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Week 6 notes

by: Emma Notetaker

Week 6 notes CELL 2050

Emma Notetaker
GPA 3.975

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week 6 of notes
Dr. Meadows
Class Notes
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This 8 page Class Notes was uploaded by Emma Notetaker on Thursday October 6, 2016. The Class Notes belongs to CELL 2050 at Tulane University taught by Dr. Meadows in Fall 2016. Since its upload, it has received 2 views. For similar materials see Genetics in CELL at Tulane University.

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Date Created: 10/06/16
Friday, September 30, 2016 Week 6 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 • 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) 1 Friday, September 30, 2016 • 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 • 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 2 Friday, September 30, 2016 • 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 • 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 3 Friday, September 30, 2016 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) 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”) 4 Friday, September 30, 2016 • 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 • 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 • • 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 5 Friday, September 30, 2016 • 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 • • 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 6 Friday, September 30, 2016 • 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 • • 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: 7 Friday, September 30, 2016 • 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 8


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