Bio Exam 4

Extensions to Mendel

Mendel's model of inheritance assumes that: -each trait is controlled by a single gene -each gene only has two alleles -there is a dominant-recessive relationship between the alleles Most genes do not meet this criteria

Meselson and Stahl

-bacterial cells were grown in a heavy isotope of nitrogen, N15 -cells were switched to a media containing lighter N14 -DNA was extracted from the cells at various time intervals -Conservative model=rejected (2 densities were not observed after round 1) -Semiconservative model=supported (consistent with all observations; 1 band after round 1; two bands after round 2) -dispersive model=rejected (1st round results consistent; 2nd round did not observe 1 band)

DNA replication requires 3 things

-something to copy: parental DNA molecule -something to do the copying: enzymes -building blocks to make copy: nucleotide triphosphates

DNA polymerase III

synthesizes DNA; forms the phosphodiester bond between adjacent nucleotides in a new DNA acid chain in 5' to 3' direction

RNA polymerase II

transcribes mRNA

RNA polymerase I

transcribes rRNA

RNA polymersae III

transcribes tRNA

DNA polymerase I

erases primer and fills gap; has a proofreading function that corrects errors in replication

Codominance

no single allele is dominant and each allele has its own effect

Semiconservative

old/new + old/new

Conservative

old/old + new/new

Dispersive

mixed old and new on each strand

Complementity of bases

A forms 2 hydrogen bonds with T G forms 3 hydrogen bones with C gives consistent diameter

ABO gene encodes a surface protein

Allele A makes A protein Allele B makes B protein Allele O makes no protein -alleles A and B can be present on the cell surface at the same time -they are codominant (AB) -Allele O is recessive to both A and B alleles

Chargaff's Rules

Erwin Chargaff determined that: -amount of adenine=amount of tymine -amount of cytosine=amount of guanine -always equal proportion of purines (A and G) and pyrimidines (C and T)

DNA ligase

Primer RNA removal and replacement; joins the ends of DNA segments; DNA repair; catalyzes the formation of the final bond connecting the two precursors

Double helix

-2 strands are polymers of nucleotides -phosphodiester backbone-repeating sugar and phosphate units joined by phosphodiester bonds -wrap around 1 axis -Antiparallel

E. coli polymerases

-DNA polymerase I (pol I): acts on lagging strand to remove primers and replace them with DNA -DNA polymerase II (pol II): involved in DNA repair processes -DNA polymerase III (pol III): main replication enzyme -all 3 have 3' to 5' exonuclease activity- proofreading -DNA pol I has 5' to 3' exonuclease activity

Semidiscontinuous

-DNA polymerase can synthesize only in 1 direction -Leading strand synthesized continuously from an initial primer -lagging strand synthesized discontinuously with multiple priming events (okazaki fragments)

Replication Process

-DNA polymerase cannot link the first nucleotides in a newly synthesized strand ( RNA polymerase (primase) constructs an RNA primer) -DNA polymerase adds nucleotides to 3' end (leading strand replicates toward replication fork; lagging strand elongates from replication fork; okazaki fragments)

Chromatin= DNA and associated proteins

-DNA winds around histone proteins (nucleosomes) -other proteins wind DNA into more tightly packed form, the chromosome -unwinding portions of the chromosome is important for mitosis, replication, and making RNA

Lagging strand synthesis

-Discontinuous synthesis (DNA pol III) -RNA primer made by primase for each Okazaki fragment -All RNA primers removed and replaced by DNA (DNA pol I) -Backbone sealed (DNA ligase) -Termination occurs at specific site (DNA gyrase unlinks 2 copies)

Helicase

unwinds the double helix at the replication fork to separate the parental strands

Prokaryotic Replication

-E. coli model -single circular molecule of DNA -replication begins at one origin of replication -proceeds in both directions around the chromosome -replicon: DNA controlled by an origin

Eukaryotic DNA replication

-Eukaryotes usually have multiple, large chromosomes -multiple origins of replication -Complicated by: larger amount of DNA in multiple chromosomes Linear structure -Basic enzymology is similar (requires new enzymatic activity for dealing with ends only)

Discovery of the genetic code

-Francis Crick and Sydney Brenner determined how the order of nucleotides in DNA encoded amino acid order -Codon- block of 3 DNA nucleotides corresponding to an amino acid -introduced single nucleotide insertions or deletions and looked for mutations (frameshift mutations) -indicates importance of reading frame

Elongation

-Grows 5' to 3' direction as ribonucleotides are added -Transcription bubble-contains RNA polymerase, DNA template, and growing RNA transcript -After the transcription bubble passes, the now transcribed DNA is rewound as it leaves the bubble

Eukaryotic pre-mRNA splicing

-Introns- non-coding sequences -Exons-sequences that will be translated -Small ribonucleoprotein particles (snRNPs) recognize the intron-exon boundaries -snRNPs cluster with other proteins to form spliceosome (responsible for removing introns)

DNA primase

-RNA polymerase that makes RNA primer -RNA will be removed and replaced with DNA

RNA

-all synthesized from DNA template by transcription -messenger RNA (mRNA) -Ribosomal RNA (rRNA) -Transfer RNA (tRNA) -Small nuclear RNA (snRNA) -signal recognition particle RNA -Micro-RNA (miRNA)

The prokaryotic translation initiation complex consists of

-an mRNA molecule -a special initiator tRNA charged with N-formylmethionine -a small ribosomal subunit

Phosphodiester Bond

-bond between adjacent nucleotides -formed between the phosphate group of one nucleotide and the 3' -OH of the next nucleotide The chain of nucleotides has 5'-to-3' orientation

Hammerling Experiment

-cells of green alga were cut into pieces and observed -discovered hereditary information is stored in the cell's nucleus -nucleus in the base determines type of cap regenerated

DNA structure

-composed of nucleotides -5 C sugar called deoxyribose -Phosphate group (attached to 5' carbon sugar) -Nitrogenous base (adenine, thymine, cytosine, guanine -Free hydroxyl group (-OH) (attached to 3' carbon of sugar

Central Dogma

-created by Francis Crick -Information only flows from DNA--->RNA---->protein -transcription= DNA---> RNA -translation= RNA---> proteins -retroviruses violate this order using reverse transcriptase to convert their RNA genome into DNA

James Watson and Francis Crick

-deduced the structure of DNA using evidence from Chargaff, Franklin, and others -did not perform any experiments themselves -proposed double helix structure

Beadle and Tatum

-deliberately set out to create mutations in chromosomes and verify that they behaved in a Mendelian fashion in crosses -studied Neurospora crassa -looked for nutritional mutations (had to have minimal media supplemented to grow) -looked for fungal cells lacking specific enzymes (the enzymes were required for the biochemical pathway producing the amino acid arginine; they identified mutants deficient in each enzyme of the pathway) -one-gene/one-enzyme hypothesis has been modified to one-gene/one-polypeptide hypothesis

Replication as a process

-double stranded DNA unwinds -the junction of the unwound molecules is a REPLICATION FORK -a new strand is formed by pairing complementary bases with the old strand -two molecules are made. Each has one new and one old DNA strand

Telomerase

-enzyme makes telomere of lagging strand using and internal RNA template (not the DNA itself) (leading strand can be replicated to the end) -telomerase developmentally regulated (relationship between senescence and telomere length -cancer cells generally show activation of telomerase

Replisome

-enzymes involved in DNA replication form a macromolecular assembly -2 main components: -Primosome (primase, helicase, accessory proteins) -Complex of 2 DNA pol III (one for each strand)

DNA repair

-errors due to replication (DNA polymerases have proofreading ability) -Mutagens- any agent that increases the number of mutations above background level (radiation and chemicals) -importance of DNA repair is indicated by the multiplicity of repair systems that have been discovered

How is the process of translation initiation different between eukaryotes and prokaryotes?

-eukaryotic mRNAs do not have a ribosome binding sequence -In eukaryotes, the initiating amino acid is methionine, not N-formylmethionine

Promoter

-forms a recognition and binding site for the RNA polymerase -found upstream of the start site -not transcribed -Asymmetrical: indicate site of initiation and direction of transcription -promoter=nucleotide sequence 5' to the transcription start site, which is the initial binding site of RNA polymerase and transcription initiation fact -promoter recognition by RNA polymerase is a prerequiste for transcription initiation -many promoters contain a similar DNA sequence= TATAAT= "TATA" box (-10) is a consensus sequence of many promoters -consensus promoter sequence at -35= TTGACA -transcription termination sites are inverted repeat sequences which can form loops in RNA=stop signal

Autosomal Recessive Inheritance

-heterozygotes carry the recessive allele but exhibit the wildtype phenotype -males and females equally affected -may skip generations

Watson-Crick model of DNA replication

-hydrogen bonds between DNA bases break to allow strand separation -Each DNA strand is a template for the synthesis of a new strand -Template (parental) strand determines the sequence of bases in the new strand (daughter)=complementary base pairing rules

Incomplete dominance

-indicates the heterozygous phenotype is distinct from either homozygous phenotype -typically intermediate to the homozygous phenotype

stages of replication

-initiation: always occurs at the same site -elongation: majority of replication spent in elongation -termination: exact details unclear

Hershey and Chase

-investigated bacteriophages: viruses that infect bacteria -bacteriophage was composed of only DNA and protein -wanted to determine which of these molecules is the genetic material that is injected into the bacteria -Bacteriophage DNA was labeled with radioactive phosphorus P32 -Radioactive molecules were tracked -Conclusion: DNA is the genetic material

Transcription bubble

-it contains an RNA-DNA hybrid, about 9 nt long -it moves along the DNA at a rate of about 50 nt/sec -the growing RNA strand protrudes from the bubble

Autosomal Dominant Inheritance

-located on the non-sex chromosomes -heterozygotes exhibit the affected phenotype -males and females are equally affected -Affected phenotype does NOT skip a generation

Termination

-marked by sequence that signals "stop" to polymerase -causes the formation of phosphodiester bonds to cease -RNA-DNA hybrid within the transcription bubble dissociates -RNA polymerase releases the DNA -DNA rewinds -Hairpin

DNA polymerase

-matches existing DNA bases with complementary nucleotides and links them -All have several common features: -add new bases to the 3' end of existing strands -synthesize in 5' to 3' direction -require a primer of RNA

Multiple Alleles

-may be more than 2 alleles for a gene in a population -ABO types of blood in humans (3 alleles) -# of alleles possible for any gene is constrained but usually more than two alleles exist for any gene in an outbreeding population

Multiple replicons

-multiple origins of replications for each chromosome -not sequence specific; can be adjusted

Excision repair

-nonspecific repair -damaged region is removed and replaced by DNA synthesis 3 steps: 1. recognition of damage 2. removal of the damaged region 3. Resynthesis using the information on the undamaged strand as a template

Polygenic inheritance

-occurs when multiple genes are involved in controlling the phenotype of a trait -the phenotype is an accumulation of contributions by multiple genes -these traits show continuous variation and are referred to as quantitative traits ex. human height

DNA synthesis

-one strand of the newly made DNA is synthesized continuously= leading strand -lagging strand is made in small precursor fragments= okazaki fragments -the size of okazaki fragments is 100-200 base pairs

Transcription- RNA polymerase

-only one of two DNA strands (template or antisense strand) is transcribed -non-transcribed strand is termed coding strand or sense strand -in both bacteria and eukaryotes, the polymerase adds ribonucleotides to the growing 3' end of an RNA chain (synthesis proceeds in 5' to 3' direction) -Components of the prokaryotic core RNA polymerase: One beta subunit, one beta prime subunit, two idential alpha subunits

Recessive pedigree- albinism

-pedigree for form of albinism due to a nonfunctional allele of the enzyme tyrosinase -males and females affected equally -most affected individuals have unaffected parents

Rosalind Franklin

-performed xray diffractoin studies to identify the 3D structure -discovered that DNA is helical -using Maurice Wilkin's DNA fibers, discovered that the molecule has a diameter of 2 nm and makes a complete turn of the helix every 3.4 nm

Which of the following enzymes involved in DNA replication are found at the replication fork in all three types of cells (bacterial, archaeal, and eukaryotic)?

-polymerases -helicase -sliding clamp -clamp loader -primase

Primosome

-primase and helicase -initiates strand synthesis by forming RNA primer; primase synthesizes RNA primers

Alternative splicing

-produce multiple transcripts from the same gene in eukaryotes -single primary transcript can be spliced into different mRNAs by the inclusion of different sets of exons -15% of known human genetic disorders are due to altered splicing -35-59% of human genes exhibit some form of alternative splicing -explains how 25,000 genes of the human genome can encode the more than 80,000 different mRNAs

Operon

-prokaryotic transcription is coupled to translation -mRNA begins to be translated before transcription is finished -operon: grouping of functionally related genes, multiple enzymes for a pathway, can be regulated together

The eukaryotic initiation complex consists of

-promoter -transcription factors -RNA polymerase II

Archibald Garrod

-recognized that alkaptonuria is inherited via recessive allele -proposed that patients with the disease lacked a particular enzyme -these ideas connected genes to enzymes

Avery experiment

-removed almost all lipid and protein from bacteria, and found no reduction in transforming activity -DNase destroyed all transforming activity

Initiation phase

-requires more factors to assemble both helicase and primase complexes onto the template, then load the polymerase with its sliding clamp unit -Primase includes both DNA and RNA polymerase -Main replication polymerase is a complex of DNA polymerase epsilon (pol epsilon) and DNA polymerase delta (pol delta)

Gel Electrophoresis

-separate DNA fragments by size -Gel made of agarose -submersed in buffer that can carry current -subjected to an electrical field -moves negative to positive -larger fragments move slower, smaller move faster -DNA is visualized using ethidium bromide of fluorescent dyes

Prokaryotic transcription

-single RNA polymerase -initiation of mRNA synthesis does not require a primer -Requires: promoter, start site, termination site (all together is a transcription unit)

Prokaryotic Transcription

-single RNA polymerase exists as core and holoenzyme -the core enzyme consists of two identical alpha subunits, one beta subunit and a beta' subunit. Active site is formed by the beta and beta' subunit. Two alpha subunits hold the complex together and bind regulatory molecules -Holoenzyme is core enzyme + sigma subunit. the sigma subunit recognizes promotorer elements at -35 and -10 and binds to the DNA

Differences between prokaryotic and eukaryotic transcription

-single factors allow recognition of start site in prokaryotes but there is a whole host of transcription factors in eukaryotes -termination in prokaryotes is caused by the formatino of the hairpin loop. This stretch of A-U base pairing makes the RNA-DNA hybrid less stable and it falls off the enzyme -In eukaryotes, the end of transcription is not defined by RNA polymerase since the transcript undergoes modification-capping at the 5' end and addition of the poly-A tail at the 3' end

Leading strand synthesis

-single priming event -strand extended by DNA pol III -Processivity- beta subunit "sliding clamp" to keep it attached -it is the number of nucleotides that are added before the polymerase dissociates from the template (in a single template binding event). -Increase in DNA replication efficiency increases with increase in processivity

Telomeres

-specialized structures found on the ends of eukaryotic chromosomes -composed of short repeated sequences of DNA -protect ends of chromosomes from nucleases and maintain the integrity of linear chromosomes -gradual shortening of chromosomes with each round of cell division (unable to replicate last section of lagging strand)

Photorepair

-specific repair mechanism -for one particular form of damage caused by UV light -Thymine dimers: covalent link of adjacent thymine bases in DNA -Photolyase: absorbs light in visible range and uses this energy to cleave thymine dimer

RNA Synthesis

-the nucleotide sequence in the transcribed mRNA is complementary to the base sequence in DNA -RNA is copied from the template strand which is 3' to 5' in the 5' to 3' direction=antiparallel -RNA synthesis does not require a primer and proceeds by the addition of nucleotides to form mRNA chain

Eukaryotic transcription differs from prokaryotic transcription:

-three RNA polymerase enzymes: 1. RNA POL I transcribes rRNAs 2. RNA POL II transcribes mRNAs 3. RNA POL III transcribes tRNAs -initiation complex forms at promoter -each polymerase recognizes a different promoter structure -mRNAs are modified after transcription

Coiling

-unwinding DNA causes torsional strain -Helicases- use energy from ATP to unwind DNA -Single-strand binding proteins (SSBs) coat strands to keep them apart -Topoisomerase prevent supercoiling ( DNA gyrase is used in replication)

Transcription steps

1. DNA is used as a template for creation of RNA using the enzyme RNA polymerase 2. RNA polymerase reads the nucleotides on the template strand from 3' to 5' and creates an RNA molecule that looks like the coding strand 3. the new RNA molecule is formed by incorporating nucleotides that are complementary to the template strand

Steps for replication

1. DNA polymerase III enzyme is active on each strand. Primase synthesizes new primers for the lagging strand 2. The "loop" in the lagging strand template allows replication to occur 5' to 3' on both strands, with the complex moving to the left 3. When the polymerase III on the lagging strand hits the previously synthesized fragment, it releases the beta clamp and the template strand. DNA polymerase I attaches to remove the primer 4. The clamp loader attaches the beta clamp and transfers this to polymerase III, creating a new loop in the lagging strand template. DNA ligase joins the fragments after DNA polymerase I removes the primers 5. After the beta clamp is loaded, the DNA polymerase III on the lagging strand adds bases to the next Okazaki fragment

DNA repair categories

1. Specific repair: targets a single kind of lesion in DNA and repairs only that damage 2. Nonspecific: use a single mechanism to repair multiple kinds of lesions in DNA

replication steps

1. heliase protein binds to DNA sequences called origins and unwinds DNA strands 2. Primosome makes a short segment of RNA complementary to the DNA, a primer. 3. DNA polymerase enzyme adds DNA nucleotides to the RNA primer 4. Leading strand synthesis continues in a 5' to 3' direction 5. Discontinuous synthesis produces 5' to 3' DNA segments called Okazaki fragments

splicing steps

1. snRNA forms base-pairs with 5' end of intron, and at branch site 2. snRNPs associate with other factors to form spliceosomes 3. 5' end of intron is removed and forms bond at branch site, forming a lariat. The 3' end of the intron is then cut 4. Exons are joined: spliceosomes disassembles 1. The 5' end of an intron is cleaved 2. The 5' end of the intron is attached to the 2' OH of the branch point A 3. The 3' end of the first exon displaces the 3' end of the intron 4. The intron is released as a lariat

Eukaryotic mRNA modifications

In eukaryotes, the primary transcript must be modified to become mature mRNA: 1. Addition of a 5' cap: protects from degradation;involved in translation initiation 2. Addition of a 3' poly A-tail: created by poly-A polymerase; protection from degradation 3. Removal of non-coding sequences (introns): Pre-mRNA splicing done by spliceosome

I^A

adds galactosamine

I^B

adds galactose

I

adds no sugar

Environment effects on phenotype

degree of allele expression may depend on the environment ex. coat color in Himalayan rabbits and Siamese cats -allele produces an enzyme that allows pigment production only at temps below 30 degrees Celsius

Griffith Experiment

documented movement of genes from one organism to another (transformation) -movement of material can alter the genetic makeup of the recipient cell

Continuous Variation

greater number of genes influencing a character, more continuous the expected distribution of a character will be

The sliding clamp of a DNA polymerase

holds the polymerase to the DNA template

ABO blood groups

human gene that encodes enzyme that adds sugar molecules to lipids on the surface of RBCs

Pleiotropic Effects

individual alleles often have more than one effect on the phenotype

Epistasis

one gene interferes with the expression of another gene ex. coat color in Labradors -H gene is epistatic to the ABO gene -H protein attaches the A or B protein to the cell surface -without H protein the A or B antigens can not be attached to the cell -hh genotype=no H protein -all hh genotypes have the phenotype of type O

Replication fork

partial opening of helix forms replication fork

DNA gyrase

relieves torque; topoisomerase II introduces a double stranded break ahead of the replication fork and swivels the cleaved ends to relieve the stress of helix unwinding

Single-strand binding protein

stabilizes single stranded regions of DNA at the replication fork

What subunit of DNA polymerase III forms the sliding clamp?

the beta subunit

The enzymes in the replisome are active on

the leading and lagging strands

Replication

the process of making new copies of DNA molecules