Genetics Module 3

Friedrich Miescher

The first to isolate DNA in 1869 from white blood cells. He called it “nuclein”. He noted that it was slightly acidic and high in phosphorus.

Albrecht Kossel

In 1901, discovered that DNA has four nitrogenous bases (Adenine, Guanine, Cytosine, and Thymine).

Phoebus Levene

Found that DNA is made of nucleotides (A sugar, phosphate group, and base). Proposed tetra nucleotide structure of DNA.

Why did most researchers at first believe that protein was the genetic material in DNA?

They are very abundant (about 50% of cell dry weight). Proteins have lots of variability. Structure of nucleic acid was too simple (at the time) to account for necessary variability. Most researchers interested in transmission genetics easily accepted this with little evidence.

Griffith, 1928

Performed the “Transformation” experiment. (DID NOT establish the transforming principle) Virulent strain of streptococcus pneumonia appears smooth (S). The avirulent strain does not have a polysaccharide capsule so it appears rough (R). Heat killed S-cells do not cause death. Live, virulent S cells found when R strain was transformed to virulent strain by heat killed S-cells. Conclusion: A substance in the heat-killed virulent bacteria genetically transformed the type IIR bacteria into live, virulent Type IIS bacteria.

Avery, MacLeod, and McCarty, 1944

Discovered that DNA is the “Transforming Principle”. They treated samples of virulent bacteria with enzymes: RNase, Protease, and DNase. Sample treated with RNase resulted in Type IIIS and Type IIR bacteria. Sample treated with Protease resulted in Type IIIS and Type IIR bacteria. Sample treated with DNase resulted in Type IIR bacteria. Conclusion: Because only DNase destroyed the transforming substance, the transforming substance is DNA.

Transformation

Taking something from strain and putting it in another to make a change.

Hershey and Chase, 1952

DNA is the genetic agent in bacteriophages. 35S is in protein. 32P is in DNA. Label pages, infect unlaced cells. DNA entered E. coli cell, not protein. Labeled DNA found in progeny phages too. Conclusion: DNA, not protein, is the genetic material in bacteriophages.

Heinz Fränkel-Conrat and Bea Singer, 1956

RNA is the genetic material in Tobacco Mosaic Virus (TMV)

Yanofsky, 1964

Discovered the collinearity between DNA and protein. Studied tryptophan synthesis genes and proteins. Established that changes in DNA and protein were collinear (e.g. if the gene changed, the protein changed)

Central Dogma \*picture

DNA → DNA replication (Information is transferred from one DNA molecule to another).

DNA → Transcription (Information is transferred from DNA to an RNA molecule. Same nucleotide language, just putting it into different form. Imagine DNA as spoken language and you’re transcribing/writing that spoken language down) → RNA (the written version of the language) → Translation (Information is transferred from RNA to a protein through a code that specifies the amino acid sequence. Imagine translating that written language into amino acid language.) → Protein

Revisions to the Central Dogma \*picture

DNA →

Rosalind Franklin and Maurice Wilkins, 1953

Using X-ray diffraction, concluded that DNA was helical. Found 3 important regularities: 3.4 angstoms (0.34 nm), 34 angstroms (3.4 nm), helical structure 20 angstroms (2 nm) wide.

Erwin Chargaff, 1950

Found that A=T and G=C. However, (A+T)/(G+C) does not equal 1.

James Watson and Francis Crick, 1953

Base pairing between two strands of nucleotides allows for constant width of helix and agrees with Chargaff’s base composition rules.

Pyrimidines

Cytosine, Uracil, and Thymine (Spells CUT so like CUT the Py)

Purines

Adenine and Guanine. (Remember it like Pure silver; the notation for silver is AG)

Always read and write DNA in a _________ direction

5’ to 3’

DNA has a _______ charge

negative

Which nucleic acid is this? \*picture

RNA

Which nucleic acid is this? \*picture

DNA

How do you distinguish between RNA and DNA?

Ribonucleic acid (RNA) is not missing an O (OH). Deoxyribonucleic acid (DNA) is missing an O; only has a H. DNA has thymine. RNA has uracil.

Is this a purine or pyrimidine? \*picture

Purine

Is this a purine or pyrimidine? \*picture

Pyrimidine

Nucleoside \*picture

Only contains a sugar and base

Nucleotide \*picture

Contains a sugar, base, and phosphate group

Describe the nucleotide structure.

1’ carbon attaches to the base.

2’ carbon distinguishes DNA (-H) from RNA (-OH)

5’ carbon attaches to the phosphate group of the previous nucleotide

3’ carbon bonds to next 5’ phosphate group of new nucleotide

Complementary base pairing between 2 strands of nucleotides allows for ______________

A constant helix width

A and T

Adenine and Thymine using 2 hydrogen bonds

G and C

Guanine pairs with Cytosine using 3 hydrogen bonds

Hydrogen bonds in the double helix

Hydrogen bonds are relatively weak attractions between positively and negatively partially charged molecules. They are not the main force joining the two strands of DNA.

Polymerization of Nucleotides

Phosphodiester bonds connect the sugar-phosphate backbone. 3’ end of one nucleotide bonds to the 5’ end of the next one. (ALWAYS adding on the 3’ end) A phosphodiester linkage connects the 5’ phosphate group and the 3’ -OH group of adjoining nucleotides. The strands run in opposite directions; they are antiparallel.

Model for DNA structure

3\.4 nm distance for a full helical turn. 0.34 nm distance between nucleotides. Hydrophobic bases in the center. Hydrophilic backbone on the outside. Strands are antiparallel. Plectonic coil that can’t be pulled apart (have to unwind it first). Major and minor grooves.

What maximizes the hydrophobic interactions of a DNA molecule?

Base stacking

What is the main force in stabilizing the double helix of DNA?

Hydrophobic interactions

What does hydrogen bonding provide?

It provides specificity (e.g. it ensures that A pairs with T and G pairs with C). G-C has 3 H-bonds. A-T has 2 H-bonds.

Which DNA form is the DNA model of Watson and Crick?

DNA form B-DNA (10 bp/turn) and right-handed. Z-DNA is the only left-handed coiled DNA.

Can DNA structure change in the cell?

Yes

Explain electrophoresis.

Fragments migrate from negative to positive. Smaller pieces move further than larger pieces. Migration distance is inversely proportional to the log (DNA fragment size).

Explain quantification of DNA using UV Absorption.

UV light is absorbed by the ring structures in bases. Relative max. absorption at 260 nm is used to quantify DNA. Absorption increases by 40% when DNA is denatured.

Denaturing the DNA

This means breaking the hydrogen bond and hydrophobic interactions with heats or chemically which allows the two strands to separate.

Hyperchromic Effect

An increase in UV absorption (40%) due to denaturation (exposed bases absorb more UV light)

DNA melting curve

A melting curve of DNA showing Tm and possible molecular configurations for various stages of melting. Tm = Midpoint of Thermal Denaturation. 50% of DNA is denatured. High GC content results in higher Tm.

For a given DNA molecule, cooling the solution of DNA ________

will stabilize the DNA and make it more likely to be double-stranded.

Tobacco Mosaic Virus (TMV) RNA packaging

RNA virus, each layer has 17 identical proteins, develops helical structure as RNA and proteins interact.

Bacteriophage Lambda Virus DNA packaging

Winds the DNA on itself back and forth, so it can fit inside the head particle.

E.coli Chromosome

Is associated with RNA and positively charged proteins. Circular RNA folds into itself and keeps doing this process until it can fit inside the cell.

How much DNA is in the human body?

A human diploid cell has 2 meters of DNA. Chromosomes range from 1.7 cm for the Y and up to 8.5 cm for chromosome 1. the human body has 50 trillion cells or 100 trillion meters of DNA.

Chromatin

The complex of DNA, chromosomal proteins and RNA within the nucleus

Euchromatin

Lighter staining parts of the chromosome during interphase. Actively transcribed genes. Condenses and relaxes (stretched out).

Heterochromatin

Darker staining parts of chromosome. Have fewer genes, usually condensed, usually not involved in crossing over, and replicates late in S phase.

Constitutive Heterochromatin

Always heterochromatin (ex. centromeres)

Facultative Heterochromatin

May be euchromatic sometimes (ex. X chromosome that is Barr body)

Width of naked DNA is ____

2 nm. Wind it around twice on nucleosome that has histone molecules (to shorten length)

Width of histone molecules is _____

11 nm. Wrap nucleosome cores around each other to change the width to 30 nm. begin the loops, this eventually leads to butterfly shape of chromosome.

________ Histones package eukaryotic DNA

Highly conserved

Describe the purpose of histones.

Histones are basic proteins and have lots of positively charged amino acids. This allows them to bind electrostatic ally to negatively charged phosphates in DNA. Histones are highly conserved proteins especially H3 and H4. Individuals with mutated histones die out before ever reproducing. Plants and animals have 102 amino acids in H4, with 100 identical. This means that 100 out of 102 amino acids positions are critical for the histone.

11 nm Fiber Chromatin Structure

This is when the naked DNA is wrapped around the nucleosome. 11nm fiber = tandem nucleosomes; “beads on a string”. Deduced by sing micrococcal nuclease digestion.

Nucleosome

Nucleosome core + 53 bp linker DNA (200 bp/nucleosome)

Nucleosome core

Core histones + 147 bp DNA. Core histones = 2 each of H2A, H2B, H3, and H4

Describe 30 nm in situ (dead cell) Chromatin Structure Model.

Helical model of nucleosome coiling to form 30 nm fiber. Nucleosomes arranged in zig-zagging ribbon that twists or supercoils.

Describe 5-24 nm in vivo Chromatin Structure Model.

Chromatin strands are 5-24 nm. Smaller than proposed 30 nm in situ model. Disordered organization. chromatin strands are closer together in mitotic cells.

Nuclear Matrix

Fibrous network throughout the nucleus that anchors a series of DNA loops. DNA stays tethered from various places within the nucleus; they have their own territories.

______________ in DNA induces supercoiling

Torsional stress

Supercoiling

Occurs when DNA coils back on itself when it is overwound or underwound.

Positive Supercoiling

Over-rotated in same direction as DNA coil so left-handed supercoil compensates. (e.g. you continue over twisting right-handed twists in order to compensate, you create supercoils in the left-handed direction)

Negative Supercoiling

DNA is underwound (not enough right-handed twists) so right-handed supercoil compensates.

Topoisomerase

Alter torsional stress in DNA by cutting the DNA backbone. Typically remove supercoiling, but in some cases, some can induce supercoiling. Controlling the amount of torsional stress is important in processes like replication and transcription.

_________________ are the most compacted chromatin.

Mitotic chromosomes; have to extremely compacted so they can faithfully be divided into daughter cells. banding patterns along chromosomes are from various DNA staining techniques.

Bands

Characteristics for a strain of organism and can be used to identify specific chromosomes.

Chromatin compaction can be visualized in massive polytene chromosomes during Interphase due to ______

Puffs and Bands

Endopolyploidy

Several rounds of DNA replication without separation of replicated chromosomes

Puffs

Areas where the DNA is loosely coiled so that transcription can occur (lightly stained)

Some species have centromeres with ___________

Conserved DNA sequences; Must attach to spindle fibers for proper segregation into daughter cells. Yeast have a conserved DNA sequence at the centromeres, but most organisms do not.

______ rather than DNA sequence identifies many centromeres

Chromatin; Most centromeres are heterochromatic with short DNA sequences repeated many times. Surprisingly, no specific sequence is found in all centromeres. A centromere specific histone H3 (CenH3) that replaces H3 in centromeres of humans, flies, and Arabidopsis that results in a change in chromatin structure to allow binding of spindle fibers.

Telomeres

Provides stability for ends of chromosomes s tat chromosomes are not degraded by exonucleases (enzymes that degrade free DNA). Prevent chromosomes from joining each other at the ends due to ligase activity. Provide proper replication of end of chromosome. Although sequences vary from species to species, they are oriented GC pairs toward end of chromosome. Human telomeric sequence is 5’-TTAGGG-3’; repeated 300-5000 times.

Telomere t-loop Structure

Note GC rich area at end and presence of 3’ overhand with extra copies of telomeric sequence. 3’ overhang can loop and pair with another part of chromosome to protect ends (forms t-loop; hides the ends by “tucking them” into themselves). Displaced strand (the one tucked) is protected by proteins.

What are the 3 hypotheses for DNA replication?

Conservative Replication, Dispersive Replication, and Semiconservative Replication

Conservative replication

During the first replication, there is one original DNA helix and one new DNA helix. During second replication, there is one original DNA helix and three new DNA helixes.

Dispervise Replication

During first replication, there is a two helixes that contain new and original DNA. During second replication, there is less of the original DNA and more of the new DNA in the helixes. The original DNA decreases every time.

Semiconservative replication

During the first replication, there is two helixes that contain new and original DNA. During second replication, there is two DNA helixes that contain original DNA and new DNA and two DNA helixes that contain just new DNA. The original DNA will always be present.

Meselson and Stahl DNA Replication Experiment

They grew E coli in 15N media for many gens and sampled the DNA then they transferred the cells to 14N media. And let replicate for several gens and sample DNA after each sample. The new DNA should contain 14N (light) and the old DNA should contain 15N (heavy). The weight of the DNA was observed after every generation. The heavier generations were at the bottom and the lighter were at the top. This showed that DNA replication is semiconservative.

DNA Replication

DNA replication uses an odds trend as a template to direct synthesis of a new complementary strand. The resulting double-stranded DNA molecule is half old DNA and half new DNA.

Autoradiography

Help examine chromosomes during replication. 3H labeled Thymine-only present for one cell division. (labels just one strand of dsDNA on each chromatid) then pattern radioactivity observed as cells continue to divide.

Models of Semi-Conservative Replication

All replication starts at a Replication origin and proceeds until the entire Replicon (unit that is replicated together) is replicated.

Theta Replication \*picture

Common in bacteria and other circular DNA molecules. Results in 2 circular molecules. Replication can proceed in both directions from the origin: Uni and Bidirectional.

Process: 1. Double stranded DNA unwinds at the replication origin.


2. Produces single-stranded templates for the synthesis of new DNA. A replication bubble forms, usually with a replication fork at each end.
3. The forks proceed around the circle.
4. Eventually two circular DNA molecules are produced.

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Rolling Circle Replication \*picture

The F factor and some viruses (e.g. lambda) (circular) Results in many circular molecules. The replication fork continues around many times producing many strands that are used as templates to synthesize many double-stranded circular DNA molecules.

Linear Eukaryotic Replication

For eukaryotic chromosomes, which have multiple origins for replication along their length (eukaryotic replicons are 20,000-300,000 bp long). The origins start at different times. it replicates bidirectionally from each origin. there are tow replication forks. They will eventually run into each other, creating a double-stranded molecule. Unique procedure for replicating ends involving telomerase.

Theta, Rolling Circle, Linear Eukaryotic Replication

DNA Template, Breakage of Nucleotide Strand, # of Replicons, Unidirectional or Bidirectional

Theta replication

Circular, No, 2, Uni or Bi

Rolling circle replication

Circular, Yes, 1, Uni

Linear Eukaryotic

Linear, No, Many, Bi

DNA Poly I, II, III, IV

5’→ 3’ Polymerase Activity, 3’→ 5’ Exonuclease Activity, 5’→ 3’ Exonuclease Activity

DNA Poly I

Yes, Yes, Yes

DNA Poly II

Yes, Yes, No

DNA Poly III

Yes, Yes, No

DNA Poly IV

Yes, No, No

3’ → 5’ Exonuclease Activity

Backing up and removing added DNA

5’ → 3’ Exonuclease Activity

Laying down new DNA and cutting the old

DNA Polymerase III

Responsible for most DNA synthesis. Proofreads with 3’ → 5’ exonuclease activity. If it makes a mistake, it can back up.

DNA Polymerase I

Discovered first. 5’ → 3’ exonuclease activity to remove RNA primers. Also proofreads with 3’ → 5’ exonuclease activity. Removes things in front of it.

__________ are used to study essential genes.

Conditional mutants; If you want to study the phenotype of having a defective protein being produced, then you raise the temperature and observe essential genes. If not conditional, cell would die when T is raised.

Phosphodiestor Bond Formation requires ____________

Nucleoside Triphosphates. Reaction catalyzed by enzyme: DNA polymerase (add on the 3’ end).


2. In replication, the 3’-OH group of the last nucleotide on the strand attacks the 5’ phosphate group of the incoming dNTP.
3. Two phosphates are cleaved off. The energy from the cleavage is required to make the phosphodiestor bond.
4. A phosphodiestor bond forms between two the nucleotides.

DNA polymerase can only add nucleotides ______

5’ to 3’

Continuous Synthesis

Produces the leading strand

Discontinuous Synthesis

Produces the lagging strand. Okazaki fragments are 1000-2000 its in prokaryotes and 100-200 nts in eukaryotes.

Label the Theta Model.