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Genetics Study Guide Exam 2

by: Emma Notetaker

Genetics Study Guide Exam 2 CELL 2050

Marketplace > Tulane University > CELL > CELL 2050 > Genetics Study Guide Exam 2
Emma Notetaker
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Comprehensive study guide including book and lecture notes.
Dr. Meadows
Study Guide
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This 48 page Study Guide was uploaded by Emma Notetaker on Sunday October 16, 2016. The Study Guide belongs to CELL 2050 at Tulane University taught by Dr. Meadows in Fall 2016. Since its upload, it has received 48 views. For similar materials see Genetics in CELL at Tulane University.


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Date Created: 10/16/16
Wednesday, October 12, 2016 Exam 2 Chapter 8 - Chromosome Variation • chromosome morphology: position of centromere • submetacentric - almost in the middle • metacentric - in the middle telocentric - on the end • • acrocentric - almost on the end • karyotyping: • chromosomes prepared from actively dividing cells • halted in metaphase • chromosomes arranged according to size banding: different stains • • G bands: Giesma stain, rich in adenine-thymine (A-T) base pairs • Q bands: quinacrine mustard • C bands: centromeric heterochromatin • R bands: rich in cytosine-guanine base pairs • 1. rearrangements duplication: segment of chromosome is duplicated • • caused by unequal crossover • types • tandem: duplicated chromosome segment is adjacent to original • displaced: duplicated segment is some distance from original (can be on same chromosome or different - NOT the same as translocation because there is MORE DNA) • reverse: sequence of duplicated is inverted relative to sequence • effects: • in alignment during meiosis, duplicated region (mutation) must loop out so that homologous sequences can align • ex: gene duplication in drosophila causes eye to become smaller, bar shaped eyes - as the mutation develops, eyes get smaller and smaller • example of unequal crossover: red and green opsin genes • chromosomes do not align properly (red gene matched with green gene), which results in unequal crossover (2 chromosomes misalign) • one resulting chromosome has 2 green opsins, and the other chromosome has no green opsins if the male offspring inherits this X chromosome, he will not have any green opsin • and will be red-green colorblind • unbalanced gene dosage: altered protein levels (zebrafish example) • development processes often require the interaction of many genes • development may be effected by relative amount of protein product • duplication and other mutations may produce extra copies of some (not all) genes this alters relative amounts of interactive products • • if amount of one product increases but others don’t, developmental problems can occur • deletion: loss of a chromosomal segment • caused by unequal crossover 1 Wednesday, October 12, 2016 • large deletions easily detected - during pairing, normal chromosome loops out • in prophase I, the normal chromosome loops out in order for the homologous sequences of the chromosomes to align • effects: • imbalances in gene product - loss of protein product • expression of normally recessive gene (pseudodominance) if the dominant gene has been deleted in heterozygote • haploinsufficiency: single gene is not enough to produce wild type (even though one wild type is present, still get mutant) • inversion: segment of the chromosome is turned 180 degrees • caused by breaks in DNA and crossover • depends on involvement of the centromere • paracentric: centromere not included • pericentric: includes centromere (more problematic) • inversions in meiosis: • homozygous inversion: no problems during meiosis (haven’t changed amount of DNA, so can go through process okay) • heterozygous paracentric: • homologous sequences align only if there is an inversion loop • reduced recombination (gametes form nonviable offspring) • inversion loop forms in prophase 1 with a single crossover within inverted region • results in one chromatid with 2 centromeres (dicentric) and one without (acentric) • centromeres separated in anaphase 1, which breaks dicentric and degrades eccentric • now 2 gametes have nonrecombinant chromosomes (one wild and one with inversion), 2 have nonviable recombinants (missing genes) • heterozygous pericentric • inversion loop forms in prophase I • if crossing over occurs in inverted region, 2 of the chromatids have too many copies of some genes and none of others • in anaphase 1 these separate to form 4 gametes • 1 normal nonrecombinant, 2 nonviable recombinant and 1 nonrecombinant with pericentric inversion • abnormal gametes formed in pericentric inversion • translocation: segment of one chromosome moves from one to non homologous chromosome OR to another place on the same chromosome • nonreciprocal: genetic material moves from one to another without any exchange • reciprocal: 2 way exchange of segments between chromosomes • Robertsonian translocation: • short arm of one acrocentric chromosome is exchanged with long arm of the other • this creases a large metacentric chromosome with a small fragment that fails to segregate (usually lost) • essentially end up losing a whole chromosome • meiosis: • because each chromosome has sections that are homologous to 2 other chromosomes, often form a cross-like configuration in prophase 1 (because homologous segments in odd places) 2 Wednesday, October 12, 2016 • can separate in different ways during anaphase I: • alternate segregation (diagonal cut) - this creates viable gametes • adjacent-1 segregation: horizontal cut - nonviable gametes (some genes present in 2 copies and some not present) • adjacent 2-segregation: vertical cut - nonviable gametes (some genes present in 2 copies and some not present) • evolution: translocation event in which 2 long arms of great apes joined to form large metacentric chromosome (reason why we have fewer chromosomes, but our’s are larger) • 2. aneuploidy: change in number of individual chromosomes • causes: • deletion of centromere during mitosis and meiosis • Robertsonian translocation (lose a chromosome) • nondisjunction during mitosis and meiosis (loss and gain of chromosomes) • types: • nullisomy: loss of both members of a homologous pair (2n - 2) • monosomy: loss of single chromosome (2n-1) • trisomy: gain of single chromosome (2n+1) • tetrasomy: gain of 2 homologous chromosomes (2n+2) • nondisjunction in meiosis I: • both homologous chromosomes go into one cell, other cell gets neither • ends up with 2 trisomic gametes (2 from one parent, 1 from other) and 2 monosomic gametes (0 from one gamete, 1 from the other) • nondisjunction in meiosis II: • fail to separate equally into the 4 daughters • after fertilization, end with 1 trisomic, 1 monosomic and 2 normal diploid daughter cells • nondisjunction in mitosis: • ends with one monosomic cell and one trisomic cell • after cell proliferation, there are many of these cels • ex: diploid organism has 2n = 36 chromosomes. How many chromosomes in trisomic member? 36 + 1 —> 37 • plants: Jimson weed have many trisomic phenotypic types • humans: sex chromosome aneuploids • Turner syndrome: XO • Klinefelter syndrome: XXY • humans: autosomal aneuploids: • Trisomy 21: Down syndrome • primary (most common): 75% random nondisjunction in egg formation • familial: Robertsonian translocation between chromosomes 14 and 21 • carriers: long arm of 21 with short arm of 14 —> subsequent 14/21 short arm is lost • —> 2/3 of offspring will be healthy (1/2 of these are carriers - SO 1/3 of offspring are carriers) • —> when this is inherited by child (1/3 probability), that’s when Down syndrome will occur • other chromosomal recombinations result in aborted embryos • LOOK AT CHART SLIDE 35 • Trisomy 18: Edward syndrome • Trisomy 13: Patau syndrome • Trisomy 8 3 Wednesday, October 12, 2016 • uniparental disomy: both chromosomes inherited from the same parent • originates as a trisomy, but one chromosome is lost early in development and the remaining 2 chromosomes are from one parent • older mothers are more likely to give birth to a child with Down syndrome • 3. polyploidy: change in number of chromosome sets • autopolyploidy: all chromosome sets from a single species • via nondisjunction • mitosis: no cell division occurs to make autotetraploid (4n) - all chromosomes were copied but no separation occurred • meiosis: nondisjunction in meiosis I produces 2n gamete which fuses with 1n to make autotriploid (3n) • autotriploid during meiosis I: multiple possibilities starting with 3n cell • 2 homologous chromosomes pair while the other segregates randomly • all 3 pair and segregate randomly • none pair and all 3 go into same cell • some resulting gametes have extra chromosomes, some have none • —> this results in unbalanced gametes, which lead to sterile or nonviable offspring • allopolyploidy: chromosome sets from 2+ species - hybridization • hybridization between 2 diploid species produces hybrid with 6 non homologous chromosomes that don’t pair and segregate properly (still diploid because same number of chromosomes and lives via asexual mitosis • —> this results in unbalanced and nonviable gametes • nondisjunction leads to doubling of all chromosomes, producing an allotetraploid (2n = 12) • functionally diploid (because everything has one and only one homologous partner) • can still produce viable gametes due to normal chromosome pairing and segregation • example: species A has 2n = 16 chromosomes and species B has 2n = 14. How many chromosomes would be found in an allotriploid of these 2 species? • triploid is 3n • by definition, allopolyploid must have genomes from 2+ species • allotriploid could have 1n from A and 2n from B (1*8) + (2*7) = 22 • OR 2n from A and 1n from B (2*8) + (1*7) = 23 • significance: • increase in cell size • larger plant attributes - genetically engineered to be larger • evolution: may give rise to new species • table 8.3 • chromosomal instability is a general feature of cancer cells • deletions, inversions, translocations • ex: reciprocal translocation between chromosome 9 and 22 causes chronic myelogenous leukemia • potential cancer causing agent leads to a more active protein —> leads to unregulated cell division • ex: Burkitt lymphoma: potential cancer causing agent expressed in B-cells Bacterial and Viral Genetics • advantages of using bacteria/viruses for genetic studies 4 Wednesday, October 12, 2016 • rapid reproduction • many progeny • haploid genome allows all mutations to be expressed directly • asexual reproduction simplifies isolation of genetically pure strains • growth in lab is easy, little space needed • genomes are small and around 90% DNA encodes proteins • techniques are available for isolating and manipulating their genes • medical importance • can be genetically engineered to produce substances of commercial value • all prokaryotes are unicellular and lack membrane bound nucleus/organelles • eubacteria • archaea - extremes • characteristics of bacteria • diverse shapes and sizes • some photosynthetic • replication prior to binary fission • techniques for study of bacteria • prototrophic bacteria: wild type • grow on minimum medium - can produce everything else they need • auxotrophic: mutant (lacks 1 or more enzymes essential for synthesizing essential molecules) • require complete medium: contains all substances required by all bacteria including auxotrophic • can’t grow on minimum medium • bacterial growth in lab • sterile liquid medium (minimum or complete) incubated with bacteria so it grows and divides • shake medium with bacteria to mix • take growth culture and grow it on gelatin-like agar plates (can contain different growth factors and amino acids) • add dilute solution of bacteria and growth factor to petri dish • spread bacterial solution evenly • incubate for 1-2 days —> genetically identical cells in colony - this leads to pure colonies of bacteria • ex: mutant bacteria may be isolated based on nutritional requirements (trying to find colonies that don’t use leucine as amino acid) • plate bacteria on medium containing leucine • both leu+ and leu- colonies grow • replica plate the colonies by pressing velvet surface to plate • cells adhere to velvet • press onto new petri plates - cells from each colony transferred onto new ones • leucine auxotrophs (leu-) are recovered from the colony on supplemented medium and cultured for future study • medium with leucine has both leu+ and leu- growing • medium without leucine has only leu+ bacteria growing (because these produce their own leucine) • —> colony growing only on the supplemented medium has a mutation in the gene that encodes the synthesis of essential nutrient • bacterial genome: 5 Wednesday, October 12, 2016 • single, circular DNA molecule/chromosome • plasmids: extra chromosome • small, circular DNA • replicate INDEPENDENT of chromosome replication • can have many copies, many types • usually not essential for function but can play roles in important processes in life cycle and cell growth • replication • begins at ori site(origin of replication) • strands separate and replication occurs in both directions • eventually produces 2 circular DNA molecules • with new and old strands • replicate independent of chromosome replication - OWN sets of DNA • episomes: freely replicating plasmids • some genes regulate plasmid transfer to other cells • other sequences regulate insertion into bacteria chromosome • some genes control plasmid replication • F factor (fertility): controls mating and genetic exchange between bacteria • has OWN set of DNA • conjugation: direct transfer of DNA from one bacterium to another • direct transfer via connection tube • one-way traffic from donor cells to recipient cells • NOT reciprocal exchange of genetic info • cytoplasmic bridge forms between donor and recipient cell • DNA replicates and transfers from one cell to the other - replicating itself while doing this • usually whole chromosome not replicated, which leads to crossover events and creation of recombinant chromosome (rest of DNA is degraded) • Lederberg and Tatum experiment • does bacteria exchange genetic info? • 2 bacteria strains - 1 cannot synthesize 3 necessary nutrients, the other cannot synthesize a separate 3 • —> SO neither can grow on minimal medium on their own • when strains mixed and plated, they can grow because of the genetic exchange • if mutations were responsible, some colonies would have been able to grow on the first plates - must have been crossover • similar experiment in U tube separated by filter that allows mix of medium but not bacteria • nothing happened - must have been interaction between actual bacteria • Fertility (F) factor: contains own ori and other genes for conjugation (such as sex pili) • F factor required in order to exchange information • F+ cells: donors with F factor • one DNA strand of the F factor is nicked at the origin and separates (begins replicating to replace the nicked part) • goes into recipient cell where is begins regulating • F- cells: recipient cells without F factor • F+ cell will give plasmid to these, makes them become F+ • conjugation can take place ONLY between a cell that possesses F and a cell that lacks F • sex pili: connection tubes (cytoplasmic bridges) • Hfr cells: high frequency strains: donor cells with F factor integrated into the bacterial chromosome 6 Wednesday, October 12, 2016 in F+ cells • • crossover between F factor and chromosome - F becomes integrated • transfer from Hfr cell to F- cell • F is nicked and the 5’ end moves into the F- cell (containing F factor and some bacterial genes) transferred strand replicates and crossing over takes place between donated Hfr • chromosome and original chromosome of F- • may lead to recombination of alleles and linear bacteria degraded • STILL usually end with F- cell, but end with recombinant that may be able to grow on minimal medium − + • the F cell almost never becomes F or Hfr because the F factor is nicked in the middle in the initiation of strand transfer, placing part of F at the beginning and part at the end of the strand to be transferred. • F’ cells: contain F plasmids carrying some bacterial genes inside it formed by crossing over within Hfr - when F factor leaves, may carry some bacterial • genes • during conjugation, F factor with gene is transferred to F- cell • produces partial diploid with 2 copies of gene • merozygotes: partial diploid bacterial cells with F plasmid carrying some bacterial genes Type F factor characteristics Role in Conjugation F+ present as separate circular DNA donor F- absent recipient Hfr present, integrated into bacterial high frequency donor chromosome F' present as separate, circular donor DNA with some bacterial genes integrated Conjugating cell types present after conjugation F+ x F- 2 F+ cells (f- becomes F+) Hfr x F- one Hfr cell and one F- (no change usually) F’ x F- 2 F’ cells (F- becomes F’) mapping bacterial genes with interrupted conjugation • • distance between genes measured by the time required for DNA transfer from Hfr cells to F- cells • interrupt conjugation at set time intervals • experiment: • Hfr cell: has F factor with regular threonine and leucine, but missing strep resistant gene • F- cell: with strep resistance gene (some missing), missing some threonine and leucine • grown together, add streptomycin to media which kills off all Hfr cells • F- cells die bc don’t have thr and leu • ONLY survivor is f- cell that got the required genes (thr/leu) and the strep resistant genes 7 Wednesday, October 12, 2016 • transfer times indicate the order and relative distances between genes and can be used to construct a genetic map • **basic unit of distance is 1 minute • direction of gene transfer: • different Hfr strains of a given species of bacteria have the F factor integrated into the bacterial chromosome and in different orientations • transfer always starts with F; orientation of F determines the direction of transfer • **although the starting point and direction of transfer may differ between two strains, the relative distance in time between any two pairs of genes is constant!!! • natural gene transfer and antibiotic resistance • resistance comes from genes located on R plasmids that can be transferred naturally • R plasmids have evolved in the past 60 years since antibiotics • transfer of R plasmid not restricted to bacteria of the same/related species • transformation: bacterium takes up free DNA from the medium • recombination takes pace between introduced genes and bacterial chromosome • can start from DNA fragments • competent cells: have ability to take up DNA • transformants: cells that receive genetic material • cotransformed: cells transformed by 2+ genes • process: • one strand of double-stranded DNA fragment enters cell, other is hydrolyzed • single stranded fragment pairs with chromosome and recombination takes place • remainder of single strand is degraded • when cell replicates and divides, one resulting cell is transformed and other is not • mapping via transformation • DNA from donor is fragmented and fragments taken up by recipient • when cell replicates, one of the resulting cells is transformed and other is not • after entering cell, donor DNA becomes incorporated into chromosome through crossing over • closer = more likely to be on same DNA fragment and be recombined together • **rate of cotransformation inversely proportional to the distance between genes • linked gene principles still apply: • if two genes are close together on the same fragment, any two crossovers are likely to take place on either side of the two genes, allowing both to become part of the recipient chromosome. • if the two genes are far apart, there may be one crossover between them, allowing one gene but not the other to recombine with the bacterial chromosome. • **higher rate of cotransformation, closer the genes are** • example: DNA from a bacterial strain with his- leu- thr- is transformed with DNA from his+ leu+ thr+. Some leu+ thr+ cells and some his+ thr+ cells found, but NO his+ leu+ cells. Which genes farthest apart? — his and leu • bacterial genome sequences • horizontal gene transfer: DNA exchange between different species by nonproductive mechanisms • virus: replicating structure • DNA or RNA, single or double, linear/circular, ALWAYS with protein coat • bacteriophage: virus infection in bacteria • virulent phages: • reproduce through lytic cycle - ALWAYS kill host 8 Wednesday, October 12, 2016 • phage binds to bacterium and its DNA enters host, hijacks cell • host DNA digested, phage DNA replicated • host cell transcribes and translates phage DNA which makes new phage proteins • phage-encoded enzyme causes cell to lyse, new phages now escape to go infect others • temperate phages: inactive prophage - phage DNA integrates into bacteria chromosome • lysogenic cycle • phage binds to bacterium and DNA enters cell • phage DNA integrates into bacterial chromosome to become a prophage • prophage replicates (maybe for many cell divisions) • prophage may separate someday to enter lytic cycle • transduction: bacterial viruses (bacteriophage) transfers DNA from one bacterium to another • phage latches on and injects DNA • phage replicates, taking up bacteria DNA • bacterial cell lyses and now virus infects a new bacterium carrying bacterial DNA from the other one • results in combination of DNA (some phage DNA combines with bacteria’s) • usually between bacteria of the same or closely related species • makes bacteriophage with new genetic makeup - when cell lyses, phages contain other bacteria’s DNA and can infect a new cell • phage no longer will lyse new cells after integrating the new DNA • generalized: any gene may be transferred • bacteria infected with phage and chromosome fragmented • some bacterial genes incorporated into phages • cell lysis releases transducing phages • phage can transfer bacterium to another bacterium, recombination may take place to make a transducer bacterial cell • can map genes: • donor cell infected with phage - lysis leads to phage with good portions of DNA • infects cells lacking the DNA portions • produces single transductants, cotransductants and nontransductant • rate of cotransduction is inversely proportional to distance between genes • limited size of a phage particle results in only ~1% of the bacterial chromosome can be transduced (fairly rare event). • only genes located close together on the bacterial chromosome will be transferred together, or cotransduced • specialized: only a few genes are transferred • experiment: 2 strains separated in U-tube and actually got growth (due to phage) • gene mapping in phages experiment: how to determine position of gene on phage chromosome? • infect bacteria with 2 different strains of T2 phage (r-h+ and r+h-) • r+: small, clear plaques, r-: large, cloudy plaques • h+: infects type B E. coli, h-: only infects type B/2 • makes nonrecombinant and recombinant (due to crossing over) • some nonrecombinant, some clear and large, some cloudy and small • progeny plates on 2 types of E. coli cells, count cells • % recombination allows for gene mapping 9 Wednesday, October 12, 2016 • retrovirus: RNA viruses that have been integrated into host genome • reverse transcriptase: synthesizing DNA from RNA • protein coat encapsulates single stranded RNA • viral envelope glycoprotein allows viruses to attach to/ infect cells • core-shell proteins • RNA is single stranded • all have 3 genes in common: • gag – viral protein coat • pol – reverse transcriptase and integrase • env – viral envelope glycoproteins • process: • virus attaches to host cell at receptors in membrane • viral core enters host • viral RNA uses reverse transcriptase to make complementary DNA and viral RNA degrades • reverse transcriptase synthesizes 2nd DNA strand • viral DNA enters the nucleus and is integrates into the host chromosome —> forms provirus • on activation, proviral DNA transcribes viral RNA which is exported into cytoplasm • viral RNA translated in cytoplasm • viral RNA, proteins, new capsids and envelopes are assembled • assembled virus buds from cell membrane and is released • ex: HIV-1: attacks helper T cells or T lymphocytes (needed for immune function) • HIV’s reverse transcriptase is very error prone resulting in high mutation rates - vaccines hard to make • flu: rapid changes occur through genetic recombination (new strains) • 3 types: influenza A, B, C • mostly A: divided into subtypes based on HA and NA surface of virus • exchange of strains make new ones • simultaneous infection of different strains causes exchange of genetic material from different strains Chemical Nature of the Gene • genetic material must contain complex info - stores lots of information must replicate faithfully - DNA copied at each division • • must encode phenotype - instructions in DNA • must be able to vary - species and individuals have different genetics • early studies: KNOW people and experiments • Mendel 1866 • Miescher: nuclein (DNA + protein), later named nucleic acid (1869) Kossel: DNA contains 4 nitrogenous bases (late 1800s) • • Griffith demonstrates transforming principle (1928) • Avery, MacLeod and MCCarty demonstrate that transforming principle is DNA (1944) • Chargaff’s rules about regularity in base ratios of DNA (1948) • Watson and Crick devise structure (1953) • Griffith: discovery of the transforming principle 10 Wednesday, October 12, 2016 • can an extract from dead bacterial cells genetically transform living cells? • type IIIS virulent bacteria injected into mouse —> mouse dies, virulent bacteria recovered • type IIR nonvirulent bacteria injected into mouse —> mouse lives, no bacteria recovered • heat-killed type IIIS virulent bacteria injected into mouse —> mouse lives, no bacteria recovered • mixture of IIIR and heat-killed type IIIS injected —> mouse dies —> type IIIS virulent bacteria is recovered (mouse acquired genetic virulence) • conclusion: substance in heat-killed virulent bacteria genetically transformed type IIIR bacteria into live, virulent type IIIS • Avery, MacLeod and McCarty: identification of the transforming principle • what is the chemical nature of transforming substance? • heat-killed virulent bacteria, homogenized and filtered • samples treated with different enzymes • RNase kills RNA • protease destroys proteins • DNase destroys DNA • the treated samples were added to cultures of IIR • cultures with protease have transformed IIIS bacteria • cultures with RNase have transformed IIIS bacteria • culture treated with DNAse didn’t have IIIS! • —> the transforming substance is DNA • identification of the viral transforming principle • T2 bacteriophage attaches to E. coli and injects chromosomes • bacterial chromosome breaks down and the phage chromosome replicates • expression of phage genes produce phage structural components • progeny phage particles assemble • bacterial wall lyses, releasing progeny phages • Hershey-Chase experiment • which part of phage serves as genetic material? DNA or protein? • E. coli infected, in 2 conditions: • one in medium with 35S (radioactive form of sulfur) • taken up by phage protein, now containing S but not P • one with 32P (radioactive phosphorus) • taken up by phage DNA, now containing P but not S • phages with S or P infect unlabeled E. coli, shear off protein coat in blender and separate protein from cells via centrifuge • results • S recovered in the fluid with the virus coats - NO radioactivity found • —> protein has not been transmitted to progeny • P form pellets in the bottom of the tube - progeny phages are radioactive, indicating that DNA was transmitter • —> DNA is genetic material in bacteriophages (not protein) • Chargaff’s Rule • base composition % of DNA from different sources and ratios of bases • A and T in 1:1 ratio, as are C and G • William Astbury and Rosalind Franklin discovered the diffraction pattern of DNA separately • x-ray diffraction revealed DNA structure • crystals of substance are bombarded with xrays, which are diffracted • spacing of atoms within crystal determined pattern 11 Wednesday, October 12, 2016 • Watson and Crick’s discovery of 3D structure (chemistry was already worked out • built models with all the current information (using Rosalind Franklin’s diffraction) • phosphate + sugar + base = nucleotide • DNA consists of two strands of nucleotides • run in opposite directions (are antiparallel) • wind around each other to form a right-handed helix • sugars and phosphates on the outside, bases in the interior • RNA as genetic info • in most organisms DNA carries genetic info but in some, RNA carries it • Fraenkel-Conrat and Singer experiment: Tobacco mosaic virus • Type A and B tobacco mosaic virus (TMV) degraded to yield RNA and coat proteins • RNA of one type missed with protein of the other to make hybrid (reciprocal) • tobacco infected with hybrids • type of RNA in hybrid parent TMV determined RNA and protein of progeny • —> RNA is the genetic material of TMV • primary structure of DNA • 3 parts: sugar, phosphate, base • 5 carbon sugars • ribose: OH group attached at 2’ carbon (makes it more unstable) • deoxyribose H group attached at 2’ carbon • purines: adenine and guanine • pyramidines: cytosine, thymine (DNA), uracil (RNA) • nucleoside: deoxyribose/ribose sugar + base • nucleotide when the phosphate group is added to 5’ carbon • nucleotides contain phosphate group with negative charge (makes DNA acidic) • bases always attached to 1’ carbon via covalent bonds (share electrons) • phosphate attached at 5’ carbon • secondary structure of DNA • double helix • polynucleotide strand: string of nucleotides • backbone formed through phosphodiester bonds between phosphate and sugars • covalent bonds • phosphodiester linkage connects 5’ phosphate group and 3’ OH group of adjoining nucleotides via covalent bonds • unattached phosphate at 5’ end of sugar • free OH group attached to 3’ carbon atom of the sugar • DNA runs in 5’ —> 3’ direction • **nucleotides can only be added at the 3’ end • hydrogen bonds between nucleotides - non-covalent • T-A pairs have 2 hydrogen bonds • C-G pairs have 3 (stronger interaction than between T and A) • antiparallel, complementary DNA strands • antiparallel refers to the act that they run in opposite directions • 10 base pairs per 360 degree rotation • 3.4 nanometers between major grooves • .34 nm between each base pair • B-DNA (right handed) is the predominate form of DNA • minor grooves and major grooves alternate • these are important for protein binding to DNA 12 Wednesday, October 12, 2016 • secondary structures of DNA: may play important roles in the functions of RNA • B form: right-handed • A form: shorter and wider • Z form: left handed (very rare) • secondary structures of RNA (form more often because single-stranded) • hairpin structure: forms a stem loop • occurs when sequences of nucleotides on the same strand are inverted complements • stem structure: complementary sequences are contiguous • complex RNA: multiple hairpins, stems, etc. • special structure in RNA • single-stranded • form same phosphodiester bonds between 5’ phosphate and 3’ hydroxyl group • in RNA, uracil replaces thymine • RNA has ribose sugar (OH at 2’ carbon) • phosphodiester linkage connects 5’ phosphate with 3’ OH of adjoining nucleotides • special structured DNA: • H-DNA: 3 stranded (triplex) formed when DNA unwinds and one strand pairs with double0stranded DNA from another part • often in long sequences of only purines or only pyrimidines • common in mammalian genomes • DNA methylation: • methyl groups added to nucleotide bases • related to gene expression in eukaryotes • affects 3D structure Chromosome Structure and Organelle DNA • DNA must be tightly packed to fit in small spaces - tertiary structure • usually around 10 bases per turn/rotation • supercoiling: makes DNA take up less space, allows loops to form relaxed state: ~10 bases per turn/rotation • • hard to pack into small cell in this state • positive supercoiling: DNA is overrotated and the helix twists on itself • negative supercoiling: DNA is underrotated - helix twists on itself in the opposite direction • topoisomerases: enzymes responsible for adding and removing turns in the coil • ex: DNA molecule 300bp long has 20 rotations. if relaxed, there would be 30 rotations • • because it has 20 (which is <30) molecule is negatively supercoiled • bacterial chromosomes have single circular DNA molecule • bacteria and eukaryotic DNA fold into loops stabilized by proteins which prevents free rotation of the ends • negative supercoiling takes place within loops advantages: • • allows easier access to DNA during replication and transcription • can be packed into higher spaces • chromatin: DNA complexed with protein (2 types - euchromatin and heterochromatin) 13 Wednesday, October 12, 2016 Characteristic Euchromatin Heterochromatin Condensation less condensed more condensed (DNA very tight) location on chromosome arms at centromere, telomeres and other specific places type of sequences unique sequences (many repeated sequences (only if proteins made in these regions) permanent) presence of genes many genes few genes (only if permanent) when replicated throughout S phase late S phase transcription often infrequent (DNA cannot be accessed bc so heavily condensed) crossing over common uncommon • histones: major protein component of chromatin • small (+) proteins (due to abundance of positive amino acids) • • (+) charges attract (-) on the phosphates of DNA and hold DNA in place • 5 major types: H1, H2A, H2B, H3 and H4 • **H1 not part of nucleosome but plays important role of locking DNA in place • neutralizing histone’s positive charges would cause the histone proteins to separate from the DNA • this would cause changes in transcription because DNA is now more accessible to transcription factors • chromatin structure: • nucleosome: DNA complexed with histones • 8 histone proteins (2 copies each of H2A, H2B, H3 and H4 in protein complex) • DNA wrapped around each protein complex 1.65 times • linker DNA is what connects each histone (between the “beads”) • 200 base pairs wrap around the nucleosome, linker DNA is about 45 base pairs if small amount of nuclease added, cleaves “string” between core histones of • nucleosomes • releases individual “beads” • larger nuclease destroys unprotected DNA, leaving a core of proteins attached to 145-147 bp of DNA DNase can only chop up exposed DNA (if wrapped around histones, will be safe) • • each histone has a (+) tail that interacts with the (-) charges on phosphates of the DNA, keeping the DNA and histones tightly associated • histone H1 (one per nucleosome) s not part of the nucleosome but still plays a role in locking the DNA in place • **if less of a histone, it’s likely not inside the core • chromatosomes: nucleosome + H1 histone • high order chromatin structure • nucleosomes fold up with H1 to produce 30 nm fiber fibers form loops around 300 nm long • 14 Wednesday, October 12, 2016 • fibers compressed and folded to make 250 nm wide fiber • tight coiling of fibers makes chromatid of chromosome • changes in structure • polytene chromosome puffs • sites of intense transcription • made by repeated rounds of DNA replication with no cell division • localized swelling where chromatin has a related structure = active transcription • so big because it needs to allow transcription factors • DNase I sensitivity: correlates with gene activity • question: is chromatin structure altered during transcription? • use embryonic globin gene (made early in development) and adult globin genes aD and aA • before hemoglobin synthesis, none of the globin genes are sensitive to DNase I digestion (likely because wrapped around histone) • after globin synthesis has begin, all genes are sensitive to DNase I but the embryonic global gene U is the most sensitive • because this area has been unbound for transcription • in the 14 day old, when only adult hemoglobin is expressed, adult genes are most sensitive and the embryonic gene is insensitive • now, adult genes are being transcribed so the DNase can get in and chop it up • globin genes in the brain (doesn’t produce globin) remain insensitive throughout development (control group) • —> sensitivity of DNA to digestion by DNase I is correlated with gene expression • suggests that the chromatin structure changes during transcription • more susceptible during activity (not as tightly wrapped around histone • epigenetic changes: methylation - capable of being reversed and often due to environmental changes • centromere structure: constricted region of a chromosome (heterochromatin) where spindle fibers attach • critical in mitosis/meiosis • chromosome break makes 2 types of fragments (one with centromere and one without) • if no centromere, cannot attach to spindle fibers and will be lost from nucleus • telomere structure: ends of chromosome • stabilize and provide a means to replicate the ends of linear chromosomes • sequences here can be repeated 1000s of times • special proteins bind to the G-rich single-stranded sequence (3’ overhang - one strand longer than the other) • protects the telomere from degradation • protects ends of chromosomes from sticking together • T-loops can also prevent degradation • eukaryotic DNA contains several classes of sequence variation • organisms differ in amount of DNA per cell (C value) • cannot be explained by differences in organismal complexity (corn has higher C value than humans) • denaturation - melting temp • separation of DNA double strands • the more H bonds, the higher the melting temp 15 Wednesday, October 12, 2016 • ex: more energy to separate CG pairs (3 H bonds) than AT pairs (2 H bonds) • renaturation: hydrogen bonds will form again between complementary base pairs, producing double-stranded DNA • denaturation of DNA by heating is reversible • types of DNA sequences in eukaryotes • unique sequence DNA (seen 1-2% in the genome - many proteins) • repetitive DNA • moderately repetitive: 150-300 bp long • tandem repeat sequences (one after another) • interspersed repeat sequences: short (SINEs: Alu element) or long (LINEs (1000-2000 bp long) • highly repetitive: less than 10 bp long • microsatellite DNA • organelle DNA has unique characteristics • mitochondria and chloroplasts both have their own DNA • encodes some polypeptides used by the organelle, rRNA and some tRNA • endosymbiotic theory: mitochondria and chloroplasts were once free-living bacteria (advantageous because they make ATP) • anaerobic eukaryotic cell engulfed aerobic eubacteria through endocytosis • aerobic part inside evolved into mitochondria • similar process in chloroplasts (endocytosis of photosynthesizing eubacterium) • evidence: • modern single-celled eukaryotes (protists) are hosts to endosymbiotic bacteria • mitochondria and chloroplasts are similar in size to present-day eubacteria and possess their own DNA • antibiotics that inhibit protein synthesis in eubacteria but do not affect protein synthesis in eukaryotic cells also inhibit protein synthesis in these organelles. • sequences in mtDNA and cpDNA are more closely related to sequences in the genes of eubacteria than they are to those found in the eukaryotic nucleus. • both organelles similar to eubacteria and DNA within them similar too • uniparental inheritance of organelle-encoded traits • animal mtDNA inherited mainly from female • replicative segregation: • each cell has 100s-1000s of organelles • heteroplasmic cells: organelles segregate randomly • 2 types of DNA contained in organelles, which segregate randomly in cell division • these then replicate and segregate randomly again….again…etc. • ex: myoclonic epilepsy and ragged red fiber syndrome (MERRF): • muscle twitches, epilepsy • completely linked to mutation in mitochondrial DNA • 20-year-old person who carried this mutation in 85% of his mtDNAs displayed a normal phenotype • cousin who had the mutation in 96% of his mtDNAs was severely affected • traits encoded by mtDNA • petite mutations in yeast - slow growth, defective ATP production • neurospora (fungus) mutation - slow growth, defective ATP production • human diseases: MERRF, LHON, NARP, KSS, CEOP • in plants, cytoplasmic male sterility • mitochondrial genomes are very small and vary greatly between species 16 Wednesday, October 12, 2016 • human mtDNA: • circular • 16569 base pairs • encodes 2 rRNAs (ribosomal), 22 tRNAs (transfer), and 13 proteins • highly economical in its organization • few noncoding nucleotides between genes • almost all mRNA codes for proteins • yeast mtDNA: • 5x size of humans • 2 rRNA, 25 tRNA, 16 polypeptides • lots of noncoding sequences • flowering plant mtDNA • extensive size variation • can loop • part of the extensive size variation can be explained by the presence of long sequences that are direct repeats • evolution of mitochondrial DNA • high mutation rate in vertebrate mtDNA • number of genes and organization remains relatively constant (even though there is a high mutation rate) • ideal for reconstructing patterns of evolution in humans and other organisms • small size and abundance • rapid evolution of mtDNA sequences in some organisms facilitates the study of closely related groups • maternal inheritance of mtDNA and lack of recombination makes it possible to trace female lines of descent • abundance of mtDNA, thousands of copies per cell • damage to mitochondrial DNA associated with aging • many human genetic diseases associated with mtDNA appear in middle age or later • oxidative phosphorylation capacity (ATP synthesis) declines with age • those with mutations in mtDNA start life with decreased oxidative phosphorylation capacity • mechanism of age-related mtDNA damage unknown • chloroplast genome: • many traits associated with chloroplasts exhibit cytoplasmic inheritance (not nuclear genes) • shown to have their own DNA in 1963 • sequence very similar to DNA found in cyanobacteria • through time, genetic info has moved between nuclear mitochondrial and chloroplast genomes • many proteins found in modern mitochondria and chloroplasts are now encoded by nuclear genes • more stable as nuclear DNA? • fewer mutations? • sequences of nuclear genes that encode organelle proteins are most similar to eubacterial counterparts 17 Wednesday, October 12, 2016 DNA Replication and Recombination • genetic information must be accurately copied each time cell divides • one error/million bp —> 6400 mistakes every time a cell divides replication also takes place at high speeds, very efficiently • • E. coli replicates 100 nucleotides/second • proposed DNA replication models • conservative • both strands of original DNA serve as template strands, which gives rise to completely new helix • the parental helix is conserved throughout the whole process • dispersive • different bits of the whole molecule are used as templates • each new strand has choppy mixture • semiconservative: actual model - all DNA goes through this • one strand of the parent used for one new cell, the other strand used for the other • after second replication, 2 completely new helices Meselson and Stahl’s experiment: 2 isotopes of nitrogen • 14 15 • N common, N rare and heavy form • E. Coli were grown first in 15N media, then transferred to 14N • cultured E. Coli cells were subjected to equilibrium density gradient centrifugation • spun in centrifuge for days 15 14 • density gradient develops within tube ( N moves to bottom, N moves to top) • which model of DNA replication applies to E. coli? • DNA from bacteria from 15N appeared as single, heavy band ( N original parent) • DNA transfered to normal nitrogen: 14N • after one round of replication, DNA appeared as single band at intermediate weight • is it were conservative, there would’ve been a heavy band and a light band • after 2nd round, DNA was 2 bands - one light and the other intermediate • intermediate made up of original and newly synthezied light ones are completely new ones • • IF this were dispersive, it would all have been intermediate band throughout all rounds • samples taken after additional rounds continued to appear in 2 bands • lighter band becomes thicker because there are more and more completely new ones Repplcaatono DNA template Breakage of number of unidirectional/ products Moodell nucleotide replicons bidirectional strand Theta circular no either 2 circular 1 molecules Rolling circle circular yes 1 uni one circular molecule and one linear molecule that may circularize 18 Wednesday, October 12, 2016 Replication DNA template Breakage of number of unidirectional/ products Model nucleotide replicons bidirectional strand linear linear no many bi 2 linear molecules • modes of replication replicons: units of replication • • replication origin (one in bacteria, many in eukaryotes) • theta replication: • usually in bacteria • circular DNA, E. Coli • single origin of replication - makes single stranded templates for new DNA synthesis • double-stranded DNA unwinds at replication origin • bubble forms with a replication fork at each end • usually bidirectional replication • at each fork, synthesis of leading strand proceeds continuously in the same direction as unwinding synthesis of lagging strand is discontinuous in opposite direction • • leading and lagging strands in circular formation • products: 2 circular DNA molecules • rolling circle replication • viruses • F factor of E. coli • single origin of replication • replication initiated by break in one of the nucleotide strands • DNA synthesis begins at 3’ end of broken strand • inner is used as template • 5’ end of broken strand is displaced cleavage or circle releases single stranded linear DNA and double-stranded circular • DNA • linear DNA may circularize and serve as a template for synthesis of complementary strand • —> product: multiple circular DNA models • continuous DNA synthesis begins at 3’ end of broken strand • as the DNA molecule unwinds, the 5’ end is progressively displaced • all continuous • linear eukaryotic replication • eukaryotic cells • 1000s of origins at each origin, DNA unwinds to produce replication bubble • • synthesis takes place on both strands at each end of the bubble as forks proceed outward and fuse • typical replicon: ~200,000 - 300,000 bp • produces 2 linear DNA models • requirements of linear eukaryotic replication • template strand • raw material: nucleotides • enzymes and other proteins 19 Wednesday, October 12, 2016 • synthesis of new DNA: • new DNA synthesized from dNTPs (deoxyribonucleoside triphosphates) • 3’-OH group attacks 5’ - phosphate group of the incoming dNTP • 2 phosphates cleaved off • phosphodiester bond forms between 2 nucleotides • direction of replication: • DNA polymerase: enzymes that synthesize DNA • adds nucleotides only to 3’ end of growing strand • replication from 5’ —> 3’ • because the strands are antiparallel, DNA synthesis occurs in different directions on each strand • continuous replication: synthesis in the same direction as unwinding • on lower strand, DNA synthesis proceeds continuously in the 5-3 direction (same direction as unwinding) • when lower runs out of template, this one starts again • leading strand • discontinuous replication: synthesis in the OPPOSITE direction of the unwinding • on upper template strand, DNA synthesis begins at the fork and proceeds in the direction opposite that of unwinding (soon runs out of template) • has to restart replication multiple times - gaps form • short fragments of DNA produces by discontinuous synthesis are called Okazaki fragments • lagging strand • result of antiparallel nucleotide strands • bacterial DNA replication • initiation: • 245 bp in oriC (single origin replicon) • initiator proteins (DnaA) recognize oriC, and bind there • causes a short stretch of DNA to unwind • this allows helices and other single-stranded binding proteins to attach to single stranded DNA • unwinding: • initiator protein • DNA helicase binds to lagging strand template at each fork and moves in 5’-3’ direction • breaks hydrogen bonds and moves replication fork • this separates the DNA • single strand binding proteins (SSBs) • bind to single strand and stabilize exposed DNA • DNA gyrase (topoisomerase) • relieves strain ahead of the fork • elongation: • starts with synthesis of primers • primase synthesizes short stretches of RNA nucleotides • makes a 3’ OH group so DNA polymerase can add DNA nucleotides • on leading strand, primer required only at 5’ end of new strand • because replication is continuous • on lagging strand, new primer needed at the beginning of each Okazaki fragment • because replication discontinuous • primers: existing group of RNA nucleotides with 3’-OH group 20 Wednesday, October 12, 2016 new nucleotide can be added here • • usually 10-12 nucleotides long • DNA polymerase is the primase • removal of RNA primer: DNA polymerase I • nicks connected after primers are removed by DNA ligase DNA polymerase I: removes RNA primer • • 5’-3’ polymerase and 3’-5’ exonuclease activities • ALSO has 5’-3’ exonuclease activity • removes RNA primers and replaces them with DNA nucleotides by synthesizing in 5’-3’ direction • DNA ligase seals the nick • DNA polymerase III: bacterial elongation • multi-protein complex, large • main workhorse of replication 5’-3’ activity adds nucleotides in 5’-3’ direction • • 3’-5’ exonuclease activity allows it to remove nucleotides in 3’-5’ direction • this enables it to correct errors • backs up to removes excess nucleotides, then resumes 5’-3’ activity)\ • DNA ligase: connects nicks after RNA primers are removed seals gap with phosphodiester bond between 5’-P group of the initial nucleotide and 3’-OH • group of the final nucleotide • catalyzes the formation of phosphodiester bond without adding another nucleotide to strand • fidelity of replication: error rate < 1 mistake per billion nucleotides • proofreading • DNA polymerase I • 3’-5’ exonuclease activity removes incorrect pairs • mismatch repair: corrects errors after replication requires ability to distinguish old and new strands of DNA (enzymes need to determine • which of the wrong pairs to remove) • termination: replication fork meets termination protein Commpponnent Function initiator protein binds to origin and separates strands of DNA to start replication DNA helicase unwinds DNA at replication fork single-stranded binding proteins attach to sing stranded DNA and prevents secondary structures from forming DNA gyrase moves ahead of replication fork, making and resealing breaks in the double helical DNA to release torque that builds up DNA primase sunthesizes a short RNA primer to provide 3’-OH group provided by primer DNA polymerase III elongates new nucleotide strand from 3’ OH group provided by primer 21 Wednesday, October 12, 2016 Component Function DNA polymerase I removes RNA primers and replaces them with DNA DNA ligase joins Okazaki fragments • eukaryotic DNA replication • autonomously replicating sequences (ARSs) • 100-120 bps (ie origin of replication) • origin-recognition complex (ORC) • bind to ARSs to initiate DNA replication • replication licensing factor: approval of DNA replication • MCM: minichromosome maintenance - binds DNA and initiates replication as a helices on all origins eukaryotic DNA polymerase • • DNA polymerases in eukaryotic cells DNA polymerase 5’—> 3’ nuclease 3’-5’ exonuclease cell function activity activity alpha yes no initiation of n


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