Genetics exam 1
what is Chromatin structure?
-The nucleosome -Chromatosome -Linker DNA -High-order chromatin structure 30 nm fiber 300 nm loops 250 nm wide fiber
Watson & Crick - (1953)
1 page paper in the British journal Nature "Molecular Structure of Nucleic Acids: A Structure for Deoxynucleic acids" 1962 won Nobel prize along with Maurice Wilkins
Replication in Prokaryotes
1. DNA unwinds at ori C 2. produce single stranded templates for synthesis of new DNA. Replication bubble forms, usually w/ replication fork at each end 3. forks proceed around circle 4. eventually 2 DNA circular molecules are produced CONCLUSIONS: products of theta replication are two circular DNA molecules
what is the Eukaryotic chromatin hierarcheal structure?
1. Short region of helix = 2NM 2. Beads on a string of chromatin =11NM 3. chromatin fiber of packed nucleosomes =30NM AKA solenoid fiber 4. section of chromosome in extended form =300NM 5. condensed section of chromosome = 700NM 6. entire mitotic chromosome = 1400 NM
Mendel's interpretation of his results
1. The inheritance of the characteristic traits was predictable 2. Each form of a character is controlled by a certain heritable factor 3. Obtained results are best explained by the existence of such factors in pairs 4. Each parent only passes to its progeny one of its two factors for the character CHARACTER = GENE e.g. seed shape FACTOR = ALLELE = certain form of character e.g. dented or round DOMINANT and RECESSIVE factors (alleles) HOMOZYGOTES and HETEROZYGOTES GENOTYPE 1:2:1 and PHENOTYPE 3:1
Watson and Crick's scale model
1. The laws of chemistry should be obeyed 2. The polynucleotide must be coiled in a way so that it's atoms would not be placed too close together 3. Take into account the results of other investigations into the structure of DNA 4. Account for Chargaff's discoveries of equimolarity between the nucleotides; A=T & C=G 5. Respect the X-ray Diffraction Pattern provided 6. Photograph 51 provided from Franklin & Wilkins showed DNA as a helix 7. Calculations were done based on this picture to reveal the helix's two periodicities of 0.34 nm and 3.4 nm
Replication in Eukaryotes
1. each chromosome contains numerous origins 2. at each origin, DNA unwinds and makes replication bubble 3. DNA synthesis takes place on both strands at each end of bubble as forks repel outwards 4. eventually forks run into each other segments FUSE 5. producing 2 identical linear DNA CONCLUSIONS: products of Eukaryotic DNA replication is 2 linear DNA molecules
What happens if we apply nuclease to Chromatin?
1. nuclease cleaves string betwn beads 2. released beads attached to 200bp of DNA 3. Nuclease destroys unprotected DNA btwn beads 4. leaves core of proteins attached to 145-147bp of DNA
DNA strand
A-T pairs have two hydrogen bonds G-C pairs have 3 hydrogen bonds Phosphodiester bond connects 5' phpsphate group and 3' OH group In RNA, Uracil replaces Thymine Phosphodiester bonds are examples of covalent bonds
August Weismann
Acquired characteristics (e.g. muscle gain/ loss resulting from exercise) are not inherited - germ plasm theory
Donohue
Advised Watson and Crick that the base tautomers in DNA are most likely to be the Keto form and not the Enol form they had been modelling
In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod
Announced that the transforming substance was DNA Only DNA worked in transforming harmless bacteria into pathogenic bacteria
Chromatin (eukaryotes)
At interphase, eukaryotic chromosomes uncoil and decondense into a form called chromatin During interphase, chromatin is dispersed throughout nucleus During cell division, chromatin coils and condenses back into visible chromosomes
What About Replication in Eukaryotes?
Autoradiograph analysis shows multiple origins of replication on eukaryotic chromosomes Large eukaryotic genomes contain thousands of origins of replication separated by 40,000 to 50,000 base pairs The human genome contains more than 10,000 origins DNA replication rate varies among different types of cells Linear eukaryotic replication - Eukaryotic cells -Thousands of origins - A typical replicon: ~ 200,000-300,000 bp in length
How does bacterial DNA differ from eukaryotic DNA?
Bacterial DNA is not complexed with histone proteins and is circular
Major and Minor Grooves
Base-pair stacking creates gaps between the sugar-phosphate backbones that partially expose the nucleotides The major groove, approximately 12Å wide, alternates with the minor groove, approximately 6Å wide These grooves are regions where DNA binding proteins can make direct contact with nucleotides
Recent important milestones
Berg's construction (1972) of the first recombinant DNA molecule in vitro. Boyer and Cohen's first cloning (1973) of a recombinant DNA molecule. Invention by Mullis (1986) of the polymerase chain reaction (PCR) to amplify specific DNA sequences
What additional Support for Bidirectional Replication was there?
Biochemical studies of DNA polymerase of E. coli show that the polymerase can incorporate about 1000 nucleotides per second into the new DNA strand At this rate of synthesis, the entire genome can be replicated in approximately 33 minutes This corresponds well with the generation time of E. coli; unidirectional synthesis would require twice as much time
What are some Chemical Modifications of Histones?
Chemical modifications are important to genetic function Histone tails provide potential targets along chromatin fiber for chemical modifications: 1. Acetylation 2. Methylation 3. Phosphorylation
Prokaryotes (theta) vs. Eukaryotes (linear) Replication
Circular / Linear no breakage of nucleotide strand/ no breakage 1 replicon / many replicons unidirectional OR bidirectional / unidirectional products is 2 circular/ products is 2 linear
1944: Oswald Avery, Colin MacLeod and Maclyn McCarty
DNA can transform bacteria, demonstrating that DNA is the hereditary material
Meselson-Stahl experiment
DNA extracted from E-coli grown for many generations on a heavy 15N isotope of nitrogen will specifically sediment in a salt gradient DNA extracted from E-coli transferred back to normal 14N containing media one can infer the mechanism of DNA replication after each cell division
Additional Evidence That DNA Is the Genetic Material
DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group 1950, Erwin Chargaff reported that DNA composition varies from one species to the next This evidence of diversity made DNA a more credible candidate for the genetic material
How does DNA synthesis get started?
DNA polymerase III can not simply start synthesizing a new strand It can only elongate from existing one (i.e. a free 3'OH group is required) How does DNA synthesis get started? A specialized RNA polymerase called 'DNA primase' can simply start the synthesis of a new strand using the template DNA strand as a guide These 11-12 nucleotide RNA primers then provide the free 3'OH required by DNApol III to replicate the rest of the DNA
Origin and Directionality of Replication in Bacterial DNA
DNA replication is most often bidirectional, proceeding in both directions from a single origin of replication in bacterial chromosomes Eukaryotic chromosomes have multiple origins of replication
Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment - 1956 Fraenkel-Conrat & Singer - 1957
Demonstrated that RNA is the genetic material of TMV. RNA can serve as genetic material in viruses Created hybrid virsuses; progeny particles were of RNA type
DNA Contains Several Classes of Sequence Variation, what are they?
Denaturation - Melting temperature Renaturation Types of DNA sequences in eukaryotes - Unique sequence DNA Gene family: Similar but not identical copies of unique DNA sequences that arose through duplication of an existing gene Repetitive DNA • Moderately repetitive DNA: 150 ~ 300 bp long • Highly-repetitive DNA: less than 10 bp long
Rosalind E. Franklin
Discovered by experimentation/optimisation that DNA could exists in a dehydrated 'A-form' and a fully hydrated 'B-form' Wilkins + Franklin were using X-ray crystallography to study molecular structure Analysis of the diffracted X-rays detected on the photographic film yields structural information about the crystalised sample 1) DNA was helical and most likely a double helix consisting of 2 anti-parallel strands 2) Phosphates were on the outside of the helicies with the bases on the inside 3) The distance between bases (3.4A), the length of the period (34A i.e. 10 bases per turn of helix) and the rise of the helix (36 degrees) interpretation of DNA X-ray diffraction was hampered by the presence of both hydration forms in the crystal before franklin 1958, Rosalind Franklin died of cancer aged 37
Sequence Variation
E COLI - 4,640,000 bp HUMAN - 3,200,000,000 bp Salamander - 760,000,000,000 bp
Martha Chase & Alfred Hershey
E.coli infected by 'bacteriophage' bind to cell surface causing multiplication of progeny virions inside cell that are released upon cell lysis Experiment used bacteriophage T2 infection as model -DNA labeled with 32P (DNA is very phosphorus rich) -Protein coat labeled with 35S (amino acids w/ sulphur) Hypothesis: either DNA or protein would carry injected/ relayed genetic instructions inside the infected E-coli cell Only DNA entered cell but both new DNA and protein coats synthesized and incorporated into new viruses indicating that DNA had the genetic information for synthesis of both of these viral components
Somatic cell division
Each homologous chromosome pair is replicated The sister chromatids are separated into the daughter cells during mitosis The genetic information is not mixed progeny cells are genetically the same as the parental cell (and diploid)
What about Nucleosomes?
Electron microscopic observations of chromatin revealed fibers composed of linear array of these spherical particles. Resemble beads on a string Are condensed several times to form intact chromatids.
Acetylation
Enzyme histone acetyltransferase (HAT) Addition of acetyl group to positively charged amino group on side chain (lysine) changes net charge of protein by neutralizing positive charge The addition of the acetyl group neutralizes this positive charge and hence reduces the binding between histones and DNA, leading to a more open structure which is more accessible to the transcriptional machinery. Histone acetylation therefore leads to transcriptional activation.
Frederick Griffith in 1928
Griffith worked with two strains of a bacterium, one pathogenic and one harmless When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic He called this transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNA Concluded that the type II-R had been "transformed" into the lethal III-S strain by a "transforming principle" that was part of the dead III-S strain bacteria. Griffith's Experiment did not prove that DNA was responsible for transformation! The III-S strain DNA contains the genes that form the smooth protective polysaccharide capsule. Former II-R strain bacteria were now protected from the host's immune system and could kill the host
Elucidating the Nature of the Genetic Material
H. Morgan's group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material 1. Biologically useful and stable source of information 2. Ability to replicate accurately and transmitted across generations 3. Capable of change 4. Express itself to make other biomolecules
Nucleosome
H2A, H2B, H3, H4
Modern Genetics
In the last century, genetics is important biological tool, using mutants to gain an understanding of processes. This work has included: a. Analyzing heredity in populations. b. Analyzing evolutionary processes. c. Identifying genes that control steps in processes. d. Mapping genes. e. Determining products of genes. f. Analyzing molecular features of genes and regulation of gene expression
Bacterial DNA Replication
Initiation -oriC (single origin replicon) -an initiation protein (DnaA in E.coli) Unwinding -DNA helicase -Single-strand-binding proteins (SSBs) -DNA gyrase (topoisomerase) 1. DNA helicase binds to lagging strand at each replication fork and moves 5'-3' breaking H bonds and moving replication fork 2. single strand binding proteins stabilize exposed single stranded DNA 3. DNA gyrase relieves strain ahead of replication fork
Which Proteins are required for Replication?
Initiator proteins - bind to origin and separate strands to initiate replication Helicase- Unwinds DNA at replication fork single strand binding proteins - attach to single strand DNA and prevent secondary structure forming Gyrase- ahead of replication fork, making and resealing breaks in DNA to release torque that build ups as result of unwinding at fork Primase- synthesizes short RNA primer to provide a free 3' OH group for attachment of DNA nucleotides Polymerase III - elongates new strand from 3' OH group provided by primer (11-12 nucleotides) Polymerase I - Removes RNA primer and replaces with DNA Ligase - joins okazaki frags by sealing breaks in in sugar phosphate backbone of newly synthesized DNA
Eukaryotic DNA
Is complexed with HISTONE proteins that together form more and more ordered structures of CHROMATIN resulting in chromosome
John Cairns
John Cairns reported the first evidence of bacterial origins of replication in 1963 Cairn's work showed expansion around the origin of replication, forming a replication bubble, once replication gets under way in bacteria At each end of the replication bubble is a replication fork; replication is complete when the replication forks meet
Euchromatin VS Heterochromatin
Less condenses / more condensed on chromosome arms / at centromeres, telomeres, etc.. unique sequences / repeated sequences many genes / few genes replicated thru the S phase / replicated LATE S phase Transcripted often / transcripted infrequent Crossing over common / crossing over uncommon APPLIES TO CONSTITUTIVE HETEROCHROMATIN
1900: rediscovery of Mendel's work by Robert Correns, Hugo de Vries, and Erich von Tschermak
Major event
1902: Archibald Garrod discovers that alkaptonuria, a human disease, has a genetic basis.
Major event
2001: Sequence of the entire human genome is announced.
Major event
All DNA Replication Takes Place in a Semiconservative Manner
Modes of Replication - Replicons: Units of replication + Replication origin - Theta replication: circular DNA EX: E. coli; single origin of replication forming a replication fork, and it is usually a bidirectional replication
What is the result of chromatin hierarcheal structure?
Net result is that a eukaryotic (human) cell's DNA is packaged into a mitotic chromosome 10,000 fold shorter than it extended length!
What is Nucleotide Base Stacking?
Nucleotide base pairs are spaced along the DNA duplex at intervals of 3.4Å This tight packing leads to base stacking, the offsetting of adjacent base pairs so that their planes are parallel This leads to a twist in the double helix
Supercoiling
Positive supercoiling (over rotate) Negative supercoiling (under rotate) bacterial chromosomes are covalently closed, circular DNA duplexes, as are almost all plasmid DNAs Double-stranded circular DNA form supercoils if the strands are underwound (negatively supercoiled) or overwound (positively supercoiled) two strands are wound about each other once every 10 bp
What are Histones?
Positively charged proteins associated with chromosomal DNA in eukaryotes Contain large amounts of lysine and arginine Makes electrostatic bonding to negatively charged phosphate possible Five main types of histones: H1 - Lysine rich H2A - slightly Lysine rich H2B - slightly Lysine rich H3 - Arginine rich H4 - Arginine rich
Eukaryotic cell cycle
Prophase Metaphase Anaphase Telophase
Replication Initiation in Bacteria
Replication in E. coli requires that replication-initiating enzymes locate and bind to oriC consensus sequences Enzymes DnaA, DnaB, and DnaC bind at oriC and initiate DNA replication DnaA binds first, bends the DNA, and breaks hydrogen bonds in the A-T rich sequences DnaA first binds the 9-mer sequences, bends the DNA, and breaks hydrogen bonds in the A-T rich sequences of the 13-mer region DnaB is a helicase that uses ATP energy to break hydrogen bonds of complementary bases to separate the strands and unwind the helix DnaB is carried to the DNA helix by DnaC
Replication Origins (Ori C)
Replication origins have sequences that attract replication enzymes The origin of replication sequence of E. coli is called oriC, and it contains about 245 bp of A-T rich DNA The origin is divided into three 13-bp sequences followed by four 9-bp sequences
Bacterial ori C
Replication origins of bacterial species have similar (conserved) but not identical sequences Comparison within and between species leads to identification of consensus sequences, the nucleotides found most often at each position of DNA in the conserved region The 13-mer and 9-mer sequences of oriC are conserved—they play an essential role in replication
Eukaryote Ori C
Saccharomyces cerevisiae (yeast) has the most fully characterized origin-of-replication sequences The multiple origins of replication are called autonomously replicating sequences (ARS) ARS organization and sequence is similar throughout the yeast genome Replication origins of other eukaryotes are less well characterized
DNA replication theories
Semi conservative conservative dispursive
The Central Dogma
TRANSCRIPTION DNA -> RNA TRANSLATION Ribosomes, tRNAs (expanded later) PROPERLY FOLDED PROTEIN executes its function in cell
Thomas Hunt Morgan
The 'father' of modern experimental genetics test the chromosome theory of inheritance Drosophila melanogaster Chose the fruit fly: reproduced quickly and prolifically, inexpensive to keep in the lab, only had four pairs of chromosomes, including morphologically distinct sex chromosome pairs (XX & XY) Therefore more than one gene could be associated/ transmitted with one chromosome Morgan proposed (and later proved) that chromosomes represented linear arrays of genes that reside at specific locations (loci) along their length.
The Twisting Double Helix
The DNA double helix has an axis of helical symmetry, an imaginary line that passes lengthwise through the core of the helix The diameter of the molecule is 20Å, The diameter results from the fact that each complementary base pair (A and T or G and C) is 20Å wide One helical turn = 34A/ 3.4 NM AKA 10 base pairs
Helicase causes a problem?
The action of helicase at the replication fork places great strain on the DNA double helix ahead of it because the two ends of the helix cannot freely rotate with respect to each other An enzyme called 'DNA topisomerase I' relieves this strain by catalysing a break in the phosphodiester backbone of one DNA strand allowing the two ends of the helix to rotate relative to each other Unwinding the helix causes torsional strain. The enzyme DNA gyrase can relieve supercoiling
What is antiparallel orientation?
The antiparallel arrangement of two strands of double helix is essential for forming stable H bonds Brings the partial charges of complementary nucleotides into alignment If two strands were to align in parallel, the charges of complementary nucleotides would repel each other
Erwin Chargaff's rules
The base composition of DNA varies between species In any species the number of A and T bases are equal and the number of G and C bases are equal 1. Base composition studies indicated double-stranded DNA consists of ~50% purines (A,G) and ~50% pyrimidines (T, C) 2. amount of A = amount of T and amount of G = amount of C 3. %GC content varies from organism to organism
Allels
The expression of these alleles leads to two forms of proteins. 1. Both alleles and thus both forms of proteins can be exactly the same and then they execute the function in the same way, i.e. we can not distinguish between them 2. two alleles can differ in certain way and the two produced forms of proteins can execute the function in different way. 3. can be nonfunctional and then the function is carried in cell only by the other, functional form the two different forms of alleles can be in dominant or recessive manner
Hydrogen Bonds
The nucleotides of DNA strands are connected by noncovalent bonds to each other a.k.a hydrogen bonds. Individually, hydrogen bonds are weaker than a single covalent bond, such as a phosphodiester bond. But since we have so many hydrogen bonds in the DNA, the two DNA polymers are very strongly connected
1901 Danish botanist Wilhelm Johannsen
The words 'gene', "Phenotype and "Genotype" coined
Genes and Alleles
There are two copies of each chromosme in the nucleus of somatic cells and hence two copies of each gene. Therefore the cell is said to be diploid Pair of homologous chromosomes, one from father, one from mother
what is Chromatin Remodeling?
To accommodate DNA-protein interactions, chromatin structure must change To allow replication and gene expression, chromatin must relax compact structure expose regions of DNA to regulatory proteins have a reversal mechanism for inactivity
TOPOISOMERASES
Type I - break only one strand, relaxing or twisting of the helix Type II - break both strands and pass another part of the double helix through the gap A typical bacterial chromosome consists of about 50 giant supercoiled loops of DN
What is a Pedigree?
We use a pedigree chart to keep track of how traits are passed on from generation to generation. Is a diagram that shows the history of a trait as it is passed from one generation to the next. Pedigrees indicate patterns Pedigrees identify carriers of genetic disorders Pedigrees are useful for genetic counseling
Most of the genes that encode proteins are found in?
a. unique-sequence DNA.
Back to Mendel
an Augustinian monk who later became the abbot born in 1822, Czech math & science teacher, and became interested in botany (plants) Height (tall or short), Flower position (on side of stem or on top of stem), Flower color (white or purple), Seed color (green or yellow), Seed shape (round or wrinkled), Seed pod color (yellow or green), Seed pod shape (inflated or constricted) 'true-breeding varieties' for each characteristic that had persisted for several generations Pollen taken from a pea plant exhibiting one form of the character was used to pollinate the flower carpels (that had its own anthers removed to prevent self pollination)
Geoffroy St. Hilaire
argument for evolution in 1844: Vestiges of a Natural Creation.
Z-DNA h
as a left-handed helix sugar-phosphate backbone zigzags back and forth
Why Do We Need SSBPs?
can hinder DNApolymerase Single-strand binding proteins facilitate DNApol's progress cooperative protein binding straightens region of chain
Centromere structure
constricted region of a chromosome where spindle fibers attach
1940s Barbara McClintock
describes mobile genetic elements in maize
1953: James Watson and Francis Crick
determine the structure of the DNA molecule, which leads directly to knowledge of how it replicates
1904: Gregory Bateson
discovers linkage between genes. Also coins the word "genetics".
Linus Pauling
discovery of alpha helix and beta sheet structure of proteins quantum chemistry resonance work and the discovery of sickle cell anemia as a molecular disease Proposed a 3-chain helix with a sugar phosphate backbone core and nucleic acid bases facing outward
Telomere structure
ends of chromosomes provide a means to replicate the ends of linear chromosomes T- loop G rich single stranded overhang
meiosis
generation of germ cells (i.e. sperm or eggs) Recombination between pairs of homologous chromosomes leads to mixing of the genetic material (cross over) After completion of meiosis The resulting haploid gametes are genetically distinct from each other and their parental cells This mechanism of chromosomal cross-over is the driver of genetic diversity
Central Dogma of Molecular Biology - Francis Crick 1958
genetic flow of information in a cell starts with DNA 'instructions' and passes through RNA 'intermediates' that dictate the synthesis of 'functional' protein
B-DNA
has a right-handed helix. sugar-phosphate backbone forms smooth continuous ribbon. Negative supercoiling introduces a torsional stress that favors unwinding of the right-handed B-DNA double helix, while positive supercoiling overwinds such a helix Both forms of supercoiling compact the DNA so that it sediments faster upon ultracentrifugation or migrates more rapidly in an electrophoretic gel in comparison to relaxed DNA
What is a Trait?
is a specific characteristic that is unique. Examples are hair color, eye color, handedness, etc.
1871: Friedrich Miescher
isolates "nucleic acid" from pus cells.
Plasmids
naturally occurring, self-replicating, circular, extrachromosomal DNA molecules found in bacteria Plasmids carry genes specifying novel metabolic capacities advantageous to the host bacterium. Various animal virus DNAs are circular as well.
Marshall Nirenberg & Heinrich Matthaei
produced RNA solely of uracil, which only occurs in RNA. added this synthetic poly-uracil RNA into E-coli which contained the DNA, RNA, ribosomes They added DNase, so that no additional proteins would be produced other than that from their synthetic RNA Then added 1 radioactively labeled amino acid, the building blocks of proteins, and 19 unlabeled amino acids to the extract
1977 Phillip Sharp and Richard Roberts
protein-coding genes are carried in segments
1910: Thomas Hunt Morgan
proves that genes are located on the chromosomes (using Drosophila).
Pulse-chase labeling
provided the first evidence of bidirectional replication in 1968 (Huberman and Riggs) Cells were exposed to high levels (pulse) and then low levels (chase) of a radioactive tracer The autoradiographs produced show the locations where radioactivity is incorporated into the DNA - both replication forks
1866: Gregor Mendel
publishes Experiments in Plant Hybridization, which lays out the basic theory of genetics. It is widely ignored until 1900.
1859 Charles Darwin
publishes The Origin of Species, which describes the theory of evolution by natural selection. This theory requires heredity to work relied on same evidence for evolution that Lamarck did (such as vestigial structures and artificial selection through breeding) complexity evolved simply as a result of life adapting to its local conditions from one generation to the next "The emergence of characteristics (phenotypes) that confer a reproductive advantage will be more likely to be passed onto the next generation and hence be retained within populations (and vice-versa)." Evolution of beaks shape and size allowing exploitation of specific food sources, conveys a selective advantage BUT absence of any detail on the mechanisms underpinning its existence
1926: Hermann J. Muller
shows that X-rays induce mutations.
1966: Marshall Nirenberg
solves the genetic code, showing that 3 DNA bases code for one amino acid
what is Heredity?
the passing on of traits from one generation to the next.
