Genetics Exam 2

Genetic Material

contains information that is expressed to determine all traits & functions of an organism

is stable, but must also have the ability to change (variation in many traits without lethality to organism)

is carried on chromosomes (contains proteins & DNA)

Order of Experiments to Disprove “Genes are mad of protein” hypothesis

1. Griffith (1928) ; Avery et al (1944) → streptococcus pneumoniae strains & transformation

2. Hershey & Chase (1952) → bacteriophage T2 & radioactive labeling

Griffith & Avery et al Experiments

Streptococcus pneumoniae = bacteria that causes pneumonia in humans & lethal in mice

2 strains

• smooth (S)

  • virulent (infectious)

  • wild-type

  • cells enclosed in polysccharide capsule

• rough (R)

  • nonvirulent (easy to engulf & destroy)

  • mutant

  • no polysaccharide capsule

Griffith:

• transformation: conversion of rough → smooth

• living cells necessary for virulence

Avery, Macleod, and McCarty continued Griffith’s work

• they knew genetic material must be either protein, RNA, or DNA

• concluded transforming principle is made of DNA

• transformed bacteria remained virulent in subsequent generations, so they concluded that transformed traits = heritable

Hershey & Chase

bacteriophage T2 = virus that infects bacteria ; DNA core surrounded by protein coat

they knew T2 is ~50% protein and 50% DNA

infection steps:

1. adsorption - phage attaches to bacteria via tail fibers

2. phage material enters bacteria - production of new viruses within bacterial cell

   1. phage material must be the genetic material of the phage because it is necessary to make a new phage (molecule of inheritance!)

3. bacterial lysis - new phage progeny is released

radioactive labeling experiment steps

• preparation

1. grow sample of phage in presence of E. coli

2. separate into two samples

   1. add ³⁵S (radioactive sulfur) into one media

   2. add ₃₂P (radioactive phosphorus) into the other

3. results in two labeled phages (phosphorus - DNA labeled & sulfur - protein labeled)

• infection

1. infect two separate fresh E. coli samples (each with one of the labeled phages)

2. blend - shears protein coats & anything that did not enter the bacterial cell

3. spin - infected bacteria will pellet (concentrate & collect on bottom & separate from media)

4. found that the phage with DNA labeled had radioactive pellets and the other didnt → DNA is genetic material

Components of DNA (base unit & what that’s made of)

basic building block of DNA = nucleotide

deoxyribose - 5C sugar (one less O than ribose: ribose has OH on 2’ and deoxyribose has H)

Base Pairs & Chargaff’s Rule

Purines (nitrogenous base with two rings)

• adenine (A)

• guanine (G)

Pyrimidines (nitrogenous base with one ring)

• thymine (T)

• cytosine (C)

Chargaff’s Rule:

• total pyrimidine (T+C) = total purine (A+G)

• T = A & G = C

• A + T is not equal to G + C

Pairings:

• A-T

• G-C

Rosalind Franklin & Maurice Wilkins Findings

• DNA is made of 2 parallel parts

• DNA is helical

• Helix contains 10 nucleotides per turn

• measured helix diameter

Watson & Crick Findings

figured out DNA is a double helix

• two strands of DNA wound around each other

• each strand is one chain of nucleotides

• 5’ phosphate of one nucleotide is linked to 3’ OH of another (antiparallel)

• two strands of DNA of double helix held together by H bonds between complementary bases (A + T & G + C)

recognized DNA structure is important for replication

• complementary strands (each strand can be a template for the other)

• specific base-pairing (only one sequence is possible from a template)

Three Models for Mechanism of DNA Replication

add to this???

DNA Backbone

made of sugar & phosphate (covalently bonded)

phosphate has a (-) net charge, giving DNA a negative charge since backbones is on the outside of the helix

Major vs Minor Grooves

Major: area between turns of double helix

Minor: distance between 2 back bones of anti-parallel strands

Three Forms of DNA

A Form

• less hydrated

• more compact (10.9 base pairs per turn)

• right-handed

• typically seen in vitro

  • in vitro = in controlled lab conditions (for this situation: high salt conditions)

• B Form (most common)

  • more hydrated

  • less compact (10 base pairs per turn)

  • right-handed

  • typically found in vivo (in living organism/cell)

  • proposed by Watson & Crick

• Z form

  • left-handed

  • zig-zag phosphate backbone

  • seen in short DNA pieces with alternating purine/pyrimidine

  • seen in small localized regions (ex: regions of big chromosome)

Oligonucleotide

short chain of nucleotides

single-stranded DNA

Denaturing

separating 2 strands of DNA

aka “melting”

can be done by heating DNA, which disrupts H bonds between strands

G/C-rich DNA has higher melting temp than A/T-rich DNA because G/C has 3 H-bonds & A/T has 2 H-bonds

Annealing

when 2 complementary single strands of DNA bind back together via H-bonds

DNA vs RNA

DNA

• deoxyribose (2’ C has an H)

• A, G, C, T

• double-stranded

• more stable (relative to RNA)

RNA

• ribose (2’ C has an OH)

• A, G, C, U

• single-stranded

• less stable (relative to DNA)

Special Structures found in Single-Stranded Nucleic Acids

Hairpin

• sequences on same strand are inverted complements

• sequence between inverted repeats forms loops

  

Stem

• hairpin without loop

Complexity of RNA

molecules fold into complex 2˚ & 3˚ structures

RNA is synthesized as single stranded nucleic acid

hairpins, bulges, stems, loops, etc fold onto each other (these structures typically are binding sites for proteins)

3 Models for Mechanism of DNA Replication

1. Conservative: template strands create new strands - template strands recombine & new strands combine

2. Semi-Conserative: template strand binds with the new strand it made

3. Dispersive: pieces of template strand synthesize new strands - recombination is random, results in mixed strands

Meselson-Stahl Experiment

proved semi-conservative model for prokaryotes

E. coli - circular chromosome

¹⁵N = heavy isotope of nitrogen that labels DNA

¹⁴N = “normal” nitrogen

used density gradient centrifugation at different points in time

time = 0 → ¹⁵N-containing ds DNA is extracted and goes through gradient centrifugation → result = one band of heavy DNA

time = 1 (after E. coli is exposed to ¹⁴N & one replication occurs - all new dna = ¹⁴N) → DNA extracted, gradient centrifugation = 1 intermediate band between light and heavy (supports semi-conservative OR dispersive)

time = 2 (another cycle of replication in ¹⁴N) → DNA extracted, gradient centrifugation = 1 band of all light & 1 band of intermediate (consistent with semi-conservative)

Density Gradient Centrifugation

DNA is extracted form cells, cut into pieces & added to a gradient of Cesium Chloride (CsCl) - higher density towards bottom

samples are centrifuged at high speed

at equilibrium, DNA concentrates within the gradient where its density is equivalent to that of CsCl

See DNA by staining with ethidium bromide & shining UV light

Taylor Experiment

proved semi-conservative model in eukaryotes

studied bean root tips - followed cells through 2 rounds of mitosis - labeled DNA with tritriated (³H) thymidine (radioactive)

started with unlabeled chromosome in G1 & added ³H-thymidine → chromosome goes through S, G2, and prophase → autoradiography at metaphase = both sisters are radiolabeled (consistent with semi-conservative OR dispersive)

anaphase occurs & they follow one sister through the next round of mitosis

sister in G1 → unlabeled thymidine is added, sister goes through s-phase, g2, and prophase → autoradiography in metaphase = only one chromatid is labeld (consistent with semi-conservative! if dispersive, then both sisters = labeled)

Autoradiography

Method Taylor used in his experiment that proved eukaryotic DNA replication is through the semi-conservative method

done during metaphase of mitosis - exposes DNA to film, dark areas show radioactivity

picture shows DNA molecules with both sister chromatids labeled

Things needed for DNA Synthesis

1. template (ssDNA) - single stranded DNA

2. substrate (dNTPs) - deoxyribonucleotide triphosphates (A, G, C, T)

3. cellular machinery (enzymes, etc) to read template and synthesize new DNA strands

• DNA polymerase

  • DNA Pol I & III

• initiator protein

• helicase

• SSBs

• topoisomerase (DNA gyrase)

• RNA polymerase (primase)

• DNA ligase

• Telomerase

Initiator Protein

starts process binds to oriC & begins melting DNA at A/T rich regions (easier to melt than G-C)

Origin of Replication (OriC)

where replication begins

eukaryotic chromosomes have many

only 1 in E. Coli - OriC

sequence is recognized by initiator proteins

Helicase

continues to melt DNA (breaks down H-Bond)

DNA Polymerase

lots of types in eukaryotes

5 types in prokaryotes (only need to know DNApol I & III)

makes new DNA strand 5’ → 3’

needs -OH group to begin

DNApol I

3 activities

• 5’ → 3’ exonuclease activity - destroys RNA primer

• 5’ → 3’ polymerase activity - replaces RNA primer with DNA

• 3’ → 5’ exonuclease activity - allows proofreading

DNApol III

prokaryotic enzyme

does most of DNA synthesis

elongates RNA primer by adding new DNA (5’ → 3’ polymerase activity)

very fast (~1,000 nucleotides/sec)

3’ → 5’ exonuclease activity to correct mistakes

• corrects mistakes for the nucleotide that was just added to new DNA strand - destroys incorrect nucleotide & corrects mistake

RNA polymerase

prokaryotic enzyme

type of RNApol - primase

doesn’t need free 3'‘-OH group to work

makes RNA primer using ssDNA as template

RNA primer provides free 3’-OH for DNApol to add onto

Single-Strand-Binding Proteins

keeps DNA melted

non-specifically binds to ss DNA to stop H-bonds from reforming

Topoisomerase

DNA Gyrase - type of isomerase for prokaryotes

• works ahead of replication fork to reduce strain due to helix undwinding

• relieves supercoiling by cleaving dsDNA of sugar-phosphate backbone, allowing it to unwind & recreates those covalent bonds before helicase gets there to melt the DNA

• because as DNA helicase unwinds DNA @ fork, it sets up supercoiling

topoisomerase can either undo supercoiling or cause it

DNA Ligase

binds okazaki fragments together

creates covalent bond between 5’ phosphate & 3’ OH to join 2 nucleotides in newly synthesized DNA

Replication Licensing Factor (RLF)

controls timing of replication initation in eukaryotes

keeps track of where replication already happened & where it needs to happen because eukaryotes have multiple OriCs that open at different times

contains several proteins that bind to OriC during G1 phase of cell cycle

marks an OriC as ready for DNA replication

once replication begins, different proteins will bind to mark that OriC as already replicated

Telomerase Enzyme

extends DNA at chromosome ends

synthesizes highly repetitive DNA in telomeres

has protein & RNA components

• protein part - synthesizes new DNA

• RNA part - acts as template for DNA synthesis

-telomerase moves down & continues extending 3’ overhang

-primase makes primer on 5’ prime end

• DNA polymerase fills in , ligase seals nick

• primer is removed

Telomerase is expressed in germ cells (give rise to gametes) & stem cells (immortal cells that provides new cells for processes like wound healing)

Telomeres & Replication

Telomeres contain condensed highly repetitive DNA that do not code for anything - impossible to replicate end of lagging strand - each time you go through replication (without telomerase) the chromosome should be getting smaller because end does not replicate (typically not good because you lose the info at the end of the strand)

• repeat sequence depends on sequence of telomerase RNA (all eukaryotes have a version of telomerases, but different RNA sequences)

2 Types of DNA Replication in Prokaryotes

circular chromosome replication: theta replication

plasmid replication: rolling circle replication

Theta Replication in Prokaryotes

starting at oriC, DNA is melted so single strands act as templates

DNA synthesis happens simultaneously in both directions

1 replication bubble made of 2 replication forks

product = 2 circular chromosomes (each with 1 old & 1 new DNA strand)

Leading Strand

since DNApol makes DNA 5’ → 3’, each fork will have only 1 strand made continuously

strands coming straight out from oriC

Lagging Strand

made discontinuously

short pieces = okazaki fragments

strands going toward oriC

The problem at ends of linear chromosomes & what fixes them

When RNA primer 5’ end is removed at end of DNA strand, there’s no 3’-OH for DNApol to use

very end of chromosome cannot be replicated (end of lagging strand)

chromosome should get shorter after every round of replication, but telomerase prevents this

Replication in Eukaryotes

eukaryotic chromosomes have many DNA packaging proteins bound to DNA

• histones

• must be synthesized during DNA replication

many replicons (point of origin & area of DNA replicated)

slower than prokaryotes

many types of DNApol

1 bubble (2 forks) from each origin

bidirectional DNA synthesis

final product = 2 linear chromosomes (1 old + 1 new DNA strand)

Two Levels of DNA Packaging in Prokaryotes

formation of loop domains

• each loop is held in place by DNA binding proteins

• keeps chromosomes organized - most of the time, related genes are present near each other, so expressing these types of genes only needs one loop to be turned on (ex: genes that code for a biochemical pathway all in one loop)

supercoiling

• happens within loop domains

• condenses DNA even further

Levels of DNA Packaging In Eukaryotes (Simplest to Complex)

DNA double helix (2nm) → nucleosome / chromatosome (11nm) → 30nm → 300nm → 250-nm-wide fiber (700nm) → chromosome (1400nm)

chromatin = DNA + proteins

chromosome = chromatin that is highly condensed

Two Types of Chromatin in Eukaryotes

Heterochromatin

• remains highly condensed (tightly packed) throughout cell cycle

• no gene expression here

• major state of human Y chromosome

• can be constitutive (always condensed ; telomeres/centromeres) or facultative (condensed at certain times ; x-inactivation)

Euchromatin

• packaging changes during cell cycle (based on whether or not gene needs to be expressed & where in the cell cycle we are in)

• contains expressed genes

• major state of most genes

• only this type found in prokaryotes

Centromeres

made up of heterochromatin

part of chromosome that attaches to spindle fibers - acts as a protein binding sit

required for proper segregation of chromatids

no genes present here; DNA sequence is highly repetitive (# of repeats on each chromosome varies)

DNA remains tightly packaged throughout cell cycle

Differences in Replication in Eukaryotes vs Prokaryotes

prokaryotic replication

• A continuous process that can happen rapidly, even while the cell is dividing

eukaryotic replication

• Strictly restricted to the S phase (synthesis phase) of the cell cycle

General Process of DNA Replication (one oriC in view)

SEMI-CONSERVATIVE

1. Initiation

• initiator proteins bind to origin of replication & begin process of melting DNA into two ss DNA

• 2 DNA helicases continue to melt DNA away from origin of replication & towards forks (two diff directions)

• SSBs bind to single strands to prevent re-annealing (rebinding/reforming H bonds)

• topoisomerase relieves the strain of supercoiling ahead of replication fork by cutting & resealing the phosphate backbone

2. elongation

• primase synthesizes RNA primer to provide free 3’-OH

• DNA polymerase attaches to free 3’-OH & reads template strand from 3’ to 5’ direction to build new strand in 5’ to 3’ direction

  • leading strand: synthesized continuously in same direction that replication fork opens

  • lagging strand: synthesized discontinuously in short segments away from the opening fork (okazaki fragments)

3. termination & processing

• primer removal & replace with DNA (prokaryotes = DNApol I)

• DNA ligase seals nicks in phosphodiester backbones of okazaki fragments (turns them into a continuous strand)

• EUKARYOTES ONLY: telomere maintenance - telomerase uses internal RNA template to replicate ends of chromosomes to prevent gene loss

Telomeres & Single Stranded Overhang

made up of heterochromatin & also is a protein binding site

POT (protection of telomere) proteins bind single stranded overhand (3’ overhang due to end of chromosome’s inability to be replicated)

• provides stability

• prevents ends from degrading and/or sticking together

• overhang = single stranded nucleic acid that will get destroyed by nucleases in cell if no proteins bind to it for protection or if no other protection mechanisms occur (destruction = shorter chromosome & sometimes loss of vital information)

  • sometimes folds over to form triple-stranded structure (SEE PICTURE)

DNA remains tightly packaged throughout cell cycle

Packaging of Euchromatin

dynamic! level of packaging changes throughout cell cycle

FINISH SLIDE 275!!!!