-an experimental procedure that has demonstrated that DNA determines genotype/hereditry

transformant -

-the changed recipient when a recipient is exposed to the DNA extracted from a donor, and proceds to integrate it into its genome

1)faithful replication... every cell has same genome

2)must have informational content to incode the constellation of proteins expressed by an organism

3)must be able to change on rare ocasion to accomodate mutations

-however, must be stable so that organisms can rely on its encoded information

-DNA contains a phosphate, a deoxyribose (sugar), and four nitrogenous bases (adenine, guanine, cytosine, and thymine).

-adenine/guanine have double ring structure and are called purines

-cytosine and thymine have a single ring structure, and are called pyrimidine

 -backbone of each strand is a repeating phosphate-deoxyribose sugar polymer

phosphodiester bonds

-these are the sugar phsophate bonds in the backbone

-the carbons of the sugar groups are numbered 1' through 5'

-a phosphodiester bond links the 5' to the 3'

-in double stranded DNA molecules, the two backbones are in opposite, or antiparallel orientation (one is 5' to 3' and the other 3' to 5')

 the bases are attached to the 1' carbon of each deoxyribose sugar in teh backbone of each strand and face inward toward a base on the other strand

*****

A-T

G-C

 these are hydrogen bonds!!!!

*****

denaturation (melting)

-when heat causes two strands of DNA to separate

-DNAs with higher G/C content require higher temperatures to melt them because of the greater attraction of the GC pairing

major groove/minor groove - can be seen in the models

-a single strand of nucleotides has no helical structure; the helical shape of DNA depends entirely on the pairing and stacking of the bases in antiparallel strands

-the helix is "right handed"

-unit of measurement of DNA length is the base pair..

-a thousand base pairs is called one kilobase (kb)

-a million.. a megabase (Mb)

-each strand serves as an unambiguous template for the synthesis of its complementary strand

-an enzyme called DNA polymerase is responsibe for building new DNA strands... matching up each base of the new strand with the proper complement on the old, template strand

-***the nucleotide sequence of a DNA strand fulfills the informational requirement

-it is copied to RNA, which then becomes the blueprint for the synthesis of proteins

-regulatory region

-to regulate transcription, this region is adjacent to the transcribed region...

-it is a segment of DNA that enables transcription of that gene to be turned on or off

introns

-many eukaryotic genes contain these mysterious segments of DNA which are interspersed in the transcribed region of the gene

-they do not contain information for the functional gene product such as a protein

-they are transcribed together with the coding regions (exons), but are then excised from the initial transcript

*the correct sequence in the introns is necessary in order to generate a properly sized transcript

-in mammals, genes are generally very large and transcription units contain many exons (and hence, introns)

-there is "spacer" DNA between genes (non-information)

-in many eukaryotes some of the DNA between genes in repetitive DNA, so called because it consists of a number of identical or nearly identical units repeated in the genome

-a large proportion of repetitive DNA results from mobile genetic elements, DNA segments that can copy themselves and move within the genome

**function of repetitive DNA is a mystery

genomics

-the study of the structure and function of entire genomes

-genome size generally increases with the complexity of the group, but there is considerable variation (up to a thousand fold) within groups

-the number of genes is roughly propotional to genome size

-bacterial cells isolated from nature often contain small DNA elements that are not essential for the basic operation of the bacterial cell... these elements are called plasmids

-they are symbiotic molecules that cannot survive at all outside of cells

-they often contain genese that encode products extremely useful to the host (sexual fusion/antibiotic resistance/toxin production)

-are inherently circular, but can linear as well

-mitochondria/chloroplasts

-have introns, but generally very little intergenetic space

-genes are specific to the functions of the organelle concerned (photosynthesis/oxidative phosphorylation)

-almost no overlap exists between the genes in the organelle and nuclear genome

-

-viruses can only reproduce via infection

-they are composed of a protein coat and a core that contains the genome

-varies alot.... circular/linear/single or double stranded/RNA sometimes/etc

-very little intergenetic space... tightly packed genes

-mostly a single circular double helix chromosome DNA

-introns extremely rare!

-operon - genes whose products are physiologically related are often located together as a group, and one molecule of mRNA is made from the entire unit... the unit is termed operon

-these are very rare in eukaryotes

-

nucleoid- in bacterial cells the DNA is seen arranged in a dense clump called a nucleoid

-when cells are broken, the packing of the nucleoid is lost and DNA tumbles out in a disorganized skein

-in each cell there is about 1mm of DNA, whereas the cell is only 1micrometer in diameter

***in most species of prokaryotes, a cell has one or more copies of a single circular genome and, sometimes, one or more copies of plasmids****

diploid(2n)- carrying two sets of nuclear chromosomes (two genomes) in each cell

haploid(n)- with only one chromosome set per cell

 -most fungi and algae are haploids, most animals and flowering plants are diploid

haploid chromosome number - this is the symbol "n"

polyploids - conditions that are 3n+

ploidy - number of chromosome sets

-in humans, n=23 and somatic (body) cells are diploid = 2x23=46

-homologs - the two members of a pair are homologous chromosomes.. members of a homologous pair are substantially alike in size and gene content, carrying the same genes in relatively the same positions

-in a diploid individual carrying two different alleles, these alleles would both be located at the same relative position in the members of a pair of homologs

-one or two sets of linear nuclear hromosomes

-multiple copies of a circular mitochondrial chromosome

-multiple copies of a circular chloroplast chromosome (plants)

-and sometimes multiple copies of plasmids (some fungi and plants)

a set of chromosomes as viewed under the microscope

-the karyotype is defined by chromosome number and by other visible landmarks

genetics: experimental science of heredity (genes).. how are individual species related?

areas of interest:

1)species identity

2)individual identity

3)development

4)evolutionary relationships

1)storage of information

2)faithfully copied

3)relatively stable (not absolutely)

early 1900s- believed protein was responsible for heredity, because it is more complex and DNA is relatively simple

1928

Fredrick Griffith experiment

-Pneumacoccus (causes lethal pneumonia in mice)

-was a virulent S-strain that was lethal and smooth on plate

-was also avirulent R-strain (non-lethal) that appeared rough on plate

-the S-strain is heat killed, and then it does not harm mice

-when it is mixed with live R-cells though, it kills mice

-extract from dead s-strain makes R-strain virulent.... a non-living substance is responsible (could isolate living S-strain in blood of mice)

-S-strain surrounded by lipo-polysaccharide coat.... this protects it from host's immune system

1944

-Avery/Macleod/McCarty

-eliminated different compounds from bacteria (polysaccharide, fats, proteins, RNA)

-only elimination of DNA (by DNase) caused elimination of transforming ability of R-strain

-conclusive evidence... it is DNA

1952

-Hershey and Chase

-radioactively label nucleic acids with 32P (lots of phosphorous in DNA)

-use 35S for the proteins (no Sulfur in Nucleic acids)

-blender then centrifugation

-seperate remenants of viruses from cells

-proteins are not in bacteria... therefore it is not genetic material

-all genetic information is found within the cell

1953

-Watson and Crick

knew 3 critical things:

1)DNA composed of 4 nucleotides

2)rules for nucleotide composition

3)helical structure

1949

Chargaff determines that all nucleotides are not equally represented, as previously thought

-rather a constant ratio

rules:

1)purines (A+G) = Pyrimidines (T+C)

2)A=T and G=C

Franklin Incident

-Rosalind Franklin and Maurice Wilkins - Xray diffraction

-in the photo, there is X pattern

-says that this molecule is helical

-pattern is straight and consistent

-this means that helix width is extremely consistent

-also very specific distance inforation

-constant distance between bases

-Watson/Crick awarded Nobel Prize

-Franklin did the work... in Wilkins' lab

-ify rights.... wasn't allowed in lunch room

-not sharing results

-Wilkins frustrated, needed Rosalind to figure it out

-Watson and Crick think phosphates are internal

-Wilkins shows W/C Ros' photo

-Watson immediately understand

-Consistency of width gave clues to base pairing

-pyrimidine+pyrimidine = too thin.. doesn't match

-purine +purine = too thick

-purine + pyrimidine = compatible with Xray data (suits Chargaff's rules)

**in DNA, bases stick together due to hydrophobic effect

-hydrogen bonds exist, but not prominent

-have enough DNA to get to sun and back 100 times (10^13m)

-chromatin is the material that makes up a chromosome

1964

-Wilkins - 10nm repeating structure in chromatin

1973

-Hewish and Burgoyene

-DNase digestion of chromatin DNA

-fragments in multiples of 200 bp were observed

-naked DNA was a smear

-with chromatin, DNA was choppy at 200kb intervals

FIRST LEVEL (Kornberg and Klug 1981)

-histones-class of proteins associated with chromatin DNA

nucleosome-DNA wrapped around an octamer of proteins (histones)

octamer- 2(H2A+H2B+H3+H4)

-DNA wraps around octomer twice

-200 base pairs wrap around each one

-DNA is protected by histone... cannot be digested

-can still degest at connecting areas

SECOND LEVEL

solenoid- coil of histones stabilized by histone H1

-association with H2 histone forms this

-spirals around these

THIRD
-SAR- scaffold attachment regions

-serve as attachment points to the central scaffold

-scaffold proteins further coil solenoids to generate supercoils

-supercoils are transient for access

-we need to access this information

**chromatin = DNA + protein (scaffold)

-a chromosome is a single DNA molecule

Four Characteristics of Eukaryotic Chromosomes

1)Size

-varies significantly in size.... all 23 in humans are different sizes (less than 100million bp to 170 million bp)

2)Position of Centromere

-metacentric- middle

-acrocentric- near the end

-telocentric- at the end

-p-arm- smaller arm(south arm)

-q-arm - larger arm (north)

3)Nucleolar organizer position (rRNA genes)

-nucleolus is the region where rRNA is produced and processed 

4)Chromomere patters

-differences in thickness

-localized differences

-chromomeres (knobs)

-position is constant in homologous chromosomes

5)heterochromatin patterns 

heterochromatin- tightly packed

euchromatic- not tightly packed

6)Banding patterns (stains)

1)Single Copy Functional Genes

2)Repetitive DNA

3)Spacer DNA

VIRAL

-really tightly packed

-nonliving particle with protein coat

-nucleic acid RNA or DNA

-double or single stranded

-almost always linear

Bacterial

-single circular DNA molecule

-genes often arranged in operons (genes + single regulatory region)

-not as densely packed as viruses

EUKARYOTIC

-most complex/spaced out

-can be haploid or diploid (generally diploid)

-2n=6

2=ploidy

n=# unique chromosomes

6= total # chromosomes

Organellar DNA

-genes important for organellar function

PLASMID

-found in bacteria and some fungi

-non-essential genes

-autonomously replicating extrachromosomal DNA

-a region of chromosomal DNA that can be transcribed into functional RNA at the correct time and place

-a gene is a physical area on a chromosome

-if in a different area, then is a different gene... all about location!!! (hair color gene in same location for all humans)

-prokaryotic

-gene = transcribed DNA + regulatory regions

-coding region>RNA>protein

-regulatory regions = initiation + termination

-in eukaryotes, genes also include introns (transcribed, but not part of functional gene produce) and exons (transcribed, and remain in functional gene produce)

-mammal gene size around 17kbp

-in prok, about 1000bp

-however, mRNA produced varies very little b/w these

-density also varies... bacterial and simple eukaryotes have densly packed genes.. humans have fewer genes per segment... less dense

DNA is different from RNA because:

1)it contains ribose instead of deoxyribose

2)it is primarily single-stranded... is not double helix (duplexes can form through complementary bonds /w RNA or DNA)

3)it contains uracil in place of thymine (bases are A,C,G,U)

4)RNA is far less stable

PULSE CHASE EXPERIMENTS

Hypothesis: RNA is the intermediate, and will therefore move from the nucleus to the cytoplasm

-label molecules with specific marker

-ie. Sulfur is specific to protein, label /w 35S

-uracil specific to RNA.. label with 32P

-look to see where is radioactive label

-Pshould move from nucleus to cytoplasm

-S was found in cytoplasm

HYPOTHESIS 2

-An increase in specific RNA will precede an increase in specific protein

-bacteria infected with viral particles

-rationale was that virus injects genome into bacterium, and uses bacterial machinery to make protein

-results were 1) an increase in viral RNA precedes increase in viral protein, and 2)sequence of viral RNA is similar to viral DNA

INFORMATIONAL RNA

-provide information template for proetin synthesis (mRNA)

FUNCTIONAL RNA

-RNA that is functional as an RNA molecule and is not translated into a polypeptide

tRNA (transport amino acids to RNA during protein synthesis)

rRNA (component of the ribosome)

snRNA (involved in RNA processing in eukaryotes)

scRNA (small cytoplasmic.... protein trafficking in eukaryotes)

transcription - production of RNA from SNA template

-ineach gene, one DNA strand is used as a template for complimentary base pairing

-the other strand is the non-template strand (the coding strand)

-in each gene, the template strand might be different

****RNA IS MADE FROM 5' to 3' ALWAYS!!

-has to do with order or hydroxyl and phosphate group.... need the hydroxyl to attach

-RNA ends up identical to non-template strand (U for T)

RNA Polymerase are enzymes which drive transcription.

(pol I) - transcribes rRNA genes

(pol II) - transcribes protein coding genes

(pol III) - transcribes tRNA, snRNA, and scRNA

*RNA polymerase exists in different forms depending on the step of transcription.(E-coli)

holoenzyme- used for initiation... consists of 4 subunits... alpha, beta, sigma, and beta prime

core enzyme- used for elongation and termination, consists of only three subunits... alpha, beta, beta prime

Initiation

-a set of DNA sequences called promoter are required to initiate transcription and will determine where a mRNA molecule will start

consensus sequences:

-35 region = TTGACAT, which is recognized by sigma factor

-10 region = TATAAT, opening of the helix by the polymerase

+1 region = 1st nucleotide transcribed, often an A in CAT

Elongation

-within a few bases of +1, the sigma factor dissociates, leaving the core enzyme to continue elongation (RNA polymeraization)

-DNA template is read to create the mRNA molecule

-identity of bases is based on complementarity to the DNA template strand

-new bases are always added to the 3'OH, therefore RNA is synthesized 5' to 3'

Termination

- There ae two mechanisms used to determine where the mRNA molecule will end (3' end)

Intrinsic

-G-C rich region followed by As in the DNA template

-haripin loop formed in mRNA followed by Us

-RNA polymerase pauses due to the formation of the haripin loop (40-60bp)

-the RNA-DNA heteroduplex dissociates (due to weak U-A interactions) asthe polymerase pauses

Rho Dependent

- requires rho protein which binds at the rut site of the mRNA

-once bound to the RNA, rho moves towards the RNA polymerase

-RNA polymerase pauses at a site within the RNA

-rho catches up, and causes RNA polymerase to dissociate from the RNA

-not all genes are transcribed in all cells at al times

-numerous points at which gene expression can be controlled

-most common is at transcription initiation

-activators help RNA polymerase to initiate (positive regulation), repressors keep RNA polymerase from initiating (negative regulation)

-in E. coli -35,-10, and +1 sequences are consensus sequences

-present in many but not all promoters

-a promoter which does not have the ideal consensus sequence needs help from other proteins= activators

-access to promoter may be prevented by other proteins (repressors)

*promoters that deviate from the consensus sequence (weak promoters) may require "activator" proteins to help RNA polymerase to bind

-occurs before mRNA enters cytoplasm

1)addition of a 5' cap of 7-methylguanosine by Guanyltransferase

-this increases stability of the molecule

-will not be exported without this

2)endonuclease cuts 3' end allowing for the addition of a 3' Poly A tail by Poly A polymerase 

-endonuclease recognizes cleavage site (AAUAA... polyadenylation sequence)... and it cuts here

-mRNA is cleaved on 3' end of this sequence

3)Removing of introns and splicing together of exons (expressed sequences)

-conserved sequences at exon-intron boundary are recognized and aligned by snRNP's (protein + snRNAs) to form a splicesome

-OH group in intron attacks the 5' splice site

-5' exon is cleaved, and intron forms a lariat

-3' OH cleaves the 3' splice site, teh lariat is released, and the two exons ligated

-5' splice site is 'GU' in intron

-3' splice site is 'AG' in intron

-branch point - conserved sequences... key nucleotide is A

-during splicing, RNA is associated with a RNA/protein complex, called the splicesome

-snRNP is used for allignment of pre-mRNA for splicing

-get 3 steps of splicing (from text)

*in extreme cases, 1 gene can yield 38000 diferent products (genes) as a result of gene splicing

A polypeptide is a chain of amino acids linked by peptide bonds

-formation of peptide bonds creates water (CCNCCNCCN)

PRIMARY STRUCTURE

-sequence of amino acids from end to end

SECONDARY STRUCTURE
-arises from interactino between neighboring amino acids

-ie. H-bonds form alpha-helix

TERTIARY

-interactions b/w more distant amino acids... ie. disulfide bridges, interactions /w cofactor

-creates a 'sub-unit'

Qaternary

-interactions b/w multiple polypeptides

-dimer- two polypeptides

-tetramer- 4pp

homo- identical

hetero-different

Hypothesis: A particular DNA sequence serves as a template for each polypeptide in a linear fashion.

List expectations of hypothesis and experiments with which they were proved.

Expectaions:

1)Each protein has a unique sequence

2)Mutation in a gene causes a change in amino acid sequence

3)Sequence of nucleotides relates to sequence of amino acids

1)Determining A.A sequence

-Sanger Sequencing - cut up proteins with enzymes (proteolytic enzymes) b/w specific AA to produce specific fragments

-movement through electropheretic field depends on size and charge of fragments, allowing separation of different fragments

-movement in different buffer allows further separation

*****each polypeptide produces a unique fragment

 profile (fingerprint)

2)Do mutations alter protein sequence?

Vernon Ingram (1957) Sickle cell anemia - mutation in a single gene for hemoglobin

-hemoglobin from normal patients vs patients with the disease

-it is a result of valine in the place of another a.a.

***mutation changes a.a. sequence

3)A mutation early in the gene affects the pp. early in the chain, when near the end, translates to the end.

RNA uses 'words' of 3 nucleotides in length to code for a.a.

-But Which word (codon) = Which A.A.??

EXPERIMENT!!

 -synthesize mRNA of known nucleotides, determine what a.a. are translated

-ie. mRNA of only Us

-get polypeptide of only pheylaline

-therefore UUU=phe

- easy for 4 of codons

-then went 25% G + 75% U

-probability of GGG = 1/64 (1/4^3)

-compare resulting frequency of aa to probability of each word.... deduce words (codons)

-6 different codons for Argenine

-no unused condon

Components of Translation and tRNA

Components

-mRNA= template

-aminoacids= building blocks

-tRNA = carry amino acids

-ribosome = machinery

-protein factors= initiation factors (IF)/elongation factors (EF)/ release factors(RF)

tRNA

 -heavily modified RNA

-amino acid acceptance site (3' end) carries a particular amino acid

-anticodon is complementary  and antiparallel to codon in mRNA

-binds to this codon

*******differenttRNAs can code for different amino acids

 WOBBLE

-allows for 1 anticodon to be able to bind to multiple codons

-can have non-specific base pairing

-ie. ACG can band UGC and UGU (though UGC is perfect match)

-woble ALWAYS in third position

-5' nucleotide of anticodon is positioned to allow unusual base pairing (wobble)- anticodon can parit with more than one codon (only tRNA)

Code Degeneracy: 

1)more than 1 tRNA carrying particular a.a

2)a single tRNA can bring a.a. in response to more than one codon b/c of wobble

RULES

5'strand                          3'end codon

G                               C or U

C                               G only

A                               U only

U                               A or G

I                                U,C, A

charging of tRNA is carried out by synthetase... adding amino acid to the top

AUG- always start codon

GGAGG- small subunit (ribosome binding site) (Shine Dalgarno sequence)

-methianine is first amino acid (UAC anti-codon)... it binds to AUG

-CCUCC- rRNA attaches to Shine-Dalgarno sequence to bind

A-site - amino acyl site- where charged tRNA comes to molecule

Psite - holds in place

Esite- exit

-the whole ribosome translocates along mRNA after binding a.a. in P and A site

-3 kinds of stop codon - **no tRNA for stop codon

-release factor enters and binds at A-Site

-translation ends with dissociation of ribosomal units

fMet- first methianine to enter... it has been modified 

-it enters P-site to commence

THEORIES
!)Lock and Key Model

-enzyme and substrate fit perfectly together 

-enzyme remains unchanged

2)Induced Fit..

-enzyme changes shape... more realistic..

-enzyme returns to previous state after 

**specificity of protein structure allows for specificity of function

active site - where a reaction proceeds

mutation- change in DNA sequence

allele- alternate form of a gene (results from a mutation)

genotype - specific allele composition of a gene or set of genes

phenotype - the detectable outward manifestations of a specific genotype

wild type - normal/standard gene OR phenotype

-problematic definition for humans... can be normally what is found in population... is an arbitrary definition

- wild type denoted by superscript"+" (ie. dunce⁺ allele)

-mutant is the same, but without the +

-this is for Drosophila

-in yeast, upper case denotes wild type (ie. WEE vs wee)

-first allele discovered is wee1... then 2,3,4,5,etc

-may or may not result in mutant phenotype depending on consequences of mutation to protein function or quantity or time or place of expression

***check Fig. 3-27)

-different types of mutation based on consequence 

-null, leaky, or silent mutatino

-null -lost function... zero protein function

-mutation is promoter region (sigma can't bind)

--change in sequence of what codes for amino acid (protein) active site (sensitive area)

leaky- mutation on the edge of active site... cna also have leaky mutation in promoter

silent - no effect on function ie. wobble position could get same amino acid

-still a change in DNA sequence... w/out having effect on protein function

******intron mutation can be null!!! change the splicing region..

-could get partial splicing or cut intron out.. but can't put gene back together..

-identify gene function by 'when it isn't working'

-ie. fuse-box analogy... what button is which if all lights are working??

-if everything is functional in wild-type organism, can't know what gene does what

-**geneticists identify genes important to a biological process through mutation and identification of abnormal phenotype

-clone genes to determine what it does at the molecular level

-Gene and function are identified/named based on what happens when gene is malfunctioning

-clal genes based on what was screwed up

-ie. phenotype of flies /w mutant allele

-dunce=no short term memory

-**gene is named dunce, but functional gene allows short term memory

ANOTHER EXAMPLE

-ingest protein... series of enzymes convert it into what we need

-phenylalanin > tyrosine

-if we lose function (mutation) of this enzyme  we end up /w a build up of phe

-body then converts it to phenylpyruvic acid which can be passed through urine

-unfortunately build up affects nervous system... leads to mental retardation

-woman /w 2 retarded  children.. urine smelled

-found in urine... worked backwards from phenotype to find build up of phenylalanine

-** hundreds of mutations can cause this **
-simple fix.... consume less protein

Dominant and recessive alleles - can only be defined in a heterozygous individual

-defined based on the phenotype of a heterozygous individual (a diploid individual who has one wild type and one mutant allele)

-homozygous - gene pair of identical alleles

-heterozygous - gene pair of different alleles

YEAST

-genotype wee/wee.... relatively small cells

-if WEE/wee (hetero) the cells are considerably larger

-obviously big WEE is dominant and therefore wee is recessive

Haplo-Sufficiency

-if one copy of the wild type gene is able to confer the normal phenotype

-wild type is dominant to mutation

Haplo Insufficiency

-one copy of wild type is unable to confer the normal phenotype (wild type is recessive to allele)

WHY might one copy of the normal gene be unable to confer the wild type phenotype??

Eg. wild phenotype requires 35 units of protein

-one allele (A+) is able to produce 20 units, while the other (A) produces 0

-heterozygote produces insufficient protein to confer the wild type phenotype

-gene (A) is haplo-insufficient (mutant allele is dominant)

-Neurospora auxotrophs (wildtype = prototroph)

-created mutant strains unable to synthesize arginine (auxotrophs)

-destroyed strain's ability to grow /w arginine (X-Ray... no DNA discovery yet... didn't know details of how it worked)

-isolated 3 mutant strains (coloration)

-mutation in 3 different genes..

**at least 3 steps in arginine biosynthesis

HYP: If the stepwise pathway is correct, a unique set of compounds will rescue each step..

-had 2 compounds similar to arginine

-all 3 strains can grow when supplemented with arginine

-therefore argenine bypasses all steps... late in pathway

-other two are earlier

??>[Arg 1]>ornithine >[Arg 2]> citrolline >[Arg3]> arginine

***Based on data, each gene produces an enzyme that controls a unique step in the biosynthetic pathway

-ideal at time.... 1 gene = 1 protein.... not entirely true.. due to splicing

EXPERIMENT 2 (cross feeding)

-mutant can't produe something... NO FEEDBACK MECHANISM

-keeps producing required enzyme in hopes that it will work to create next product

-this product will be required by a mutant blocked earlier in the pathway... good thing it is being excreted..

RULE: Downstream Mutants Feed Upstream Mutants

Mechanisms:

conservative (creates new... parents strands don't change)

semi-conservative (1 parental /w 1 new strand ... hybrid)

dispersive (what come back together varies... new/old "smear"

Meselson - Stahl (1958)

-big deal experiment..

-labeled parental DNA by growing E. coli in N15 medium, for many generations

-have to go through lots of generations to label N15... grow on N15 medium

-then transferred to N14 medium

-newly synthesized DNA incorporates only using N14

-centrifuge is CsCl gradient to separate DNA of different densities... DNA types will stop @ different levels... can then separate types out...

SEMI CON!

-in first generation, parental N15/N15 hybrid in middle

-in second generation, should also get N14/N14...

-if conservative, 2nd gen should get N14/N14 and N15/N15

-if dispersive, then would be a smear

WHY SEMI CONSERVATIVE?

-efficient

-few mistakes

-linked to cell division

1)tRNA

-matches a codon in the mRNA to an amino acid

-each tRNA is specific to a particular amino acid

-some amino acids can be carried by tRNAs having different anticodons (accounts for some of the degeneracy in the code)

tRNA consists of:

1)anticodon loop

-anticodon loop contains 3 bases (anticodon) which are complementary to the codon

-anticodon of tRNA binds to codon through complementary base pairing

-flexibility in 3rd nuclotide (5') position of anticodon, allows this nucleotide to pair with nucleotides other than its complementary nucleotide

2)amino acid acceptance site

-3' OH becomes linked to a specific amino acid, through the action of synthetase

2)Synthetase

-each tRNA is recognized by a specific synthetase enzyme, which joins the appropriate amino acid to the tRNA, to produce a charged tRNA = amino-acyl tRNA

-required ATP

3)Ribosome

-structure which positions the mRNA, charged tRNA, and appropriate factors so that peptide bonds can be made between sequential amino acids

-consists of both RNA and protein

-RNA in ribosome interacts with mRNA through complementary base pairing

-in prokaryotes, 2 subunits, 50S and 30S = 70S

-in eukaryotes, 2 subunits, 60S and 40S = 80S

INITIATION

1. Binding of ribosome to mRNA

2.entry of first tRNA 

-30S (small) subunit of ribosome + IF translocates along mRNA

-binds to ribosome binding site (GGAGG) = Shine Dalgarno sequence

-positions the ribosome so that the first codon of the mRNA (initiation codon = AUG) is in P site of ribosome

-AUG in P accepts a specific amino-acyl tRNA

3.IF+GTP+fMet-tRNA --> enters P site

-IFs released, 50S (large) subunit joins

-at the completion of initiation, both subunits of ribosome are attached to the mRNA, with the initiation codon and fMet-tRNA in the P site

ELONGATION

1.entry of aa2-tRNA2 into A site

-required EF and GTP

-aa2-tRNA2 in A site and f-met-tRNA in P-site

2.formation of peptide bond, translocation

-peptide bond forms b/w aa2 and fMet

-releases f-Met from tRNA1

-fMet-aa2-tRNA2 (peptidyl tRNA) in A site, empty tRNA in P-site

-empty tRNA is released

-ribosome moves to position peptidyl-tRNA2 in P site

-translocation requires EF

-aa3-tRNA3 enters A site (requires EF and GTP)

Many cell toxic compounds inhibit polypeptide elongation:

-antibiotic fusidic acid - mimics EF, allowing it to bind to ribosome, but cannot translocate ribosome

-diphtheria toxin - inhibits EF, therefore stops translocation

TERMINATION

-ribosome encounters a STOP codon (UAA, UGA, UAG) (enters A site.. does not have a tRNA)

-3 codons, UAG, UAA, UGA are not recognized by tRNA = STOP codons

-these codons are recognized by RFs, which enter A site instead of a charged tRNA

-entry of RF causes release of polypeptide from P site 

Step 1

-opening the helix

-opens at the origin (A-T rich recognition sequence)

-prokaryotes have single origin, euk have multiple per chromosome)

DnaA -protein that recognizes origin and binds

DnaB- protein (helicase) that recognizes DnaA and opens helix

SSBs- single stranded binding proteins.. keep helix open, strands apart

-replication proceeds bidirectionally from the origin, to produce 2 replication forks

 -elongation in prok. ~1000 bases/second

Step 2a

-priming DNA synthesis

-DNA polymerase must attach new nucleotides to a 3' OH (as /w RNA, DNA synthesis is 5'-3')

***(learn more) 3' OH is provided by a primer of RNA .... made by primase, component of primosome

Step 2b

-synthesizing new DNA

-DNA polymerase III

-adds nucleotides (complementary to parental strand) to 3'OH

leading strand

-strand in which DNA synthesis proceeds in same direction as replication fork opens

lagging strand

-synthesis proceeds in opposite direction as replication fork opens

-is discontinuous

-requires new primers

-the sections are okazaki fragments

-DNA polymerase I removes primer and replaces is with DNA

-DNA ligase forms bond b/w an okazaki fragment and the DNA replacing the primer

TORSION GENERATED

-as helicase (DnaB) separates the two strands of DNA at the replication fork, torsion (supercoiling) is generated

-torsion must be alleviated to prevent strand breakage

-gyrase/topoisomerase cuts the DNA strands ahead of the replication fork, allows them to unwind, and then reforms the phosphdiester backbone

MISTAKES

-DNA polym makes about 1/billion mistakes, roughly

-epsilon subunit proofreads as DNA is made

-recognizes and corrects errors

-it is a subunit of DNA polym III

Telomere Problem

-chromosomes will shorten with each successive round of DNA replication

-in linear chromosomes, removal of primer on discontinuous strand leaves a gap

-telomerase adds nucleotides to the 3' end to provide a buffer zone

-adds repeated DNA sequence to 3' end using a RNA template

-provides a buffer zone for shortening

-expressed in reproductive cells and stemcells (bone marrow)

-related to ageing?

the end result

-two identical DNA molecules, each with one parental (template) and one newly synthesized strand

-in eukaryotes, each daughter molecule is a sister chromatid

Bacterial chromosomes

-replication proceeds from a single origin

Plasmids

-rolling circle replication

-one strand cut, provides 3'OH for continuous synthesis from circular strand template

-displaced 5' end is a template for discontinuous synthesis

-before DNA replication, in G1 each chromosome are "single stranded" = 1 duplex DNA molecule = 1 chromatid

-after DNA replication, each chromosome is "double stranded", consisting of 2 DNA molecules (chromatids) linked by a condensed region, called the centromere.

-genetically identical daughter cells are produced

fission - prokaryotes

mitosis - eukaryotes

***************mitosis and meiosis are the division of the nucleus, NOT the cell (this is cytokinesis)*********

1)hold chromatids together while new cells are forming

-the centromere does this... holds chromatids together until separation during mitosis

2)separate chromatids in an organized fashion

-spindle fibres - microtubules which attach to the centromeres via kinetochore and move chromatids apart

1)Interphase (G1, S, G2)

-replication of DNA, growth of cell

2)Mitosis

a)PROPHASE

-condensation of chromosomes.. become visible

-breakdown of nuclear membrane

-mitotic spindle forms (outside of nucleus)

b)METAPHASE

-formation of spindle (microtubules)

-attatchment of 2 spindles to each centromere

-movement of chromosomes to the metaphase (equatorial) plate

c)ANAPHASE

-division of centromeres

-separation of chromatids... form chromosomes

d)TELOPHASE

-migration to poles is complete

-nuclear envelope reforms

-cytokinesis may or may not immediately occur.... in animals this occurs via cleavage furrow, while in plants at the cell plate

-multiprotein complex that binds to centromere

-site of attachment for microtubules

Sexual Reproduction...

How is chromosome number maintained in offspring relative to the parents?

Compare mitosis/meiosis

-involves cell fusion

-the gamete must have 1/2 the number of chromosomes that the parent does

homologous pair- 2 chromosomes which have the array of genes, although they may have different alleles of those genes (pair at meiosis)

diploid individuals- contain a pair of each chromosome

haploid individuals- contain only one member of each homologous pair

mitosis

-produces 2 identical cells

-each cell must contain 1 copy of each chromatid

-chromatids (copies) held together by the centromere during allignment

-chromatids are separated to the poles by spindle fibres

meiosis

-produces 4 haploid cells from 1 diploid cell

-each product must receive one member of the homologous chromosome pair

-4 haploid gametes are produced at the completion of meiosis, each containing a single-stranded chromosome (1 chromatid) of one member of each homologous pair

How does meiosis ensure that each daughter cell receives one member of each homologous pair?

Division 1 - align homologous chromosomes, separate one homologue to each pole

-reductional division - chromosome number is reduced by 1/2 per cell

Division 2 - align chromosomes, consisting of 2 chromatids held together at the centromere, and separate one chromatid to each pole

-equitorial division- chromosome number is maintained

MEIOSIS I

a)PROPHASE I

-leptotene -chromosomes become visible

-zygotene - pairing of homologues, crossing over (b/w non-sister chromatids... they are bound together)

-pachytene - synaptonemal complex complete

-diplotene - slight separation to reveal chiasmata (points of crossing over)

-diakinesis - further contraction

b)METAPHASE I

-homologous pairs move to the metaphase plate

-centromeres attach to spindle

c)ANAPHASE I

-one homologue moves to each pole (random)

-chromatids remain attached by centromeres, thus do NOT separate (in mitosis, split b/w chromatids occurs)(this is why it is reduction)

d)TELOPHASE

-may not be distinct

-chromosomes become diffure?

-formation of membrane MAY occur

-cytoplasmic division MAY occur

MEIOSIS II (similar to mitosis)

a)PROPHASE II

-chromosomes contract (if they've relaxed)

b)METAPHASE II

-chromosomes at metaphase plate, attachment of centromeres

c)ANAPHASE II

-centromeres split, chromatids to opposide poles

d)TELOPHASE II

-reformation of nucleus

----

-Product of Meiosis I = 2 "cells" with one member of each homologous pair (n)

-each member consists of 2 chromatids

-Product of Meiosis II = 4 cells with one member of each homologous pair (n)

-each member now consists of a single chromatid

-both haploid and diploid lifecycles involve meiosis to form haploid cells, and fusion of those cells to generate a diploid organism

-difference is whether mitosis occurs in the haploid stage (haploid life cycle) or in the diploid stage (diploid life cycle) of the life cycle

-in both lifecycles, a diploid meiocyte produces four haploid daughter cells

-both life cycles alternate b/w diploid and haploid stages, each employ mitosis and meiosis

-difference is the stage of the life cycle at which mitosis occurs to create a "non-transient" organism

-in the haploid life cycle this occurs at the haploid stage

-in the diploid life cycle this occurs at the diploid stage