Genetics 2100 Quiz 1 Dr Yee

4 Key Characteristics of Genetic Material

1. Genetic material must contain complex information
2. Genetic material must replicate faithfully
3. Genetic material must encode the phenotype
4. Genetic material must have the capacity to vary

Important Experiments for Genetics

1928 - Fred Griffith(studied bacteria)
1944- Avery, MacLeod and McCarthy(studied bacteria)
1952- Hershey and Chase(studied viruses)

Griffith's experiment

-an experiment carried out by Griffith using the heat-killed bacteria in mice to discover that a factor in heat-killed, disease-causing bacteria can "transform" harmless bacteria into ones that can cause disease
-transform phenotype

Avery, MacLeod, McCarty

-treated filtrate with different enzymes(RNase, Protease, and DNase)
-found that DNA was the genetic info

Hershey-Chase Experiment

Used radioactive material to label DNA and protein; infected bacteria passed on DNA; helped prove that DNA is genetic material not proteins

Francis Crick and James Watson

discovered the structure of DNA

Rosalind Franklin

Woman who generated x-ray images of DNA, she provided Watson and Crick with key data about DNA
-crystallize molecules, put through X-Ray,put lead screen, have detector, and then picture is shown

Difference between 2' on DNA and RNA

- DNA has H
- RNA has OH

Purines

Adenine and Guanine (2 rings)

Pyrimidines

cytosine, thymine, uracil(1 ring)

Basepairing between DNA

-double helix, hydrogen bonding, complementary pairs and anti parallel
-G+C
-A+T
-5'-3'
-one purine to one pyrimidine

Chargaff's Rule

-[A]=[T] and [G]=[C], they pair up across from one another forming two strands also called base pairing.
-% needs to add to 100%

Complementary pairing in RNA

-will most likely pair with same strand rattler than different
-has uracil instead
-still runs antiparallel

Basepairing can occur between...

-DNAxDNA
-DNAxRNA
-RNAxRNA

Different Forma of DNA Helix

-A Form
-B Form
-Z Form

A form DNA helix

-right handed
- less H2O
-Shorter and wider than B Form
- Unlikely to exist physiologically

B form of DNA helix

-right handed
- lots of H2O
-most physiologically stable
-the predominant form in a cell

Z Form of DNA helix

-left handed
-Zigzag sugar-phosphate backbone
-Stretches of DNA
-Associated w/ transcriptionally active region

How bacteria pacts DNA

-double stranded
-circular
-DNA is localized in a distinct clump in cytoplasm called Nucleoid

How eukaryotes pact DNA

1) Beads on a string histones(H2A, H2B, H3, and H4)
-+ve charged and bind with -ve DNA
2)30nm fiber of chromatin organization
3)300nm fiber
4)700 or 250 nm fiber
5)Chromosomes

DNA methylation

-The addition of methyl groups to bases of DNA after DNA synthesis; may serve as a long-term control of gene expression.
-example of epigenetic

Central Dogma

DNA-transcription-RNA-translation-protein

3 models of DNA replication

semiconservative, conservative, dispersive

conservative replication

-Conservative replication is a theoretical method of replication where the original strands of DNA are left intact and two new strands are formed bonded together.
-1 H: 3 L duplexes

dispersive replication

-a disproved model of DNA synthesis suggesting more or less random interspersion of parental and new segments in daughter DNA molecules
-all I duplexes

semi-conservative replication

-in each new DNA double helix, one strand is from the original molecule, and one strand is new
-1 H: 1 L

Messelon-Stahl Experiment

-spun isotope that causes DNA to be heavy and light
-after DNA replication only semi-conservative model is possible

Incorporation of an deoxyribonucleotide during DNA synthesis

-alpha is closest to 5' carbon, beta and gamma are further away
-beta and gamma get cleaved off and called piral phosphate
--removal of beta and gamma phospates provide energy
-alpha phosphate binds to 3' carbon always
-antiparallel to template strand

Replication fork

-one end unwinds while the other end is intact

2 types of strands at replication fork

-Lagging strand(slower+ discontinuous)
-Leading strand(faster+ continuous)

Okazaki fragments

-fragments of the lagging strand
-100-200 nucleotides in length in eukaryotes

Origin of replication

-each chromosome contains 100's to 1000's of origins of replication
-bubble expands as replication goes on
-two replication forks present
-these start in interior sites within the chromosome

What happens when bubbles meet?

-they fuse together
-new synthesized DNA is made

Steps, enzymes and proteins required for DNA synthesis

1) Initiator proteins
2) DNA Helicase
3)Single Strand-binding proteins(SSB)
3-4)Primase
4)DNA gyrase(topoisomerase)

Initiator Proteins

-bind to origin of replication
-causes short stretch of DNA to unwind

Helicase

-cannot initiate unwinding
-binds to Initiator protein and begins to unwind DNA
-breaks H-bonds

Single-strand-binding proteins(SSB)

-attaches to single-stranded DNA
-prevents DNA from folding back on itself and creating a secondary structure

Gyrase(a topoisomerase)

-2 of them
-remove the helical tension(supercoils)that build up at the end of each replication fork

Primase Enzyme

-synthesizes a short RNA segment(primer) to initiate(or prime) DNA synthesis

RNA molecules that have catalytic activity

-Ribozymes

Types of RNA

-Ribosomal RNA(rRNA, cytoplasm, component of ribosomes)
-Messenger RNA(mRNA, nucleus and cytoplasm, caries genetic code)
-Transfer RNA(tRNA, cytoplasm, carry amino acid to ribosomes)
-Small Nuclear RNA(snRNA, nucleus, processes pre-mRNA and removes introns)
-Small Nucleolar RNA(snoRNA, nucleus, processes and assembles rRNA)
-MicroRNA(miRNA, cytoplasm, inhibits translation of mRNA)
-Small Interfering RNA(siRNA, cytoplasm, triggers degradation of other RNA molecules)

Coding and Template Strand of DNA

-usually coding/non-template strand of DNA is shown
-coding strand and RNA strand are the same
-can guess codons from coding strand

RNA synthesis from DNA Template

-only one strand of double-stranded DNA is transcribed
-RNA synthesis is complementary and antiparallel
-initiation DOES NOT require a pre-existing primer

Components of Transcriptional Unit

-Promoter(DNA sequence that recognizes and binds, determines which DNA strand is the template, determines transcription start site(+1), determines amount of RNA that is transcribed)
-upstream(left) and downstream(right)
-Terminator

Different Efficiencies for transcribing genes

-usually amount of mRNA and amount of protein made are positively correlated
-high expression-> high amount of mRNA-> high amount of protein
-low expression-> low amount of mRNA-> low amount of protein
-No expression-> nothing

Process of Bacterial Transcription

Initiation -> Elongation -> Termination

Initiation

-RNA polymerase holoenzyme (core unit + sigma factor)
binds to -10 and -35 sequences within promoter
-DNA at promoter unwinds
-RNA synthesis starts at slow rate

Elongation

-RNA polymerase undergoes conformational change
-Sigma factor dissociates
-RNA synthesis starts at faster rate

Termination

-RNA polymerase transcribes terminator sequence
-RNA synthesized from terminator sequence forms hairpin
-RNA dissociates from RNA polymerase
-RNA polymerase releases from DNA template

Experiment that showed that some regions of an
mRNA molecule may be removed after it is initially transcribed

-mix DNA containing gene X with RNA transcribed from gene X
-denature than allow to anneal
-looped out region of DNA

Intron

-Intervening or Interrupting sequences

Exon

-Expressed sequences

3 Consensus Sequences for Intron removal/splicing

-5 consensus sequence
-Branch point
-3 consensus sequence

Modifications of Eukaryotic mRNAS

-Addition of specialized cap structure to 5' end
-Addition of poly(A) tail to 3' end (polyadenylation)
-Removal of introns

Addition of specialized cap structure to 5' end

-Addition of a 7-methyguanine nucleotide (see Fig. 10.18) + other CH3 groups
-Essential function in the initiation of translation
-Increases stability of mRNA and influences the removal of introns

Addition of poly(A) tail to 3' end (polyadenylation)

-Cleavage of pre-mRNA downstream of consensus sequence (AAUAAA)
-NON-TEMPLATE addition of 50 – 250 adenine nucleotides(doesn't require complentary sequence)
-Increases stability of mRNA and increases attachment of ribosome

Removal of Introns

-Removal of introns and splicing together of exons from pre-mRNA
-Consensus sequences at 5' and 3' splice sites and at branch point
-Performed by the spliceosome: a complex of snRNAs and proteins

Translation is performed by the Ribosome

-The mRNA is "read" from 5' ! 3' by the ribosome
-Protein synthesis proceeds in the N-terminal -> C-terminal direction
-Ribosomes are ribonucleoprotein complexes ! composed of proteins + rRNA
-rRNA are one of most conserved molecules ! importance in catalytic function

Core Structure of Amino acid

-amino group
-carboxyl group
-hydrogen
-Radical Group

How are protein chains(polypeptides) synthesized

-peptide bond is formed through condensation
-H2O is removed and the amino acids become linked

Levels of Protein Structure

1)Primary(amino acid sequence)
2)Secondary(alpha-helix and beta-sheet, H-bonds interactions)
3)Tertiary (3-D shape, many interactions)
4)Quaternary(subunit organization)

Nirenberg and
Matthaei's Experiment

-add homopolymer to test tube
-incubate and allowed translation to occur
-protein was seperated and the filtered
-was repeated in 20 test tubes and each had a different amino acid
-whichever tube had the radioactive protein indicated what homopolymer coded for a specific amino acid

Characteristics of Genetic Code

-64 different codons
-61 sense codons(18 amino acids have >1 codon and 2 amino acids have only 1 codon)
-3 stop codons

Redundancy of Genetic Code

-synonymous mutation/substitution
-nonsynonymous mutation/substitution

Synonymous mutation/substitution

-A single nucleotide change (usually in the 3rd position of a codon) results in the
SAME amino acid specified in mutant and in wild-type (WT)

Nonsynonymous mutation/substitution

-A single nucleotide change results in a DIFFERENT amino acid specified in
mutant and in wild-type (WT)

Reading Frames

-Reading frame 1(first nucleotide from 5' end)
-Reading frame 2(second nucleotide from 5' end)
-Reading frame 3(third nucleotide from 5' end)
-Reading frame resets after 3

Translation Initiation

-AUG is the start codon
-In bacteria, A consensus sequence called the "Shine-Dalgarno" positions the
ribosome to start translation at the correct AUG on the mRNA and translation can start from internal AUG sites
-In Eukaryotes, ribsomes binds to cap structure on 5' end and scans from 5'->3', looks for first AUG and looks for consensus sequence called "kozak"

What can an anti-codon do?

-help determine code of codon because is complementary and antiparallel

Anti-sense codon

-31 anti-sense codon(31 tRNA)
-some tRNA can recognize more than 1 codon
-wobble pairing may occur

4 Stages of Protein Synthesis

-tRNA charging
-Initiation
-Elongation
-Termination

tRNA charging

-The binding of the correct amino acid to the specific
tRNA
-Requires aminoacyl-tRNA synthetase and ATP

Initiation

-Assembly of ribosome and other components required for translation on mRNA
-Recognition of correct AUG in mRNA

Elongation

-Amino acids are added to the polypeptide chain
-Correct amino acid for each codon on mRNA are brought by charged tRNA
-Complementary base pairing between codon on mRNA
and anti-codon on tRNA

Termination

-Protein synthesis stops at termination codon
-No tRNA corresponding to any of the STOP codons but
"release factors" bind to ribosomes
-A water molecule is added instead of an amino acid
-Translation components are released from the ribosome

Control of Chromatin Structure only in eukaryotic cells

\-Alternation of structure

\-compact→relaxed DNA

Control Levels in both eukaryotes and prokaryotes

\-transcription

\-mRNA processing

\-RNA stability

\-Translation

\-Post-transistional modification

Structural Genes

\-encoding proteins (or RNA) as the end product

Regulatory Genes

\-encoding products that interact with other DNA sequences and affect the transcription and translation of these sequences

Regulatory Elements

\-DNA sequences that are not transcribed but play a role in regulating other nucleotide sequences (i.e. promoters)

Constitutive expression

\-continuously expressed under normal cellular conditions

Regulated Expression

\-Positive Control(stimulate gene expression from basal level)

\-Negative Control( inhibit gene expression from basal level)

Explain this image

Additional info;

\-Operator: the binding site of the regulator protein ! adjacent to or overlaps with promoter

\-Polycistronic mRNA: a single mRNA that contains the coding information for >1 protein

Lac Operon of E. coli

\-Characterized by Jacob and Monod (1961)

\-The operon encodes genes for the metabolism of lactose

\-A negative inducible operon because: o it is regulated by a repressor (negative) o its default "OFF" state could be turned "ON" by an inducer (inducible by lactose/allolactose)

Role of Permease, β-Galactosidase, and Transacetylase

\-Permease transports lactose into cell

\-β-galactosidase breaks lactose into galactose and glucose

\-β-galactosidase also converts lactose into allolactose and converts it to galactose and glucose

\-Transacetylase is functions in lactose metabolism

Genotype of lac Operon

\-lacI

\-lacP

\-lacO

\-lacZ

\-lacY

\-lacA

lacI gene

\-repressor encoding gene

\-lacI → repressor gene is functional

\-lacI → repressor gene is non-functional

lacP

\-operon promoter

\-lacP+ → RNA pol able to bind if not blocked by repressor bound to operator in cis

\-lacP- → RNA pol cannot bind

lacO

\-operon operator

\-lacO+ → Repressor can bind to this operator in the absence of the inducer

\-lacOc → Repressor CANNOT bind to this operator

lacZ

\-encoding β-galactosidase

\-lacZ+ → β-gal gene is functional ! active protein can be made

\-lacZ- → β-gal gene is non-functional

lacY

\-encoding permease

lacA

\-encoding transacetylase

Common Restriction enzymes

\-BamHI(cohesive)

\-CofI(cohesive)

\-EcoRI(cohesive)

\-EcoRII(cohesive and not palindromic)

\-HaeIII(blunt)

\-HindIII(cohesive)

\-PvuII(blunt)

Difference between sticky(cohesive) ends and blunt ends

\-sticky ends are produced by staggered cuts in DNA

\-blunt ends are produced by straight cuts across DNA

Mix and Match of Strands with restriction Enzymes

\-can mix and match if compatible

\-complementary base pairing must occur

Gel Electrophoresis

\-DNA samples are placed in wells in agarose gel

\-electric current is passed through

\-DNA fragments move to positive pole

\-dye specific for nuclei is added

\-DNA fragments appear under UV light

\-first well in called “DNA ladder” or “size standard”

3 Steps of PCR

1) Denaturation

2) Annealing

3) Extension

Denaturation

\-separate ds DNA

template into single strands

Annealing

\-anneal two opposing complementary primers to template strand

Extension

\-DNA polymerase synthesizes DNA by extending from annealing primer