BIOL 106 Final Exam

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Frederick Griffith- 1928

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Frederick Griffith- 1928

  • Studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia

  • 2 strains of Streptococcus

    • S strain is virulent (causes pneumonia)

    • R strain is nonvirulent

  • Griffith infected mice with these strains hoping to understand the difference between the strains

  • Transformation

    • Information specifying virulence passed from the dead S strain cells into the live R strain cells

  • Out modern interpretation is that genetic material was actually transferred between the cells

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Avery, MacLeod, & McCarty-1944

  • Repeated Griffith’s experiment using purified cell extracts

  • Removal of all protein from the transforming material did not destroy its ability to transform R strain cells

  • DNA-digesting enzymes destroyed all transforming ability

  • Supported DNA as the genetic material

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Hershey & Chase-1952

  • 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 (32P)

    • DNA contains phosphorus, proteins do not

  • Bacteriophage protein was labeled with radioactive sulfur (35S)

    • Some amino acids contain sulfur, DNA does not

  • Radioactive molecules were tracked

  • Only the bacteriophage DNA (as indicated by the 32P) entered the bacteria as was used to produce more bacteriophage

  • Conclusion: DNA is the genetic material

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DNA structure

  • DNA is a nucleic acid

  • Polymer of nucleotides

    • 5-carbon sugar called deoxyribose

    • Phosphate group (PO4)

      • Attached to 5’ carbon of sugar

    • Nitrogenous base

      • Adenine, thymine, cytosine, guanine

    • Free hydroxyl group (-OH)

      • Attached at the 3’ carbon of sugar

  • Phosphodiester bond

    • Bond between adjacent nucleotides

    • Formed between the phosphate group of one nucleotide and the 3’-OH of the nucleotide

  • The chain of nucleotides has a 5’-to –3' orientation

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Chargaff’s Rules (1950)

  • Erwin Chargaff determined that

    • Amounts of A, G, T, C varied between different species, but not within tissues of same individual (or members of the same species)

    • Amount of adenine =amount of thymine

    • Amount of cytosine =amount of guanine

    • Always an equal proportion of purines (A and G) and pyrimidines (C and T)

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Rosalind Franklin

  • Performed X-ray diffraction studies to identify the 3-D structure

    • Discovered that DNA is helical (“a spiral structure in a macromolecule that contains a repeating pattern”)

    • Using Maurice Wilkins’ DNA fibers, discovered that the molecule has a diameter of 2 nm and makes a complete turn of the helix every 3.4 nm

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James Watson and Francis Crick-1953

  • Deduced the structure of DNA using evidence from Chargaff, Franklin, and others

  • Did not perform a single experiment themselves related to DNA

  • Proposed a double helix structure

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Double helix

  • 2 strands are polymers of nucleotides

  • Phosphodiester backbone-repeating sugar and phosphate units until joined by phosphodiester bonds

  • Wrap around 1 axis

  • Antiparallel

  • Complementarity of bases

  • A forms 2 hydrogen bonds with T

  • G forms 3 hydrogen bonds with C

  • Gives consistent diameter (which was noted w/X-Ray diffraction)

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DNA Replication

  • 3 possible models

    • Conservative model

      • Produces one entirely new molecule, while conserving the old

    • Semiconservative model

      • Produces two hybrid molecules of old and new strands

    • Dispersive model

      • Produces two hybrid molecules with each strand a mixture of old and new

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Meselson and Stahl- 1958

  • Bacterial cells were grown in a heavy isotope of nitrogen, 15N

  • All the DNA incorporated 15N

  • Cells were switched to media containing lighter 14N

  • DNA was extracted from the cells at various time intervals- mixed with CsCI & spun down in a centrifuge (resulting in heaviest DNA at the bottom)

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Conservation model=rejected

2 densities were not observed after round 1

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Semiconservative model=supported

  • Consistent with all observations

  • 1 band after round 1

  • 2 bands after round 2

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Dispersive model=rejected

  • 1st round results consistent

  • 2nd round- did not observe 1 band

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DNA replication require 3 things

  • Something to copy

    • Parental DNA molecule

  • Something to do the copying

    • Enzymes

  • Building blocks to make copy

    • Nucleotide triphosphates

  • DNA replication include

    • Initiation: replication begins

    • Elongation: new strands of DNA are synthesized by DNA polymerase

    • Termination: replication is terminated

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DNA polymerase

  • Matches existing DNA bases with complementary nucleotides and links them

  • All have several common features

    • Add new bases to 3’ end of existing strands

    • Synthesize in 5’ to 3’ direction

    • Require a primer of RNA

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Prokaryotic Replication

  • E. coli model

  • Single circular molecule of DNA

  • Replication begins at one origin of replication (oriC)

  • Proceeds in both directions around the chromosome

  • Replicon- DNA controlled by an origin

  • E. coli has at least 3 DNA polymerases

    • DNA polymerase 1 (pol 1)

      • Acts on lagging strand to remove primers and replace them with DNA

    • DNA polymerase 2 (pol 2)

      • Involved in DNA repair processes

    • DNA polymerase 3 (pol 3)

      • Main replication enzyme

    • All 3 have 3’ to 5” exonuclease activity- proofreading

    • DNA pol 1 has 5’ to 3’ exonuclease activity

  • Unwinding DNA causes torsional strain

    • Helicases: use energy from ATP to unwind DNA

    • Single-strand-binding proteins (SSBs) coat strands to keep them apart

    • Topoisomerases prevent supercoiling

      • DNA gyrase is used in replication

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Nucleases which cuts DNA internally

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Nucleases which chew away at an end of DNA

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  • 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

  • DNA is antiparallel (one strand is 5’ to 3’ & other is 3’ to 5’)

  • DNA polymerase adds nucleotides only at the 3’ end

  • Partial opening of helix forms replication fork

  • DNA primase: RNA polymerase that makes RNA primer

    • RNA will be removed and replaced with DNA

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Leading-strand synthesis

  • Single priming event

  • Strand extended by DNA pol 3

    • Processivity- β subunit forms “sliding clamp” to keep it attached

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Lagging-strand synthesis

  • Discontinuous synthesis

    • DNA pol 3

  • RNA primer made by primase for each Okazaki fragment

  • All RNA primers removed and replaced by DNA

    • DNA pol 1

  • Backbone sealed

    • DNA ligase

  • Termination occurs at specific site

    • DNA gyrase unlinks 2 copies

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  • Enzymes involved in DNA replication form a macromolecular assembly

  • 2 main components

    • Primosome

      • Primase, helicase, accessory proteins

    • Complex of 2 DNA pol 3

      • One for each strand

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Replication fork

  1. A DNA polymerase 3 enzyme is active on each strand. Primase synthesizes new primers for 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 3 on the lagging strand hits the previously synthesized fragment, it releases the β clamp and the template strand. DNA polymerase 1 attaches to remove the primer

  4. The clamp loader attaches the β clamp and transfers this to polymerase 3, creating a new loop in the lagging-strand template. DNA ligase joins the fragments after DNA polymerase 1 removes the primers

  5. After the β clamp is loaded, the DNA polymerase 3 on the lagging strand adds basses to the next Okazaki fragment

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Eukaryotic 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

  • Multiple replicons: multiple origins of replications for each chromosome

    • Not sequence specific; can be adjusted

  • Initiation phase of replication 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 ε) and DNA polymerase delta (pol δ)

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  • Specializaed structures found on the ends of eukaryotic chromosomes

  • 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

  • Replication potentially shortens the ends of eukaryotic chromosomes

  • The ends of linear chromosomes are maintained by the action of the telomerase enzyme

  • Telomeres composed of short repeated sequences of DNA

  • Telomerase: enzyme makes telomere section of lagging strand using an 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

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DNA repair

  • Mistakes during replication are detected and repaired by DNA polymerase proofreading activity

  • Proofreading by DNA polymerase corrects errors during replication

  • Mutagens: any agent that increases the number of mutations above background level

    • Radiation and chemicals

  • Importance of DNA repair indicated by the multiplicity of repair systems that have been discovered

  • Falls into 2 general categories

    • Specific repair

      • Targets a single kind of lesion in DNA and repairs only that damage

    • Nonspecific

      • Use a single mechanism to repair multiple kinds of lesions in DNA

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Photo repair

  • 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

    • Uses this energy to cleave thymine dimer

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Excision repair

  • Nonspecific repair

  • Damaged region is removed and replaced by DNA synthesis

  • 3 steps

    • Recognition of damage

    • Removal of the damaged region

    • Resynthesis using the information on the undamaged strand as a template

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Uncorrected replication errors result in mutations

  • Replication is not a perfect process. Errors occur and if uncorrected produce mutations

  • Mutations are changes in the nucleotide sequence of the DNA strands

  • Mutations can lead to changes in the protein sequence encoded by the DNA

  • There are several types of mutations

  • Point mutations

    • Silent

    • Missense

    • Nonsense

  • Frameshift mutations

    • Insertions

    • Deletions

  • Chromosome mutations

    • Insertions, deletions, translocations, inversions, fusions, duplications

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The nature of genes

  • Early ideas to explain how genes work came from studying human disease

  • Archibald Garrod- 1902

    • Recognized that alkaptonuria is inherited via a recessive allele

    • Proposed that patients with the disease lacked a particular enzyme

  • These ideas connected genes to enzymes

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Beadle and Tatum- 1941

  • Deliberately set out to create mutations in chromosomes and verify that they behaved in a Mendelian fashion in crosses

  • Studied Neurospora crassa

    • Used X-rays to damage DNA

    • Looked for nutritional mutations

      • Had to have minimal media supplemented to grow

  • Beadle and Tatum 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 (even this is overly simple)

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Central dogma

  • Information typically flows from DNA->RNA->protein

  • Transcription= DNA-> RNA

  • Translation= RNA-> protein

  • Retroviruses & some mobile elements violate this order using reverse transcriptase to convert their RNA genome into DNA

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  • DNA-directed synthesis of RNA

  • Only template strand of DNA used (the other is called the “coding strand” or “sense strand”)

  • U (uracil) in DNA replaced by T (thymine) in RNA

  • MRNA used to direct synthesis of polypeptides

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  • Synthesis of polypeptides

  • Takes place at ribosome

  • Requires several kinds of RNA

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Prokaryotic transcription

  • Single RNA polymerase

  • Initiation of mRNA synthesis does not require a primer (short stretch of nucleic acids)

  • Requires: (transcription unit)

    • 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

    • Start site

    • Termination site

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  • All synthesized from DNA template by transcription

  • Messenger RNA (mRNA)

  • Ribosomal RNA (rRNA)

  • Transfer RNA (tRNA)

  • Small nuclear RNA (snRNA)

  • Signal recognition particle RNA (SRP RNA)

  • Micro-RNA (miRNA)

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Genetic Code

  • Francis Crick and Sydney Brenner determined how the order of nucleotide 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

  • Marshall Nirenberg identified the codons that specify each amino acid (1961-66)

  • Stop codons

    • 3 codons (UUA, UGA, UAG) used to terminate translation

  • Start codon

    • Codon (AUG) used to signify the start of translation

  • Code is degenerate, meaning that some amino acids are specified by more than one codon

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Spaced codons

Codon sequence in a gene punctuated

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Codons adjacent to each other

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Code practically universal

  • Strongest evidence that all living things share common ancestry

  • Mitochondria and chloroplasts have some differences in “stop” signals

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  • Grows in the 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

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  • 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

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Prokaryotic transcription is coupled with 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

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Eukaryotic Transcription

  • 3 different RNA polymerases

    • RNA polymerase 1 transcribes rRNA

    • RNA polymerase 2 transcribes mRNA and some snRNA (most genes are transcribed mRNA)

    • RNA polymerase 3 transcribes tRNA and some other small RNAs

  • Each RNA polymerase recognizes its own promoter (e.g., RNA pol 2 recognizes TATA box at –10nt)

  • Initiation of transcription

    • Requires a series of transcription factors

      • Necessary to get the RNA polymerase 2 enzyme to a promoter and to initiate gene expression

      • Interact with RNA polymerase to form initiation complex at promoter

  • Termination

    • Termination sits not as well defined

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MRNA modifications

  • In eukaryotes, the primary transcript must be modified to become mature mRNA

    • Addition of a 5’ cap

      • Protects from degradation; involved in translation initiation

    • Addition of a 3’ poly-A tail

      • Created by poly-A polymerase; protection from degradation

    • Removal of non-coding sequences (introns)

      • Pre-mRNA splicing done by spliceosome

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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

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Alternative Splicing

  • Single primary transcript can be spliced into different mRNAs by the inclusion of different sets of exons

  • ~ 25% of known human genetic disorders are due to altered splicing

  • Up to 95% of human multi-exon genes undergo alternative splicing to encode proteins with different functions

  • Explains how 25,000 genes of the human genome can encode the more than 80,000 different mRNAs

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TRNA and Ribosomes

  • TRNA molecules carry amino acids to the ribosome for incorporation into a polypeptide

    • Aminoacyl-tRNA synthetases add amino acids to the acceptor stem of tRNA

    • Anticodon loop contains 3 nucleotides complementary to mRNA codons

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TRNA charging Reaction

  • Each aminoacyl-tRNA synthetase recognizes only 1 amino acid but several tRNAs

  • This reaction is called the tRNA charging reaction

  • Charged tRNA-has an amino acid added using the energy from ATP

  • Ribosomes do not verify amino acid attached to tRNA-so correct attachment of amino acid to tRNA is important

  • The ribosome has multiple tRNA binding sites

    • P (peptidyl) site: binds the tRNA attached to the growing peptide chain

    • A (aminoacyl) site: binds the tRNA carrying the next amino acid

    • E (exit) site: binds the tRNA that carried the last amino acid

  • The ribosome has two primary functions

    • Decode the mRNA

    • Form peptide bonds

  • Peptidyl transferase

    • Enzymatic component of the ribosome (RNA-based)

    • Forms peptide bonds between amino acids

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Translation: Overview

  • MRNA is threaded through the ribosome

  • TRNAs carrying amino acids bind to the ribosome

  • TRNAs interact with mRNA by base-paring with the mRNA’s codons

  • The ribosome and tRNAs position the amino acids such that peptide bonds can be formed between each new amino acid and the growing polypeptide

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  • In prokaryotes, initiation complex includes

    • Initiator tRNA charged with N-formylmethionine

    • Small ribosomal subunit

    • MRNA strand

  • Ribosome binding sequence (RBS) of mRNA positions small subunit correctly

  • Large subunit now added

  • Initiator tRNA bound to P site with A site empty

  • Initiations in eukaryotes similar except

    • Initiating amino acid is methionine

    • More complicated initiation complex

    • Lack of an RBS- small submit binds to 5’ cap of mRNA

  • Elongation adds amino acids

    • 2nd charged tRNA can bind to empty A site

    • Requires elongation factor called EF-Tu to bind to charged tRNA and GTP

    • Peptide bond can then form

    • Addition of successive amino acids occurs as a cycle

  • There are fewer tRNAs than codons

  • Wobble pairing allows less stringent pairing between the 3’ base of the codon and the 5’ base of the anticodon

  • This allows fewer tRNAs to accommodate all codons

  • Termination

    • Elongation continues until the ribosome encounters a stop codon

    • Stop codons are recognized by release factors which release the polypeptide from the ribosome

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Protein targeting

  • In eukaryotes, translation may occur in the cytoplasm or the rough endoplasmic reticulum (RER)

  • Signal sequences at the beginning of the polypeptide sequence bind to the signal recognition particle (SRP)

  • The signal sequence and SRP are recognized by RER receptor proteins

  • Docking holds ribosome to RER

  • Beginning of the protein- trafficking pathway

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Wobble pairing

  • There are fewer tRNAs than codons

  • Wobble pairing allows less stringent pairing between the 3’ base of the codon and the 5’ base of the anticodon

  • This allows fewer tRNAs to accommodate all codons

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DNA and RNA Extraction

  • Lysis buffer (mostly detergent) breaks down lipids in the cell and its membranes

  • After the cells are broken down, they are treated with enzyme proteases and/or ribonucleases to further break down material

    • The processes and enzymes depend on the desired outcome

  • Remaining material is centrifuged to separate it. The supernatant (liquid) containing the DNA/RNA is extracted

  • Liquid is precipitated with ethanol to create strands

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DNA Manipulation

  • Restriction endonucleases revolutionized molecular biology

  • Enzymes that cleave DNA at specific sites

    • Used by bacteria against viruses

  • Restriction enzymes significant

    • Allow the creation of recombinant DNA molecules (from two different sources)

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3 types of restriction enzymes

  • Type 1 and 3 cleave with less precision and are not used in manipulating DNA

  • Type 2

    • Recognize specific 4-12 bp DNA sequences

    • Cleave at specific site within sequence

    • Can lead to “sticky ends” that can be joined

      • Blunt ends can also be joined

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DNA ligase

  • Joins the two fragments forming a stable DNA molecule

  • Catalyzes formation of a phosphodiester bond between adjacent phosphate and hydroxyl groups of DNA nucleotides

  • Same enzyme joins Okazaki fragments on lagging strand in replication

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  • Introduction of foreign DNA from an outside source into a cell

  • Natural processes in many bacterial species

    • E. coli does not

  • Temperature shifts can induce artificial transformation in E. coli

  • Transgenic organisms are all or part transformed cells (more later...)

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Molecular cloning

  • Clone: genetically identical copy

  • Molecular cloning- isolation of a specific DNA sequence (usually protein-encoding)

    • Sometimes called gene cloning

  • The most flexible and common host for cloning is E. coli

    • Vector: carries DNA in host and can replicate in the host

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  • Small, circular chromosomes

  • Used for cloning small pieces of DNA

  • 3 components

    • Origin of replication allows independent replication

    • Selectable marker: allows the presence of plasmid to be easily identified

    • Multiple cloning site (MCS)

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Artificial chromosomes

  • Plasmids have limited insert size

  • Yeast artificial chromosomes (YACs)

  • Allow for larger insert for large-scale analysis of genomes

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Polymerase chain reaction (PCR)

  • Developed by Kary Mullis (1985)

    • Awarded Nobel Prize

  • Allows the amplification of a small DNA fragment using primers that flank the region

  • Each PCR cycle involves three steps:

    • Denaturation (high temperature)

    • Annealing of primers (low temperature)

    • DNA synthesis (intermediate temperature)

      • Taq polymerase

  • Applications of PCR

    • Allows the investigation of minute samples of DNA

    • Forensics: drop of blood, cells at base of a hair

    • Detection of genetic defects in embryos by analyzing a single cell

    • Analysis of mitochondrial DNA from early human species

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Gel Electrophoresis

  • Separate DNA fragments by size

  • Gel made of agarose or polyacrylamide

  • Submersed in buffer that can carry current

  • Subjected to an electrical field

  • Negatively-charged DNA migrates towards the positive pole

  • Larger fragments move slower, smaller move faster

  • DNA is visualized using fluorescent dyes

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DNA libraries

  • A collection of DNAs in a vector that taken together represent the complex mixture of DNA

  • Genomic library: representation of the entire genome in a vector

    • Genome is randomly fragmented w/restriction enzymes

    • Inserted into a vector

    • Introduced into host cells (e.g., E. coli)

    • Usually constructed in BACs

  • Complementary DNA (cDNA)

    • DNA copies of mRNA

    • MRNA isolated

      • Represents only actively used genes

      • No introns

  • Use reverse transcriptase to make cDNA

  • CDNA used to make library

  • All genomic libraries from a cell will be the same but cDNA libraries can be different

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Molecular hybridization

  • Technique used to identify specific DNAs in complex mixtures such as libraries

  • Also termed annealing

  • Known single-stranded DNA or RNA is labeled

  • Used as a probe to identify its complement via specific base-pairing

  • The most common way of identifying a clone in a DNA library of interest

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Southern blotting

  • Sample DNA is digested by restriction enzymes and separated by gel electrophoresis

  • Double-stranded DNA denatured into single-strands

  • Gel “blotted” with filter paper to transfer DNA

  • Filter is incubated with a labeled probe consisting of purified, single-stranded DNA corresponding to a specific gene

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Northern blotting

RNA is separated by electrophoresis and then blotted onto the filter

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Western blotting

  • Proteins are separated by electrophoresis and then blotted onto the filter

  • Detection requires an antibody that can bind to one protein

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DNA fingerprinting

  • Restriction fragment length polymorphisms

  • Generated by point mutations or sequence duplications

  • Restriction enzyme fragments are often not identical in different individuals

  • Can be detected by Southern blotting

  • STRs

    • Identification technique used to detect differences in the DNA of individuals

    • Short tandem repeats (STRs)

      • Typically 2-4 nt long

      • Not part of coding or regulatory regions

    • Population is polymorphic for these markers

    • Using several probes, probability of identity can be calculated, or identity can be ruled out

    • Also used to identify remains

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Methods of sequencing

  • All modern-day methods utilize the chain termination method (the dideoxy method) of sequencing developed by Fred Sanger (1977)

  • Types:

    • Shotgun sequencing

    • Pairwise-end sequencing

    • Next generation sequencing

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Dideoxynucleosides and deoxynucleotides

  • A dideoxynucleoside is similar in structure to a deoxynucleotide, but is missing the 3’ hydroxyl group (indicated by the box)

  • When a dideoxynucleoside is incorporated into a DNA strand, DNA synthesis stops

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Genetic Engineering: “Knockout” mice

  • Can replace a wildtype gene with mutant copy to determine function

  • “knockout” mice: known gene is inactivated

  • Effect is then assessed in adult mouse (or if lethal- the stage of development at which function fails can be determined)

  • Cloned gene interrupted by replacement with a marker gene

  • Marker gene codes for resistance to the antibiotic neomycin

  • Interrupted gene is introduced into embryonic stem cells (ES cells)

  • ES cells injected into embryo early in development

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How does regulation benefit organisms?

  • Each somatic cell contains the full genome of an organism

  • All genes do not need to be expressed simultaneously in each cell (in fact, that would be detrimental to the organism)

  • Regulation maintains efficiency

    • Energy: expressing all gene would require a massive amount of energy

    • Space: cells are kept to a manageable size

    • Time: genes can be expressed as needed and more rapidly

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General model of transcription and translation in prokaryotic organisms

  • Transcription and translation occur simultaneously in the cytoplasm

  • Regulation occurs at the transcriptional level

  • Eukaryotic transcription and RNA processing occurs in the nucleus

  • Translation takes place in the cytoplasm

  • Gene expression is regulated in following ways:

    • Epigenetic

    • Transcription

    • Post-transcription

    • Translation

    • Post-translational modification of proteins

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Prokaryotic gene regulation

  • Prokaryotic DNA is organized into a circular chromosome located in nucleoid region of cytoplasm

  • Proteins with similar function or in the same biochemical pathway are organized in blocks called operons

  • Operons are regulated by three molecule types

    • Repressors: suppress transcription

    • Activators: increase transcription

    • Inducers: may suppress or activate transcription depending upon the needs of the cell

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The trp Operon: A repressor operon

  • When tryptophan is plentiful

    • Two tryptophan molecules bind the repressor protein at the operator sequence

    • The complex physically blocks the RNA polymerase from transcribing the tryptophan genes by binding to the operator

  • When tryptophan is absent

    • The repressor protein does not bind to the operator

    • The RNA polymerase can access the operator and the genes are transcribed

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Negative regulators

proteins that bind to the operator silence trp expression

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Glucose supplies become limited in cell

  • CAMP levels increase

  • CAMP binds to the CAP protein (a positive regulator)

  • CAMP/CAP protein complex binds to an operator region upstream of the genes required to use other sugar sources

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Positive Regulators

Proteins that bind the promoter in order to activate gene expression

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The lac Operon: An inducer operon

  • In the absence of lactose, the lac repressor binds the operator, and transcription is blocked

  • In the presence of lactose, the lac repressor is released from the operator, and transcription proceeds at a slow rate

  • CAMP-CAP complex stimulates RNA Polymerase activity and increases RNA synthesis

  • However, even in the presence of cAMP-CAP complex, RNA synthesis in blocked when repressor is bond to the operator

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Inducible operons

Proteins that activate or repress transcription. Activation/repression depends on the local environment and the needs of the cell

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Eukaryotic Epigenetic gene regulation

  • Eukaryotic gene expression is more complex than in prokaryotes:

    • Transcription and translation are physically separated

    • Regulation can occur at many levels

    • 1st level begins with control of access to the DNA-epigenetic regulation-and occurs before transcription

    • Transcription factors are proteins that control the transcription of genetic information from DNA to RNA

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Epigenetic control: regulating access to genes within the chromosome

  • Organization of human genome

    • 20,000 genes

    • 23 chromosomes (thousands of genes per chromosome)

    • DNA is wound/compacted tightly with proteins (histones)

    • Expressed genes must be unwound and made available to polymerases

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Organization of DNA and Proteins

  • DNA is folded around histone proteins to create nucleosome complexes

  • These nucleosomes control the access of proteins to the underlying DNA

  • When viewed through an electron microscope, the nucleosomes look like beads on a string.

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Nucleosomes control access to DNA

  • When nucleosomes are spaced closely together (top), transcription factors cannot bind, and gene expression is turned off

  • When nucleosomes are spaced far apart (bottom), the DNA is exposed

  • With the DNA exposed, transcription factors can bind to it, allowing gene expression to occur

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Chemical modifications of histones and DNA affect gene expression

  • Chemical tags are added to histones and DNA

    • Phosphate, methyl, acetyl groups serve as tags

    • Tags are not permanent-can be added or removed

  • Acts as signals to tell histones if region of chromosome should be open or closed

  • Epigenetic regulation: “around genetics” temporary changes to nuclear proteins and DNA that do not alter nucleotide sequence but do not alter gene expression

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Histone proteins and DNA nucleotides can be modified chemically

  • Modifications affect nucleosome spacing and gene expression

  • Unwinding and opening of DNA allows transcription factors to bind promoters and other upstream regions and initiate transcription

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Eukaryotic Transcription Gene Regulation

  • Gene transcription in Eukaryotes requires RNA polymerases

  • These RNA polymerases require transcription factors (specialized proteins) to initiate transcription

  • These factors bind the promoter sequence and other DNA regulatory sequences

  • Eukaryotic RNA polymerases require transcription factors in order to initiate transcription

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  • Region of DNA upstream of coping sequence (a few nucleotide to 100’s of nucleotides long)

  • Purpose is to bind transcription factors that control the initiation of transcription

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A series of thymine and adenine dinucleotides within the promoter 25-36 bp upstream of the transcriptional start site

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Transcription factor TFIID binds the TATA box

  • This recruits additional transcription factors to form a complex at TATA box

  • RNA polymerase can bind to upstream sequence

  • RNA polymerase is then phosphorylated, and part of protein is released from DNA

  • RNA polymerase is in proper orientation for transcription

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Enhancer: a DNA sequence that promotes transcription

  • Each enhancer is made up of short DNA sequences called distal control elements

  • Activators bind to the distal control elements and interact with mediator proteins and transcription factors

  • Two different genes may have the same promoter but different distal control elements, enabling differential gene expression

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Eukaryotic Post Transcriptional gene regulation

  • RNA transcripts must be processes into final form before translation can begin-post transcriptional modification

  • This step can be regulated to control gene expression

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RNA splicing, the first stage of post-transcriptional control

Pre-mRNA can be alternatively spliced to create different proteins. This process occurs in the nucleus

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How could alternative splicing have evolved?

  • Splicing requires proper identification of introns

  • Errors in this process could lead to splicing out of an intervening exon

  • Usually, would be deleterious to organism

  • But it could produce a protein variant without loss of original protein

  • New variant might have had an adaptive advantage

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Control of RNA stability

  • The protein-coding region of mRNA is flanked by 5’ and 3’ untranslated regions (UTRs)

  • RNA-binding proteins at these UTRs influences the RNA stability:

    • Can increase or decrease the length of time mRNA is present in the cytoplasm

    • They also regulate mRNA localization and protein translation

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RNA stability and microRNAs

  • MicroRNAs (miRNAs): short RNA molecules (21-24 nucleotides) that recognize specific sequence of mRNA

  • They associate with ribonucleoprotein complex called RNA-induced silencing complex (RISC)

  • RISC/miRNA bind to and degrade the mRNA

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The initiation complex and translation rate

  1. Translation controlled by proteins that bind and initiate process (formation of initiation complex)

  2. Eukaryotic initiation factor-2 (eIF-2)- first protein to bind and form complex

  3. GTP binds to eIF-2 and this complex binds to 40S ribosomal subunit

  4. Methionine initiator tRNA brings mRNA and binds the eIF-2/GTP/40S complex

  5. GTP is converted to GDP and energy is released

  6. Phosphate and eIF-2 are released and 60S binds and translation occurs

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