crossovers between linked genes generate different tetrads • Calculating recombination frequency RF= NPD + ½ T x 100 RF= Total Tetrads We will be using the formual = all SCO + 2(DCO) x 100 TOTAL 5. Confirmation that recombination occurs at the four­strand stage 6. Tetrad analysis demonstrates that recombination is reciprocal • In a cross between strains with different alleles at two genes, each tetrad contains two of each parental type and two of each type of recombinant 7. Ordered tetrads allow mapping a gene in relation to the centromere Segregation patterns in ordered asci Segregation patterns in ordered tetrads Mitotic recombination can produce genetic mosaics Mitotic recombination is rare Initiated by • • • mistakes in chromosome replication • Curt Stern – observed “twin spot” in Drosophila – a form of genetic mosaicism • chance exposure to radiation Mitotic crossing over between sn and centromere in Drosophila • Animals contained tissues with different genotypes on the same body Twin spots in Drosophila Linkage, Recombination, and the Mapping of Genes on Chromosomes Outline of Linkage, Recombination, and the Mapping of Genes on Chromosomes • • Genes linked together on the same chromosome usually assort together • Linked genes may become separated through recombination • • The frequency with which genes become separated reflects the physical distance between them Linkage and Meiotic recombination Mapping • • Rarely, recombination occurs during mitosis • In eukaryotes mitotic recombination produces genetic mosaics Mitotic Recombination Independent assortment – Genes on different chromosomes 1. In dihybrid crosses, departures from a 1:1:1:1 ration f F1 gametes indicate that the 2 genes are on the same chromosome 2. A bias in parental genotypes in the F2 generation defines linkage 3. The % parental and recombinant classes vary with the gene pair in question. For example, examine sex linkage… A. Some genes on the same chromosome assort together more often than not Linkage: Two genes on same chromosome Linkage: Two genes on same chromosome segregate together “Crossing over” and “linkage” lead to separation of linked genes 5. Chi square test pinpoints the probability that ratios are evidence of linkage • • Deviations from 1:1:1:1 rations can represent change events OR linkage • Ratios alone will never allow you to determine if observed data are significantly Transmission of gametes is based on chance events Chi square test measures “goodness of fit” between observed and expected (predicted ) results • Accounts for sample size, or the size of the experimental population different from predicted values • The larger you sample, the closer your observed values are expected to match the predicted values • Degrees of Freedom (DF) : # of independently varying parameters minus 1 • • Null hypothesis – observed values are not different from the expected Framing a hypothesis 6. Applying the chi square test values • For linkage studies – no linkage is null hypothesis • Expect a 1:1:1:1 ratio of gametes • Alternative Hypothesis – observed values are different from expected values • For linkage studies – genes are linked • expect significant deviation from 1:1:1:1 ratio 6. Applying the chi square test to a linkage study Chi Square – Experiment 1 & 2 Chi square table of critical values B. Recombination results when crossing­over during meiosis separates linked genes • 1909 – F. Janssens observed chiasmata, regions in which non­ sister chromatids of homologous chromosomes cross over each other • T.H. Morgan suggested these were sites of chromosome breakage and change resulting in genetic recombination 1. Reciprocal exchanges between homologous chromosomes are the physical basis of recombination • 1931 – Genetic recombination depends on the reciprocal exchange of parts between maternal and paternal chromosomes • • • • Harriet Creighton and Barbara McClintock studied corn Curtis Stern studied fruit flies Physical markers to keep track of specific chromosome parts Genetic markers were points of reference to determine if particular progeny were result of recombination 2. Chiasmata mark the sites of recombination Genetic recombination between car and Bar genes on the Drosophila X chromosome 3. Recombination frequencies for pairs of genes reflect distance between them • Chiasmata mark the sites of recombination A.H. Sturtevant – Percentage of recombination, or recombination frequency (RF) reflects the physical distance separating two genes • 1 RF = 1 map unit (or 1 centiMorgan) • Unlinked genes show a recombination of 50% Genes on different chromosomes (USE CHI­SQUARE test to verify this… frequency 4. Unlinked genes show a recombination frequency of 50% Summary of linkage and recombination • • • • • • • II. Mapping: Locating genes along a chromosome • • Genes close together on the same chromosome are linked and do not segregate independently Linked genes lead to a larger number of parental class than expected in double heterozygotes Mechanism of recombination is crossing over Chiasmata are the visible signs of crossing over Farther away genes are the greater the opportunity for chiasmata to form Recombination frequencies reflect physical distance between genes Recombination frequencies between 2 genes vary from 0% to 50% Difficult to determine gene order if two genes are close together • Actual distances between gene do NOT always add up • Pair­wise crosses are time and labor intensive • Mapping: Locating genes along a chromosome 3. Limitations of two point crosses • “signature” is 4 BIG classes and 4 small classes 1. comparisons help establish relative (order to eachother) 2. Genes are arranged in a length of a chromosome gene positions line along the B. Three Point Crosses: A faster more accurate method to map genes • 1. Analyzing the results of a three point cross Look at two genes at a time and compare to parental (NCO) 1. Analyzing the results of a three point cross 2. Interference: The number of double crossovers may be less than expected • Sometimes the number of observable double crossovers is less than expected if the two exchanges are independent • Occurrence of one crossover reduces likelihood that another crossover will occur in adjacent parts of the chromosome • Chromosomal interference – crossovers do NOT occur independently • Interference is not uniform among chromosomes or even within a chromosome Coefficient of coincidence = ratio between actual frequency of DCO and expected frequency of DCO • Interference = 1 – coefficient of coincidence • • If interference = 0, observed and expected frequencies are equal • If interference = 1, no double crossovers can occur Measuring interference 3. Double recombinants indicate order of three genes Summary of three point cross analysis • • • Parental class – most frequent • Double crossovers – least frequent • • • Cross a true breeding mutant with a true breeding wild­type Analyze F2 individuals (males if sex­linked) C. Do Genetic and Physical maps • • Determine order of genes based on parentals and recombinants Determine genetic distance between each pair of recombinants Calculate coefficient of coincidence and interference Order of genes is correctly predicted by using physical maps Limitations: Distance between genes is NOT always equal to physical maps • Double, triple, and more crossovers • Only 50% recombination frequency observable in a cross • Variation across chromosome in rate of recombination correspond? • D. Genes chained together by linkage relationships are known as linkage groups Model organisms for understanding the mechanism of recombination because all four haploid products of meiosis are contained in ascus • Ascospores within ascus germinate into haploid individuals • • Saccharaomyces cerevisiae – bakers yeast • Neurospora crassa – bread mold Mapping functions compensate for inaccuracies, but not often imprecise E. Tetrad analysis in fungi Saccharaomyces cerevisiae life cycle 1. Tetrads can be characterized by the number and recombinant spores they contain Neurospora crassa life cycle of parental 2. When PD=NPD, two genes are unlinked 2. When PD=NPD, two genes are unlinked (cont’d.) 3. Linkage is demonstrated by PDs outnumbering NPDs How crossovers between linked genes generate different tetrads • Calculating recombination frequency RF= NPD + ½ T x 100 RF= Total Tetrads We will be using the formual = all SCO + 2(DCO) x 100 TOTAL 5. Confirmation that recombination occurs at the four­strand stage 6. Tetrad analysis demonstrates that recombination is reciprocal • In a cross between strains with different alleles at two genes, each tetrad contains two of each parental type and two of each type of recombinant 7. Ordered tetrads allow mapping a gene in relation to the centromere Segregation patterns in ordered asci Segregation patterns in ordered tetrads Mitotic recombination can produce genetic mosaics Mitotic recombination is rare Initiated by • • • mistakes in chromosome replication • Curt Stern – observed “twin spot” in Drosophila – a form of genetic mosaicism • chance exposure to radiation Mitotic crossing over between sn and centromere in Drosophila • Animals contained tissues with different genotypes on the same body Twin spots in Drosophila How the molecule of heredity carries, replicates, and recombines information • • • • • DNA 1869: Miescher investigated the chemical composition of the nucleus Extracted a weakly acidic, phosphorous rich material from nuclei of human white blood cells He called it “nuclein” We call it DNA (deoxyribonucleic acid) today 4 subunits belonging to class of compounds called nucleotides linked together by phosphodiester bonds • The chemical composition of DNA • Chromosomes are composed of DNA • only 4 different subunits make up DNA • Chromosomes contain less DNA than protein by weight • • 20 different subunits – greater potential variety of combinations • Chromosomes contain more protein than DNA by weight DNA Are genes composed of DNA or protein? Protein B. Bacterial transformation implicates the substance of genes • • • • DNA as Rough strain (R) was harmless • Smooth strain (S) was pathogenic 1828 – Griffith Attempting to develop vaccine for pneumonia Isolated 2 strains of Streptococcus pneumoniae Griffith experiment Griffith experiment 1. Transformation • • What happened in the 4th experiment? The harmless R cells had been transformed by material from the dead S cells • Descendents of the transformed cells were also pathogenic • 1944: Avery, MacLeod, & McCarty – determined that the DNA is the transformation material Avery, MacLeod, McCarty experiment So exactly what was the transforming material? Cell extracts treated with protein digesting enzymes could still transform bacteria • Cell extracts treated with DNA: digesting enzymes lost their transforming ability • • • Concluded that DNA, not protein, transforms bacteria Avery, MacLeod, McCarty experiment C. Hershey and Chase experiments • 1952 – Hershey & Chase provide convincing evidence that DNA is genetic material • Waring blender experiment using T2 bacteriophage and bacteria • Created labeled bacteriophage • Radioactive sulfur (S^ ) • Radioactive phosphorous (P^32) labeled DNA • • Allowed labeled viruses to infect bacteria Asked: where are the radioactive labels after Viruses that infect bacteria Bacteriophages • • Consist of protein coat & DNA core • • Inject their hereditary material into bacteria Experiment examined what was transforming part, protein or DNA? Hershey and Chase Waring blender experiment II. The Watson­Crick Model: DNA is helix • • Hershey and Chase Waring blender experiment Hershey & Chase Results a double 1957: Watson learns about X­ray diffraction pattern projected by DNA Knowledge of chemical structure of nucleotides (deoxyribose sugar, phosphate, & nitrogenous base) Chargaff’s experiments demonstrated that ratio of A & T are 1:1, G & C are 1:1 1953 – Watson & Crick propose their double helix model of DNA structure • • A.DNA’s chemical constituents A.DNA’s chemical constituents DNA’s chemical constituents Chargaff’s ratios X­ray diffraction patterns produced by DNA fibers – Rosalind Franklin and Maurice Wilkins Complementary base pairing by formation of hydrogen bonds explain Chargaff’s ratios B..DNA structure B • • DNA is a double helix Strands are anti­parallel (5’à3’ & 3’à5’) w/ a sugar phosphate backbone on outside and pairs of bases in the middle • 2 strands wrap around each other every 30 Angstroms once every 10 base pairs • 2 chains are held together by hydrogen bonds between A­T & G­C base pairs • Structurally purines (A&G) pair best with pyrimadines (T&C) Thus A pairs with T and G pairs with C also explaining Chargraff’s ratios • C. Double helix may assume alternative forms D.Four requirements for DNA to be genetic material • • • • • Cracking the genetic code • DNA replication • Mutation Must carry information Must replicate Must allow for information to change Must govern the expression of the phenotype • Gene function C.. Some viruses use RNA as the repository of C genetic information IV. DNA replication: Copying genetic information for transmission to the next generation • Complementary base pairing produces semi­conservative replication • Double Helix unwinds • Each strand acts as template • Complementary base pairing ensures that T signals addition of A on new strand, and G signals addition of C on new strand • Two daughter helices produced after replication Experimental proof of semi­conservative replication – three possible models • • Semi­conservative replication – Watson and Crick model Conservative mode: The parental double helix remains intact; both strands of the daughter double helix are newly synthesized Dispersive replication: At completion, both strands of both double helices contain both original and newly synthesized material…(lots of error possible) • Meselson­Stahl experiments confirm semi­ conservative replication The mechanism of DNA replication • Kornbuerg, a nobel prize winner and other biochemists deduced steps of replication • Initiation • Proteins bind to DNA and pen up double helix • Prepare DNA for complementary base pairing • Elongation • Proteins connect the correct sequences of nucleotides into a continuous new stand of DNA • • • • • • Pol III – produces new strands of complementary DNA – proofreading DNA strand for mistakes Pol I DNA helicase – unwinds double helix Single­stranded binding proteins – keep helix open Enzymes involved in replication Primase – creates (lays down) RNA primers to initiate synthesis Ligase – welds together Okazaki fragments Replication forks move in opposite directions In linear chromosomes, the presence of telomeres ensures the maintenance and accurate replication of chromosome ends • In circular chromosome, such as E.Coli, there is only one origin of replication • • Replication is bidirectional • A problem with circular chromosomes, unwinding and replication causes supercoiling, which may impede replication • Solution: Topoisomerase: enzyme that relaxes supercoild by nicking strands The bidirectional replication of a circular chromosome Cells must ensure accuracy of genetic information • • Basis for repair of errors that occur during replication or during Redundancy storage • • V. Recombination reshuffles the information content of DNA • Enzymes repair chemical damage to DNA Errors that occur during the replication process are RARE During the recombination events, DNA molecules break up and then rejoin Meselson and Weigle – Experimental evidence from viral DNA and radioactive isotopes Co­infected E.Coli with light and heavy strains of virus after allowing time for recombination Separated the DNA genome on a CsCl density gradient • • • Meselson and Weigle demonstrate recombination occurs by breakage and rejoining of DNA Hetero­duplexes mark the spot of recombination Hetero­duplexes mark the spot of recombination • Products of recombination are always in exact register; not a single base pair is lost or gained Two strands do not break and rejoin at the same location; often they are hundreds of base pairs apart Region between break points is called hetero­duplex • • Homologs physically break, exchange parts, and rejoin Breakage and repair create reciprocal products of recombination Recombination events can occur anywhere along the DNA molecule (it does occur in some places more than others) • Precision in the exchange prevents mutations from occurring during the process • Gene conversion can give rise to unequal yield of two different alleles. 50% of gene conversions are associated with crossing over of adjacent chromosomal regions, and 50% of gene conversions are not associated with crossing over • • • Double stranded break model of meiotic recombination Heteroduplex region Double stranded break formation Resection spoI protein breaks one chromatid on both strands 5’ end on each side of break are degraded to produce two 3’ single stranded tails First strand invasion RecA binds 3’ tail and double helix allowing invasion and migration Formation of Holliday junctions Formation of Holliday junctions Branch migration New DNA synthesis forms two X structures called Holliday junctions Both invading strands zip up and migrate while newly created heteroduplex molecules rewind behind. Interlocked non­sister chromatids disengage. Two resolutions are possible The Holliday intermediate Alternative resolutions Endonuclease cuts Holliday intermediate ...
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This note was uploaded on 02/10/2011 for the course ANTH 2015 taught by Professor Sills during the Spring '08 term at LSU.

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