PCB3063: Exam One
PCB3063: Exam One PCB3603
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This 26 page Study Guide was uploaded by Brittany Woody on Thursday September 22, 2016. The Study Guide belongs to PCB3603 at University of Florida taught by Dr. W. Brad Barbazuk in Fall 2016. Since its upload, it has received 83 views. For similar materials see Genetics in Genetics at University of Florida.
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Date Created: 09/22/16
Exam One Study Guide Chapter 1 - Genetics: maintenance and transfer of information - Each child is a hybrid of two parents plus mutations - .1% (1/1000) genetic differences between each person - Genetics allows for sexual, artiﬁcial, or other types of selection - Transmission genetics: how genes are transmitted from parents to offspring - Galapagos Islands: 19 volcanic islands distributed over about 140 miles; environment varies from island to island; Darwin recognized that wildlife was related to but distinct from that on the mainland; each island has its own distinctive species of animals and plants - Epigenesis: theory by William Harvey; states that an organism develops from the fertilized egg by a succession of developmental events that eventually transform the egg into an adult - Preformation: stated that the fertilized egg contained a complete miniature adult, called a homunculus - Cell theory stated that all organisms are basic structural units called cells, which are derived from preexisting cells - Darwin’s Finches: • at least 15 species, but clearly related based on physical structures • the beaks of the ﬁnches are different and appropriate for habitat on it’s island; all birds evolved from one parent species on mainland South America - What are underlying causes of the morphological differences in beaks between islands in Darwin’s ﬁnches? What is different in genes between ﬁnch species? • Sequence variation among individuals in a population - What biological processes can produce beaks of such different shapes, and make sure that this shape will be based on to the next generation? Variation exists within (or near) genes that affect beak morphology. Ampliﬁcation of • one morphology over another within a population could be the result selection of one morphology due to improved ﬁtness - In 2015, ﬁnch’s genomes were sequenced; associated genetic differences with beak morphology (phenotype) - Align genomes of each ﬁnch with a bird whose genome is sequenced; look for differences between each ﬁnch and the benchmark bird, and between each ﬁnch (base differences: A vs. C vs. T vs. G) - Associated presences of a base or series of bases (allele) with particular phenotypes - ALX1: this gene is expressed in craniofacial region; mutations cause facial defects in mammals; slight differences result in beak differences in ﬁnches but not drastic differences or defects Chapter 2 - 2 copies of every chromosome in every cell; any more or less will result in malfunction - Charyotype: map (picture) of each chromosome set; shows each pair of chromosomes; a set of chromosomes are nearly identical - Long string of DNA is condensed into compact chromosomes during duplication - Each chromosome has centromere where you see “pinch”; mostly made up of repetitive sequences; position of centromere helps identify chromosome number - “big arm” of chromosome is Q and “small arm” is P - Sister chromatids are pair of identical chromosomes that are created in the beginning of duplication - Each copy of a chromosome is a homologue of the other copy; not exactly the same - Cell cycle: Interphase (G1, S, G2, G0) and Mitosis (Prophase, Metaphase, Anaphase, Telophase) - Genes are duplicated during S phase and sisters are connected, condensed during mitosis - Mitosis: (somatic cells, not gametes-sex cells) one 2n cell divides into two 2n cells • Interphase: two copies of every chromosome; chromosomes are extended and uncoiled, forming chromatin Prophase: chromosomes coil and condense, breakdown of nuclear membrane, • centrioles divide and move to opposite poles, microtubules from centrioles attach to centromere - Prometaphase: chromosomes are clearly double structures; centrioles reach the opposite poles; spindle ﬁbers form • Metaphase: centromeres align at midline (metaphase plate) • Anaphase: centromeres split and daughter chromosomes migrate to opposite poles • Telophase: daughter chromosomes arrive at the poles; cytokinesis (division of cytoplasm) - Meiosis: (replication of gametes: sperm and eggs) one 2n cell divides into four 1n cells • Reduces genetic material by half, each gamete is haploid; each gamete carries one member of each homologous chromosome pair; meiosis results in unique combinations of maternally and paternally derived chromosomes within the haploid complement of the gamete; “crossing- over” results in exchange of genetic material between each member of a homologous chromosome pair, mix of mom’s and dad’s chromosomes • One 2n cell results in four 1n cell in meiosis - 1n: one copy of each chromosome, 2n: two copies of each chromosome - Mitosis: one replication; Meiosis: two replications - Mitosis results in two identical sets of DNA; during meiosis, each pair of chromosomes synapse (process is called synapsis, chromosomes connect) to form a tetrad (two copies of mom’s chromosome, two copies of dad’s chromosome), and crossing over takes place; results in recombinant DNA - Meiosis I and II both have prophase, metaphase, anaphase, telophase - Prophase I has ﬁve substages • leptonema: chromomere formation and homology search begins • zygonema: initial alignment of homologs (rough pairing), homologous chromosomes have been replicated, formation of the synaptonemal complex • pachynema: chromosomes condense further • diplonema: each pair of sister chromatids begin to separate; chiasmata are apparent • diakinesis: two controllers of each tetrad attach to the spindle ﬁbers - Primary spermatocyte undergoes meiosis I to produce two secondary spermatocytes, which undergo meiosis II to produces a total of four haploid spermatids - During oogenesis (egg duplication), the four daughter cells do receive equal cytoplasm; polar bodies do not undergo further division; primary oocyte splits cytoplasm three times but is unequal, so most cytoplasm is in 1 cell - Eukaryotic DNA is wrapped around nucleosomes (which are made up of proteins called histones); the DNA+ protein complex is called chromatin - Overall charge of DNA is negative; histones are positively charged; histone and DNA associate electrostatically Spermatogenesis and Oogenesis Mitosis vs. Meiosis One Chapter 3 - Mendel did research on pea plants; many varieties (heritable features, character variants; ﬂower color) - Features of a good organism for experimental genetics: controlled mating, fast generation time (for many data points), fecundity - Character choice: either/or (not somewhere in the middle, called true breeding plants), avoid traits that varied on a continuum, true breeding varieties - Parental generation is “P generation”; their offspring is F1, ﬁrst ﬁlial generation; offspring of F1 is F2, second ﬁlial generation - Mendel observed the same pattern of inheritance on six other pea plant characters, each represented by two traits; gave same outcome as ﬂowers; one feature is dominant, one is recessive - F1 only shows dominant trait; F2 shows ratio 3 dominant: 1 recessive - Reciprocal cross: done with both pollination of one phenotype by the other, and vice versa (not always using dominant trait holder as male in pollination, for example); results will not be sex-dependent - What Mendel called a “heritable factor”, we call a gene - Mendel proposed three postulates: (Independent Assortment) • Unit factors exist in pairs; diploid, one from mom and one from dad • In the pair of unit factors for a single characteristic in an individual, one unit is dominant and one is recessive • The paired unit factors segregate independently during gamete formation - Mendel’s segregation model accounts of the 3:1 ratio he observed in the F2 generation - Parent generation allele is WW for purple, ww for white; F1 is Ww; F2 is 25% WW, 25% ww, 50% Ww • WW and Ww appear with dominant trait (purple ﬂowers), 75% • ww appear with recessive trait (white ﬂowers), 25% • 75% : 25% = 3:1 ratio - Two of the same alleles (WW or ww) is homozygous; Two different alleles (Ww) is heterozygous - Phenotype: physical characteristic; Genotype: genetic code • Phenotype: 3 purple: 1 white • Genotype: 1 WW: 2 Ww: 1ww - Test cross: to ﬁgure out the genotype of a purple ﬂower (WW or Ww), cross it with a white ﬂower (homozygous recessive ww); if the purple ﬂower is WW, all offspring will have dominant trait and will be purple; if the purple ﬂower is Ww, half of offspring will be white (ww) and half will be purple (Ww) - Selﬁng: self-fertilization of individuals from the ﬁrst generation - Dihybrid cross: uses two traits (seed color and seed shape); if you cross one round yellow pea (dominant, dominant) with a wrinkled green pea (recessive, recessive), the result of pea colors would be 3 yellow: 1 green; the resulting pea shapes would be 3 round: 1 wrinkled; this proves that the traits are independent, they are on different chromosomes, different loci (singular is locus) GW###Gw###gW###gw GW Gw gW gw - Phenotypic ratio with both traits is 9 yellow round: 3 yellow wrinkled : 3 green round: 1 green wrinkled • 9 both dominant traits: 3 one dominant trait and one recessive trait; 3 one recessive trait and one dominant trait: 1 both recessive traits - The law of independent assortment states that each pair of alleys segregate independently of each other pair of alleles during gamete formation - GGWw: one parent must be GW, the other must be Gw - Trihybrid crosses involve three independent traits; Mendel’s laws can be applied to any number of traits; trihybrid cross Punnett square will have 64 cells - Parent generation: AABBCC x aabbcc; F1 generation will all be AaBbCc - F2 generation: parents are AaBbCc x AaBbCc • AA probability= .25; Bb probability= .5; CC probability= .25 - AABbCC probability= .25 x .25 x .5= 1/32 - 1/4 x 1/4 x 1/2 = 1/32 - What is the probability that an unaffected sibling of a brother or sister expressing a recessive disorder is a carrier (heterozygote)? (assume parents are heterozygote) • Probability that unaffected sibling is heterozygote= 1/2 • Probability that sibling is unaffected= 3/4 • (1/2) / (3/4) = 2/3 - Binomial theorem: used to determine the probability of a particular combination, rather than going through all possibilities - Chi-square test compares actual outcomes to “expected” outcomes; determines if results are close enough to expected result to be correct or if there is a “statistically signiﬁcant” difference is actual outcome compared to expectations - As sample size increases, the average deviation form expected fraction or ratio decreases; larger sample size reduces the impact of chance deviation on the ﬁnal outcome - The null hypothesis is the assumption that the data will ﬁt a given ratio, such as 3:1; assumes that there is no real difference between the measured values and the predicated values If rejected, the deviation from the expected is NOT due to chance alone and you • must reexamine your assumption (expected data) • If it is failed to be rejected, then observed deviations can be contributed to chance - When drawing a pedigree, the proband is the individual of interest; signiﬁed with a P; pedigree shows a family tree with respect to a given trait; pedigree analysis reveals patterns of inheritance - “p-value” of .05 is a commonly-accepted cut-off point - p>0.05 means that the probability is greater than 5% that the observed deviation is due to chance alone’ therefore the null hypothesis - p<0.05 means that the probability is less than 5% that observed deviation is due to chance alone; therefore null hypothesis is rejected; reassess assumptions, propose a new hypothesis - Identical twins are called monozygotic; fraternal are dizygotic - Related parents are called consanguineous; connected by a double line on a pedigree - Transposable elements have the ability to move from place to place in the genome of certain organisms - Tay-Sachs disease (TSD): recessive disorder involving unalterable destruction of the central nervous system; results from loss of activity of a single enzyme hexosaminidase A (Hex-A) Chapter 4 - Modiﬁcations of the classic 3:1 or 9:3:3:1 ratios usually result from alleles that do not adhere to simple dominance/recessive, when more than one pair of genes inﬂuences a single character, or when traits in questions are linked to the X-chromosome - Allele is an alternate form of a gene (e.g. one form responsible for white ﬂowers, another for purple) - Alleles responsible for the phenotype most often in a population (what we arbitrarily designate as ‘normal’) is the ‘wild-type’ allele; wild type alleles are often, but not always dominant (implies that most mutant alleles are recessive); mutant allele speciﬁes an altered gene product; mutation is the source of new alleles (for better or for worse) - Often a mutation causes a reduction or loss of the wild-type function. e.g. a mutation in a gene whose product is an enzyme may change conformation or active site – resulting in reduced or loss of catalytic activity. - Loss of function alleles have some function; null alleles have no function; neutral mutation has a nucleotide change that does not change amino acid sequence (i.e. UCU -> UCC both Serine). - Phenotypic traits may be inﬂuenced by more than one gene and the allelic forms of each gene involved (consider an enzymatic pathway culminating in the production of an important molecule e.g. amino acid, or pigment) - Dominant alleles are usually indicated either by: • an italic uppercase letter (i.e D vs d for WT vs. dwarf) • letters (Wr) (uppercase signiﬁes dominant form vs. wr for wrinkled) - Recessive alleles are usually indicated either by: • an italic lowercase letter (d) • an italic letter or group of letters with the + superscript (Wr ) (signiﬁes the wt wing vs. the dominant wrinkled wing) - If no dominance exists, italic uppercase letters and superscripts are used to denote 1 2 W R alternative alleles (R , R , C , C ) - Incomplete dominance (aka partial dominance): a cross between contrasting traits produces an intermediate phenotype; neither allele is dominant in incomplete (partial) dominance - Assume alleles are of a gene that makes a pigment, and white is null; only produces ½ the pigment of the homozygous wild type - Codominance: 2 alleles of a gene are responsible for producing 2 distinct and detectable gene products –> note the distinction between this case and incomplete dominance; this is a case of joint expression of 2 different alleles • e.g. MN blood group; carbohydrate antigen on the surface of blood cells – providing biochemical and immunological identity to individuals; 2 forms, N and M - Codominant inheritance is characterized by distinct expression of the gene product of both alleles . - There is a large amount of information stored within each gene, so there is no reason to assume that there are only 2 alleles per gene; simplest case of a multi-allele locus occurs when 3 alleles for a gene exist • classic and simple example: ABO blood groups; like the MN blood types, the ABO system is characterized by the presence of antigens on the surface of RBC; these are distinct from the MN antigen, and are under the control of a different locus • A and B antigens are carbohydrate groups; the I allele is responsible for an enzyme that can add the terminal sugar N-acetylgalactosamine (AcGalNH) to the H substance; the I allele is responsible for a modiﬁed enzyme that cannot add N- acetylgalactosamine but instead can add a terminal galactose; the O phenotype results from an absence of either terminal sugar GENOTYPE ANTIGEN PHENOTYPE A A I I A A I i A A B B I I B B I i B B I IB AB AB ii None O - Lethal mutations deﬁne essential genes; recessive lethal mutations usually have a loss of function mutation that creates a non-functional product; tolerated as a heterozygous where one allele will produce sufﬁcient amounts of gene product - In some cases that lethal allele will have a distinctive (though on lethal) phenotype as a heterozygote; such an allele is recessive lethal, but dominant with respect to the on lethal phenotype - Dominant lethal mutation: one copy of the allele is lethal; sometimes because one copy of the allele may not provide the critical threshold of product needed; or, the new mutation may override the function of the wild type allele; Huntington’s chorea and Progeria (1 in 4 million) - Combinations of two gene pairs involving two modes of inheritance modify the 9:3:3:1 ratio: • Mendel’s principle of independent assortment applies to situations in which two modes of inheritance occur simultaneously, provided that the genes controlling each character are not linked on the same chromosome. • The probability of each phenotype arising in a cross can be determined by the forked-line method or by Punnett square, assuming that the genes under consideration undergo independent assortment. - Many traits characterized by a distinct phenotype are affected by more than one gene; in gene interaction, the cellular function of numerous gene products contributes to the development of a common phenotype - Epistasis occurs when alleles at one gene locus masks the effect of another gene (homozygous recessive at locus 1 overrides expression at locus 2, or presence of a dominant allele at locus 1 overrides expression at locus 2); or when two gene pairs complement each other such that one dominant allele is required at each locus to express a certain phenotype • The Bombay phenotype for ABO blood groups is an example of epistasis in which the homozygous recessive condition at one locus masks the expression of a second locus; the alleles at the H locus are epistatic to the alleles at the AB blood group locus (AB alleles are hypostatic to H locus) • In each case, distinct phenotypic classes are produced – each obvious from one another; genes involved are on different chromosomes, and thus sort independently; if A exhibits complete dominance, we can represent the diploid as A-, where the ‘-’ implies that either allele (A or a) could be present without phenotypic consequence; all P1 crosses involve homozygous individuals - When studying a single characteristic, a ratio expressed in 16 (e.g., 3:6:3:4) parts suggests that epistasis is occurring - Complementation testing: a test to determine if 2 mutations with similar phenotypes are alleles of the same gene- or represent alleles of different genes - Expression of a single gene may have multiple effects; pleiotropy occurs when expression of a single gene has multiple phenotypic effects, and it is quite common; examples are Marfan syndrome and porphyria variegate - Genes present on the X chromosome exhibit unique patterns of inheritance due to the presence of only one X chromosome in males, referred to as hemizygosity (not homozygous or heterozygous) • Drosophila eye color was one of the ﬁrst examples of X-linkage described (Thomas H. Morgan); demonstrated that the inheritance pattern of the white-eye trait in Drosophila was clearly related to the sex of the parent carrying the mutant allele; concluded that the white locus is on the X chromosome - Sex-limited inheritance occurs in cases where the expression of a speciﬁc phenotype is absolutely limited to one sex; the genes are autosomal - In sex-inﬂuenced inheritance, the sex of an individual inﬂuences the expression of a phenotype that is not limited to one sex or the other; the genes are autosomal In sex-limited and sex-inﬂuences inheritance, expression of autosomal genes responsible for a certain phenotype depends on the hormone constitution of the individual Penetrance: percentage if individuals that show at least some degree of phenotype Expressivity: reﬂects range of expression of the mutant phenotype; often phenotypes range in severity despite identical genotypes - If genetic background is constant in breeding, variation must be environmental Modiﬁers of phenotype: genetic background and environmental effects; temperature sensitivity and suppression Position effect: physical location of a gene in relation to other genetic material may • inﬂuence it’s expression; can be seen if a region of a chromosome is relocated or rearranged such that it is now close to chromosomal regions that are prematurely condensed and “genetically inert,” referred to as heterochromatin - Mutations affected by temperature are called temperature-sensitive mutations, a type of conditional mutation - Nutritional mutations may prevent the phenotype from reﬂecting the genotype; phenylketonuria and lactose intolerance are human examples - Not all genetic traits become apparent at the same time during an organism life span; the age at which a mutant allele reveals a notable phenotype depends on events during the normal sequence of growth and development - As a result of genetic anticipation, some heritable disorders exhibit a progressively earlier age of onset with a increased severity in each generation (myotonic dystrophy) - In cases of genomic (parental) imprinting, phenotypic expression may depend on the parental origin of the chromosome; imprinting is thought to occur before or during gamete formation and may involve DNA methylation Chapter 5 - Un-linked genes will assort independently during meiosis - Gene loci that reside on the same chromosome are considered linked – and will co- segregate; assuming complete linkage, all gametes will be non-crossover or parental gametes - During the ﬁrst meiotic prophase a reciprocal exchange of genetic material can occur; this crossing-over phenomena occurs at the site of chiasmata; this process can result in the reshufﬂing or recombination of the alleles found on the two homologues; this results in creation of homologous chromosomes with allele combinations that differ from the parental homologs – this results in recombinant gametes - No recombination: two genes on a single pair of homologs; no exchange occurs - Recombination: two genes on a single pair of homologs; exchange occurs between two non sister chromosomes - Genes on the same chromosome are part of a linkage group; the number of linkage groups should correspond to the haploid number of chromosomes; the percentage of offspring resulting from recombinant gametes depends on the distance between the two genes on the chromosome - The ratio of phenotypes produced from a cross between two organisms heterozygous at linked loci is the linkage ratio - Recombination events will occur between many linked genes; based on work with ﬂies mutant for white eyes and yellow bodies, Morgan hypothesized: • crossing over uncouples alleles resulting in recombinant gametes • linked genes are arranged in linear sequence • variable frequency of exchange occurs between any two genes as a result of their distance - One map unit is deﬁned as 1 percent recombination between two genes on a chromosome - Map units are often called centimorgan (cM) and are relative distances, not exact ones - When a single crossover occurs between 2 non-sister chromatids, the other 2 chromatids of the tetrad are unchanged; the percentage of tetrads involved in a exchange between two genes is twice the percentage of recombinant gametes produced; even if a single crossover occurs in 100% of tetrads, only 50% of the gametes are recombinant - When two linked genes are more than 50 map units apart, a crossover theoretically can be expected to occur between them in 100 percent of the tetrads - Single crossovers can be used to determine the distance between two linked genes, but double crossovers (DCOs) can be used to determine the order of three genes on the chromosome; the expected frequency of double-crossover gametes is much lower than that of either single-crossover gamete class - Three-point mapping is a mapping cross that allows the mapping of 3 or more linked genes in a single cross; in a 3- point mapping, one parent (usually female) is heterozygous for all obi under consideration (these are the gametes we are considering) - The other parent (usually male) is homozygous recessive for all loci under consideration; this is much like a testcross • need to look at adequate number of progeny • need to make sure that genotype is implicit in phenotype; this is how we evaluate the outcome of every meiosis in the heterozygote - The non-crossover F2 phenotypes occur in the greatest proportion of offspring - The double-crossover phenotype occur in the smallest proportion - The distance between two genes in a three-point cross is equal to the percentage of all detectable exchanges occurring between them and includes all single and double crossovers - The expected frequency of multiple exchanges between two genes can be predicted from the distance between them; the coefﬁcient of coincidence (C) is the observed number of DCOs divided by the expected numbers of DCOs - Interference reduces the expected number of multiple crossovers when a crossover event in one region of the chromosome inhibits a second event near; if interference is complete, no DCO will occur - C is the coefﬁcient of coincidence= Observed DCOs/ Expected DCOs - Interference is quantiﬁed by I; I= 1- C • positive interference: fewer DCOs occurred than expected (I is greater than 0) • negative interference: more DCOs occurred than expected (I is less than 0) - As the distance between two genes increases, mapping experiments become less accurate; the discrepancy results primarily from multiple exchanges that are predicted to occur between the two genes but that are not recovered during experimental mapping - Elliptocytosis: shape of erythrocytes is an oval - Log of odds (lod) favoring linkage score analysis relies on probability calculations to demonstrate linkage between two genes in organisms in which linkage analysis relies primarily on pedigrees; LOD score accuracy is limited by the extent of the pedigree - Somatic cell hybridization involves fusion of two cells in culture to form a single hybrid cell, called a heterokaryon - Upon continued culturing of the hybrid cell, chromosomes from one of the two parental species are gradually lost until only a few chromosomes of one species remain and most chromosome are from the other species, creating what is termed a synkaryon - A panel of cell lines, each containing just a few human chromosomes, can be used for sentence testing in which the presence or absence of a speciﬁc gene product is correlated with the presence or absence of each chromosome - DNA markers and higher resolution cell lines (radiation hybrids) provide a means to very dense maps - Earliest example of DNA markers are restriction fragment length polymorphisms (RFLPs); also called micro satellites or short tandem repeats - Variations in a single nucleotide is called single-nucleotide polymorphisms (SNPs) - Crossing over involves physical exchange between chromatids; mapping in maize using cytological markers established that crossing over involves a physical exchange of chromosome regions - Recombination between mitotic chromosomes are rare, <1% - Sister chromatid exchanges (SCEs) occur during mitosis but do not produce new allelic combinations Chapter 6 - Bacteria grow rapidly; spontaneous mutation is considered the primary source of genetic variation in bacteria - Bacteria have 3 growth phases: lag phase (initial, slow), log phase (rapid with a ﬁxed time interval that results in exponential growth), stationary phase (nutrients limit growth) - Bacteria are grown in liquid culture or on a semi-solid surface (agar) - “minimal medium” contains an organic carbon source (glucose) and various inorganic ions (K+, Na+, Mg(2+), NH4+,Ca (2+)) - Growth on this medium requires the bacteria to synthesize all essential organic compounds; all bacteria that can do this are prototrophs; bacteria that lose the ability to synthesize one or more essential organic compound (through mutation) is an auxotroph - Bacteria can be counted by serial dilution; can establish populations of genetically identical bacteria by plating the appropriate dilution - Bacteria undergo conjugation, in which genetic information from one bacterium is transferred to another, and it recombines with the second bacterium’s DNA; this provides the basis for chromosome mapping methodology; the genetic recombination in bacteria (and phage) involves the replacement of one or more genes in one bacterial strain with those from a genetically distinct strain - Lederberg and Tatum experimented with multiples autotroph strains; it was highly improbable that any of the cells containing 2 or 3 mutant genes would undergo spontaneous mutation to become wild type cells, so they proposed genetic recombination had occurred - Conjugation: certain strains of bacteria were able to affect a unidirectional transfer of genetic material - In bacterial conjugation in E. coli, F+ cells serve as DNA donors and F- cells are the recipients - F+ cells contain a fertility factor (F factor) that confers the ability to donate DNA during conjugation - Recipient cells are converted to F+; this requires cell to cell contact - Physical contact is required for conjugation; conjugation is mediated by a sex pilus, which is a microscopic tubular extension of the cell; results in transfer of a mobile element- which is a small piece of circular DNA; recipient F- cells become F+ cells - F factor is a plasmid; it contains about 40 genes, most of which are involved in the transfer of genetic material - An Hfr (high-frequency recombination) strain undergoes recombination 1000x more frequently than the original F+ strains; Hfr has the F factor integrated; an Her strain can donate genetic information to an F- cell, but the recipient does not become F+ - In a given Hfr strain, certain genes are more frequently recombined than others (nonrandom) - Interrupted mating demonstrated that speciﬁc genes in an Her strain are transferred and recombined sooner than others - Time mapping: the observation of a time dependent, ordered transfer of genes suggests a metric for mapping; gene order and distance (in minutes) could be determined - Other Hfr strains also transferred genes linearly with time, but the order in which genes entered the recipient cell varied from strain to strain - Gene transfer by Hfr strains led to the understanding that the E. coli chromosome is circular - If the point of origin (O) varies between strains, then a different sequence of genes will be transferred in each case - The position of the F factor determines initial point of transfer; direction of transfer is determined by the orientation of the F factor; genes adjacent to O are transferred ﬁrst; F factor becomes the last part that can be transferred - Recipient cells are usually not converted to F+ because conjugation does not often last long enough to transfer the whole genome - In some cases, an F factor is excised from the chromosome of an Hfr strain; in the process, the F factor (referred to as F’) often brings several adjoining genes with it; transfer of an F’ to an F- cell results in a partially diploid cell called a merozygote - RecA protein plays an important role in recombination involving single-strand displacement - The RecBCD protein is important for unwinding a double-stranded DNA molecule that serves as the source for genetic recombination, RecA then facilities recombination - Plasmids contain one or more genes and replicate independently of the bacterial chromosome; F factors confer fertility - R plasmids confer antibiotic resistance; rtf is a resistance transfer factor, and r- determinants convey resistance - Col plasmids encode colicins that can kill neighboring bacteria - In transformation, small pieces if extracellular DNA are taken up by a living bacterial cell and integrated stably into the chromosome; once it is integrated into the chromosome, the recombinant region contains one host strand (present originally) and one mutant strand; because these strands are from different sources, this region is referred to as a heteroduplex; the two strands of DNA are not perfectly complementary in this region - Ideally exogenous DNA consists of 10-20K bp, this size is sufﬁcient to encode several genes - Genes that are close enough to each other to be cotransformed are linked - If two genes are unlinked, simultaneous transformation requires 2 distinct recombination events requiring two distinct segments of DNA - Bacteriophages can infect a host bacterium by injecting their DNA; this DNA serves as a template for the infected bacterium to produce progeny phage particles, which are released when the host cell is lysed (destroyed) - The number of phages produced following the ﬁction of bacteria can be determined by the plaque assay • this technique entails performing serial dilutions of virally infected bacteria, which are then poured onto agar plates • by counting the number of plaques (areas clear of bacteria) on the plates, the number of phages on the original culture can be determined • a single, small, well separated plaque was produced by a single phage infecting a bacteria; subsequent release and infection (lytic cycle) results in infection of neighboring bacteria, which eventually creates a “clearing” in the lawn - Lysogenyoccurs when: • the phage DNA integrates into the bacterial chromosome it is replicated along with the chromosome • it is passed to daughter cells • • Integrated phage is a prophage. - Bacteria containing a prophage are lysogenic and can grow and divide stably until viral reproduction is induced; viral reproduction is induced by chemical or uv treatment; phage that can either lyse the cell or become prophages are temperate phages - The Lederburg-Zinder experiment identiﬁed a ‘ﬁlterable agent’ as being responsible for recombination leading to prototrophs; only bacteria from LA-22 side produced prototrophs, not the LA-2 side (note the Davis U-tube prevents conjugation) - FA produced by L-22 only when grown in association with LA-22; grow L-22 on its own, then add to LA-22 – nothing is produced • Dnase has no effect • FA could not pass if pore size < phage size - In generalized transduction, bacterial DNA instead of phage DNA is packaged in a phage particle and transferred to a recipient host - In specialized transduction, a small piece of bacterial DNA is packaged along with the phage DNA - Generalized transduction results in the transfer of a large number of bacterial genes. - Specialized transduction results in transfer of only a few bacterial genes. - Like transformation, generalized transduction can be used in linkage and chromosomal mapping. - Genes that are close together are more often transduced simultaneously. - Most phage mutants affect plaque morphology or host range; the key to doing recombination studies in phage is to co-infect - When recombination occurs between genes it is “intergenic recombination” - Map units are determined as before # of recombinants / #total - Because we can examine so many events in phage recombination, we isolate really rare events – even intragenic recombination (within one gene); this allows you to map mutations within genes, and provides a means to ﬁne-structure map a locus - Benzer’s ﬁne structure analysis had ~20,000 mutants; initial control studies were confusing because some combinations of mutations lysed E. coli K12; Benzer reasoned that during co-infection each mutant strain provided something that the other lacked; what was happening is that some mutant combinations were ‘complementing’ each other – indicating different genes - Complementation testing put each mutation into 2 groups or “complementation groups”; this indicates two genes - the rII locus was actually made up of 2 genes - Complementation testing mapped the mutant collection to each of the two genes (he called these cistrons – his deﬁnition for the smallest functional genetic unit) - Then, he crossed A-cistron mutant to other A-cistron mutations, B-cistron mutations to B-cistron mutations. - Some of the r11 mutations were actually small deletions or various parts of each ‘cistron’; the collection of these provide an overlapping set of deletions – which are very valuable for rapid placement of mutations. - Deletion testing was used to provide a rough and quick localization of each mutation Benzer studied. - A point mutation is localized in the area of a deletion if it fails to give rise to any wild- type recombinants in complementation assays. Chapter 7 - Covered: 7.2, 7.3, 7.5, 7.6 (there are some extra slides on PowerPoint) other sections will not be tested on - XX/XO also known as Protenor mode of sex determination: butterﬂy example: females have 7 autosomes and two X chromosomes; males have only one X; depends on the random segregation of the X chromosomes into half of the male gametes during segregation (in humans, females have two Xs, male has XY) - C. Elegans have only 90 cells, so they are commonly used for genetic research; C. Elegans (nematode) also has XX/XO system; sex determination involves genes on both the X chromosomes and the autosomes; the ratio of X chromosomes to the number of sets of autosomes ultimately determines the sex - XX/XY also known as Lygaeus mode of sex determination: male passes on X or Y to determine sex (female is homogametic, has two X chromosomes); females gametes all have X chromosome zygotes with two X chromosomes are homogametous, females • zygotes with one X and one Y chromosome are heterogametous, males • - Female is not always homogametic; in some organisms the fame is the heterogametic sex; notation ZZ/ZW is used in these cases (instead of XY) - X and Y chromosomes are similar enough to pair, but recombination is unlikely - Presence of Y makes a human a male, not presence of two Xs - Klinefelter syndrome has 47 chromosomes; an extra X chromosome (XXY); mostly has male physiology due to presence of Y chromosome; some slight female characteristics but male genitalia • notation “47, XXY” means there are a total of 47 chromosomes and the defect is in the sex chromosomes which have the pattern XXY • can also be 48,XXXY; 48, XXYY; 49, XXXXY - Turnersyndrome has 45 chromosomes, only one X; (45, X) develop as females; female genitalia and some other characteristic oddities; some Turner syndrome individuals are mosaics, somatic cells display 2 different genetic cell lines - Triplo-X: 3 X chromosomes along with normal set of autosomes (47, XXX); frequently women are perfectly normal, sometimes have underdeveloped secondary sexual characteristics, sterility, or mental retardation - Tetra-X (48,XXXX) or penta-X (49,XXXXX) have been reported; in many cases the presence of an extra X chromosome disrupts the delicate balance of genetic information essential to normal female development; perhaps due to disruption in autosome to X chromosome ratio - 47,XYY: males are usually over six feet tall but otherwise normal - Two Xs are not necessary to be female, only the absence of a Y - Organism without an X cannot develop (with only Y) because many genes exist that are necessary for life are on the X chromosome - Nondisjunction during meiosis results in mutations in X and Y chromosome (incorrect number of either chromosome) - Y chromosome contains 75 genes, very small chromosome; some of these have homologous counterparts on the X chromosome, some do not - By the ﬁfth week of gestation, gonadal primordial (the tissues that will form the gonad) arise as a pair of gonadal (genital) ridges associated with each embryonic kidney; potentially hermaphroditic at this stage - Bipotential gonads: gonadal ridges that can form either ovaries or testes - PAR: pseudo-autosomal region; look like autosomes, possibly evolved from autosomes; pairing at PAR is crucial for proper segregation of X and Y meiosis in males; 95% of Y does not synapse with X - Male-Speciﬁc region of the Y (MSY): part of the chromosome other than the PAR - SRY: sex carrying region Y: located within the MSY; part of chromosome that are responsible for major shift toward male sexual development; rarely a male will have an X or two Xs but male genitalia, because they have SRY, but no other part of Y; a person with all of the Y chromosome except the SRY will have female genitalia - Testis-determining factor (TDF): protein encoded by SRY that causes the undifferentiated gonadal tissue of the embryo to form testes - Transgenic mice: produced from fertilized eggs injected with foreign DNA that is subsequently incorporated into the genetic composition of the developing embryo - Mullerian inhibition substance (MIS): also known as Mullerian inhibition hormone or MIH; cells of developing testes secrete MIS; causes regression of cells in Mullein duct; prevents formation of female reproductive tract - Palindrome: sequences of base pairs that read the same but in the opposite direction on complementary strands - Amplicon: piece of DNA that is source and/or product of natural ampliﬁcation or replication events - Dosage compensation: autosomes are present in equal numbers between cells in males and females; ratio of X chromosomes in females vs. males is 2:1 (female has 2 X chromosomes and male has 1); this creates a “genetic dosage” difference (females could produce twice the products of X linked genes vs. males • extra Xs compound this (XXY,XXX, etc); this may explain developmental disorder in these individuals • Dosage compensation refers to the balancing of genetic material from the X chromosome (so that women do not produce twice as much product as men); this could occur due to increase expression level of X linked genes in males, or decrease expression level of X linked genes in females - Barr bodies: densely staining DNA based object lying against nuclear membrane; shown to be an inactive X chromosome; present in female nuclei; having more X chromosomes results in more Barr bodies; each female shuts down one X chromosome • Dosage compensation is therefore accomplished by inactivation of one of the two X chromosomes, and genetic information that can be expressed in males and females is equivalent • Individuals with sex-chromosome syndromes might have afﬂictions despite dosage compensation because Xs are not inactivated during early stages of development; it is also possible that Xs are not entirely inactivated - Lyon hypothesis: inactivation of X chromosome occurs randomly early in development; Females inherit some X-linked disorders in a “mosaic,” normal function in some spots, impaired in others (sweat glands missing in only some parts of the body or color blindness in only some spots in the retina, for example); shows that maternal X is being expressed in some cells, and paternal X is being expressed in some cells mosaic: somatic cells display two different genetic cells lines, each exhibiting a • different karyotype - Calico cats have mosaic color patterned fur because some black-colored X chromosomes are inactive, and some tan-colored X chromosomes are inactive - Xic is X-chromosome inactivation center; a locus on the proximal end of the P arm; contains 4 genes and several regulatory units - Xist transcribes a non-coding RNA that encases X chromosome containing the actively transcribed Xist genes; encasement inactivates chromosome; the activated must have activation at the Xic locus blocked, possibly by methylation - Transgenes : genes that are artiﬁcially introduced into the organism - Metafemale: “super female”; ratio of autosomes to chromosomes exceeds 3X to 2A - Metamale: infertile; ratio XY: 3 autosomes - Intersex: sterile, express both male and female morphology - Recent studies suggest that maternal and paternal Xs must pair at the Xic loci; once two X chromosomes are linked at Xic using T6 gene, one is randomly shut down - Drosophila has a Y chromosome but it seems that the Y chromosome does not participate in sex determination; Calvin Bridges proposed that the autosomes and X chromosome collaborate on determining sex; Bridges worked in two phases: • study of offspring resulting from nondisjunction of the X chromosome during meiosis in females subsequent work with progeny of triploid (3n) female • - X non-disjunction: failure to disjoin the X during anaphase 1 (homologous pair) or 2 (sister chromatids), results in gametes that are 2n+1 or 2n-1; fertilization with a normal gamete can result in XXX,XXY,X0,0Y • XXX:super female • 0Y: fetus does not survive long after fertilization • XXY: normal female • X0: sterile female • These cases show that Y does not contain male-determining factors because Y does not cause male-ness, but there are genes related to development because X0 organisms are sterile - Triploid female (3n): three copies of each autosome and 3 Xs; result of a rare diploid egg being fertilized by normal sperm; during meiosis, 3n females produce a variety of chromosomal complements that are segregated into gametes; from these studies, Bridges was able to correlate sexual morphology with chromosome composition - Normal females have an autosome to X chromosome ratio of 1 (2 copies of each autosome and 2 X chromosomes); normal males have ratio of .5 because they have only one X chromosome - Bridges proposed that the threshold for maleness is X:A 1:2, and extra X (XX:2A) alters the balance and results in female; this suggests that the factors required for developing into a male are on the autosomes, X contains some female-determining factors; called genic balance theory - RNA splicing: portions of the RNA are removed and remaining fragments are “spliced” back together prior to translation into a protein - Similar to mammals, Drosophila female contains 2 copies of every X-linked gene relative to the male counterpart, which creates dosage problem; X inactivation is not observed, rather, male X linked genes are depressed at twice the level of the comparable genes in females - Sxl: an X-linked gene that induces female differentiation; Sxl controls 4 autosomal genes, mutations in any of these reduce the increased expression of genes on the male X, which is lethal - Sxl is inactive in XY, otherwise acts as a repressor in XX - Collectively, this cluster of gene-activating proteins is called the dosage compensation complex (DCC) - Bilateral gynadomorph: one-half of the body (left) has developed as a male and the other half (right) as a female
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