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191 Cards in this Set
- Front
- Back
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What Process of Replication does DNA undergo?
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DNA undergoes a semi-conservative mode of replication. The parental strand serves as the template.
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What is DNA Polymerase I?
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principally involved in repair of damaged DNA.
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In prokaryotes, DNA Polymerase is responsible for what?
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DNA Polymerase III is the major polymerase responsible for DNA replication.
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In eukaryotes, what polymerases are involved in DNA replication?
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there are 3 DNA polymerases that are involved in DNA replication, alpha, delta and epsilon.
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What are the 2 fundamental properties that all DNA polymerases share?
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1.)All polymerases synthesize 5' --> 3' (adding dNTP to 3' hydroxyl)
2.) DNA polymerases can add a new deoxyribonucleotide only to reformed primer strand that is hydrogen-bonded to the template. |
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How do DNA polymerases differ from RNA polymerases?
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DNA polymerases alone cannot initiate DNA synthesis, they need an RNA primer, unlike RNA polymerase, which can initiate a new strand in the absence of a primer.
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What are the discontinuous fragments of DNA on the lagging strand called?
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Okazaki Fragments
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How are Okazaki fragments joined after formation?
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With DNA ligase-- resulting in a new intact DNA strand.
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What strand is continuously synthesized?
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The leading strand
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What does primase do?
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Primase is an enzyme that synthesizes short fragments of RNA complementary to the lagging strand
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How is the RNA primer taken away in prokaryotes, from the lagging strand once the DNA strand is synthesized?
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the RNA primer is removed by polymerase I. Polymerase I acts as an exonuclease that can hydrolyze DNA or RNA in either direction.
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How does polymerase I work to remove the primer?
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as a 5' to 3' exonuclease removes ribonucleotides from 5' ends of the fragments, allowing them to be replaced with deoxyribonucleotides.
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In eukaryotes, what removes RNA primers?
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the combined action of RNase and polymerase exchanges the RNA primer for DNA.
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What does RNase do?
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RNase is an enzyme that degrades the RNA strand of the RNA-DNA hybrids.
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What fills the gaps left?
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Polymerase delta.
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What is the reaction catalyzed by DNA polymerase?
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a triphosphate comes into the primer strand and cleaves a diphosphate in order to add the nucleotide to the growing DNA strand.
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What else uses DNA polymerases?
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PCR and DNA sequencing.
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What keeps polymerase on the DNA?
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an accessory protein--sliding clamp proteins and clamp loading proteins
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What do clamp-loading (RFC) and sliding-clamp loading (PCNA) proteins do?
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they form a complex that specifically recognizes and binds DNA at the junction between primer and template.
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What other function does RFC have?
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after loading the PCNA, the clamp-loader releases the sliding clamp protein and loads the DNA polymerase into DNA at the primer-template.
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what proteins unwind DNA?
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Helicases--they are enzymes that catalyze the unwinding of parental DNA, coupled to the hydrolysis of ATP.
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Single-stranded DNA-binding proteins
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eukaryotic replication protein A [RPA] stabilizes the unwound DNA template, keeping an extended single-stranded state so that it can be copied by polymerase.
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Topoisomerase
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are enzymes that catalyze the reversible breakage and rejoining of DNA strands. There are two types of topoisomerase. Topoisomerase I breaks one strand of DNA, while topoisomerase II introduces simultaneous breaks in both strands.
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What else is topoisomerase II useful for?
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in eukaryotes seem to be involved in mitotic chromosome condensation
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What is the frequency of error in DNA replication?
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one incorrect base pair per 10^8 to 10^9, which is much lower that would be predicted simply on the basis of complementary base pairing.
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Proofreading
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The proofreading activity of DNA polymerase (delta and epsilon) is an exonuclease that operates in teh reverse direction of DNA synthesis and participates in proofreading
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Importance of proofreading
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may expplain why DNA polymerase needs a primer. When DNA is synthesized in the 5'->3' direction, the energy required is derived from hydrolysis of 5' triphosphate as it is added to the 3' hydroxyl group
if extended in the 3' end, the energy would come from 5' triphosphate triphosphate, which would eliminate proofreading because it would remove the triphosphate needed for energy. |
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Origins of Replication
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serve as binding sites for proteins that initiate the replicating process
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Origins of replication in larger genomes
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multiple origins are needed or else the process would take weeks
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Autonomously Replicating Sequences (ARS)
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their rol as origins or replication of plasmids has been verified by biochemical analysis
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What is the origin recognition complex?
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a protein complex that is required for initiation of of DNA replication.
Fig. 6.15 |
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What mechanisms are required to replicate the terminal sequences of the linear chromosomes of eukaryotic cells?
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telomeres, which consist of tandem repeats of simple-sequence DNA
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What is telomerase?
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it is a unique enzyme that is able to catalyze the synthesis of telomeres in the absence of the DNA template.
fig. 6.16 |
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What are the two general classes of DNA repair?
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1.) Direct reversal of the chemical reaction responsible for DNA damage
2.) removal of the damaged bases followed by their replacement with newly synthesized DNA. |
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What types of DNA damage are repaired by Direct Reversal of DNA damage?
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pyrimidine dimers resulting from the exposure to ultraviolet light and alkylated guanine residues that have been modified by the addition of methyl or ethyl groups.
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How are pyrimidine dimers formed?
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adjacent pyrimidines on the same strand of DNA are joined by the formation of a cyclobutane ring resulting from the saturation of the double bonds between C5 and C6.
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What do these dimers do?
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The dimers distort the structure of the DNA chains and blocks transcription or replication past the site of damage.
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What is the process that would directly repair this?
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Photoreactivation: it uses the energy of visible light to break the cyclobutane structure. (Humans cannot do this)
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What is excision repair?
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The damaged DNA is recognized and removed, either as free bases or as nucleotides, the gap is then filled by synthesis of a new DNA strand using the undamaged complementary strand as a template.
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What are the types of excision repair?
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base-excision repair
nucleotide-excision repair and mismatch repair |
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What is base excision repair?
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in which single damaged bases are recognized and removed from the DNA molecule.
For example: Uracil is occasionally incorporated in place of thymine during DNA synthesis |
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How is excision catalyzed?
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it is catalyzed by DNA glycosylase, an enzyme that cleaves the bond linking the base to the deoxyribose of the DNA backbone.
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What is the result of DNA glycosylase?
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The formation of Apurinic sites, which are formed as a result of the spontaneous loss of purine bases.
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What do apurinic/apyrimidic sites require?
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They are repaired by AP endonuclease, which cleaves the adjacent AP site
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Does DNA glycolysase recognize and other damages?
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It only recognizes specific forms of damaged bases.
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What is the wide-spread form of DNA repair?
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nucleotide-excision repair
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What does nucleotide excision repair do?
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the damaged bases are removed as part of an oligonucleotide containing the lesion
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In E.coli, what products catalyze nucleotide-excision repair?
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the products of 3 genes, UvrA, UvrB, and UvrC
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What is the UvrABC complex?
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it is a complex referred to as an excinuclease because it can directly excise an oligonucleotide.
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How are repairs in human DNA been identified?
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They have been identified by studies of individuals with disease resulting from deficiencies
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What is the most extensively covered of these diseases?
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xeroderma pigmentosum (XP)
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What is another form of nucleotide excision repair?
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Transcription-coupled repair
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What is transcription-coupled repair?
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it is specifically dedicated to repairing damage within actively transcribed genes
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What causes Cockayne's Syndrome?
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It is a disease that is caused by an error in transcription-couple repair related to two proteins, CSA and CSB
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What is the mismatch repair system?
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it recognizes mismatched bases that are incorporated during DNA replication.
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How does this work in E.coli?
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the correction takes place on the daughter strand, which has not yet been methylated. It is initiated by protein MutS, which recognizes the mismatch and forms a complex with MutL and MutH. MutH cleaves the unmethylated DNA strand at GATC. MutL and MutS act together with an exonuclease and a helicase to excise the DNA between the strand break and the mismatch, which is then filled by DNA polymerase and ligase.
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How does this work in Eukaryotes?
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the presence of single-strand breaks in newly replicated DNA appears to specify the strand to be repaired. Mut S and Mut L bind to the mismatched base and direct excision occurs.
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What diseases are related to this type of repair?
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Inherited colon cancer, it is due to a mutation that affects the homologs of MutL.
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What is translesion DNA synthesis?
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the replication of damaged DNA by specialized polymerases. it provides a mechanism by which the cell can bypass DNA damage at the replication fork, which can be corrected after replication is complete
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How does translesion work?
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normal replication is blocked by a thymine dimer, and a specialized polymerase, Pol V, recognizes this and continues DNA synthesis across the lesion. Replication can then be resumed by the normal DNA polymerase and the thymine dimer removed by nucleotide-excision.
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What does recombinational repair do?
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relies on the replacement of the damaged DNA by recombination with an undamaged molecule. It is use to repair damage during replication
fig. 6.26 |
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Types of DNA rearrangements?
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Site-specific recombination, transposition, and amplification
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What is site-specific recombination?
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occurs between specific sequences, which are usually homologous. the principal interaction is mediated by proteins that recognize the specific DNA target sequences rather than by complimentary base-pairing. This leads to programmed DNA rearrangements that can play important roles in development and the regulation of gene expression
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What are the 2 classes of immune responses?
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they are mediated by B and T lymphocytes. B secretes antibodies that react with soluble antigens, while T express cell surface proteins to interact with antigens.
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what is the structure of an immunoglobulin?
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It is composed of light chains and heavy chains
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In what animal were the immunoglobulin rearrangements studied?
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Mice
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How many combinations can be formed from the the deletions and insertions?
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10^11 distinct antibodies can be formed
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What are the regions that encode light chains?
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there are 3 regions, the V (variable), J- encodes 12-14 C-terminal amino acids, and a C-region that encodes the constant region
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Process of the rearrangement of light chains
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each light chain consists of a constant (C), a joining (J) and a variable region (V). During the development of B lymphocytes, site-specific recombinations joins a V to a J, which activates transcription and VJ is joined to the C by splicing of the introns.
Fig. 6.37 |
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Process of the rearrangement of heavy chains
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The heavy chains have an added D region, first the D and J segments join, then a V segment is joined. The introns between J and C are removed by splicing to yield heavy chain mRNA.
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What are RAG 1 and RAG 2?
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they are a two protein complex that mediates the recombination of V(D)J. They recognize the signal sequences adjacent to the coding sequences of each gene segment and initiate DNA rearrangements by introducing a double stranded break between RS and coding sequences.
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What is class-switch recombination?
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it results in the association of rearranged V(D)J regions with different heavy chain constant regions, leading to the production of antibodies with distinct functional roles.
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What are the different classes of immunoglobulins that mammals produce?
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IgM, IgG, IgE, and IgA
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IgM
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activates complement ( a group of serum proteins that destroy invading cells or viruses). They are the first line of defense against bacterial or viral infection
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IgG
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the most abundant immunoglobulins in serum, activate complement, andbind receptorson phagocytic cells and can cross placenta
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IgA
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are secreted into bodily fluid, like milk or mucus
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IgE
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are effective in protecting against parasitic infections and are also the class of antibodies responsible for allergies
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What is somatic hypermutation?
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increases the diversirt of immunoglobulins by producing multiple mutations within rearranged variable regions of heavy and light chains.
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V(D)J recombination
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see figure 6.40 (p.237)
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Class switch recombination, process
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takes place by recombination between repetitive switch (S) regions upstream of a series of C regions in heacy locus chains
Fig. 6041 |
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What is Activation Induced Deaminase (AID)?
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is expressed only in B lymphocytes and required for both class switch recombination and somatic hypermutation. It catalyzes the deamination of cytosine in DNA to form Uracil. It results in a C -->U conversion in the V region
Fig 6.42 |
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What are transposable elements?
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elements that move by transposition
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What are the 2 general classes?
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transposition via RNA and theose via DNA intermediates
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How do bacterial transposons work?
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They are DNA intermediates. There is an insertion sequence range from 800-2000 nucleotides and contain a gene transposase flanked by inverted repeats. The transposase cleaves at both ends of the transposon and introduces a staggered cut of DNA. The overhanging ends are joined to the transposon and gaps are repaired
fig. 6.43 |
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How does transposition via RNA intermediates work?
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most transposons of eukaryotes are retrotransposons, move via reverse transcriptase.
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The mechanism by which reverse transcriptase works yields what?
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Long Terminal Repeats(LTRs) that arise from the duplication of the sites on viral RNA at which primers bind to initiate DNA synthesis. The LTRs play a role in reverse transcription and is involved in integration of proviral DNA.
fig. 6.45 |
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What is the major class of tetrotransponsons?
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LINEs
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What is significant about LINEs?
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Lines have tracts of A-rich sequences thought to be derived by reverse transcription of the poly-A tails that are added to mRNAs following transcription. They are flanked by short repeats of the target-site DNA, indicating integration involves staggered cuts and repair synthesis
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Do LINEs contain LTR?
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No, the mechanism of their reverse transcription differs from that of retroviruses.
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What is the mechanism of their transcription?
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DNA target site is cleaved by endonuclease encoded by the retrotransposon. Reverse transcription, primed by broken end of the DNA target initiates within the poly-A tail at the 3' end. the other strand is primed at the target site as well.
Fig. 6.48 |
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Gene Amplification, a different type of alteration:
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it increases the number of copies of a gene within a cell. It results from repeated rounds of DNA replication, yielding multiple copies of a particular region.
fig. 6.50 |
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How are these amplified genes found in a cell?
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free extra chomosomal molecules or as tandem arrays of sequences within a chromosome. It results as an increased expression of a gene
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What is an example of gene amplification?
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In amphibian oocytes: they require an increased synthesis of ribosomalRNA, which is accomplished by amplification of ribosomal RNA genes.
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How does gene amplification correlate to cancer cells?
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in cancer, there is an abnormal gene amplification, which results in the increased growth of the cell.
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What is the structure of a gene?
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genes have 2 segments, non-coding and coding segments (introns and exons)
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How is mRNA assembled?
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mRNA is assembled by RNA splicing
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What is the purpose of RNA splicing?
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It removes the introns from the RNA and what remains is an mRNA to be translated
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What were introns first found in?
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the were found with the use of the adenovirus 2
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What was the process by which they discovered introns?
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the infected human cells with ad2, and then purified the mRNA and hybridized it with viral DNA (they were attempting to map the hexon mRNA). They discovered that there were loops in between the hybridized pieces.
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What did they find in the adenovirus?
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They fond that the gene consists of 4 exons with 3 interrupting introns, which can be see in the hybridization, the exons are separated by single-stranded DNA loops corresponding to the introns.
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What is the importance of RNA splicing?
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the presence of introns allows the exons of a gene to be joined in different combinations resulting in the synthesis of different proteins from the same gene, known as ALTERNATIVE SPLICING.
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What is the function of repetitive sequences?
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(SINEs, retrotransposons, retrovirus-like elements) play an important role in chromosome structure
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What are pseudogenes and what is their function?
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pseudogenes represent evolutionary relics that increase the size of the eukaryotic genome without making a functional genetic contribution
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What happens when a gene is duplicated by reverse transcription?
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usually yields a processed pseudogene, which is inactive and accounts for 2/3 of pseudogenes that have been identified.
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What are the complexes between eukaryotic DNA and proteins?
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Chromatin, which typically contains 2x as much protein as DNA
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What are the major proteins of chromatin?
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Histones: small proteins containing a high proportion of basic amino acids that facilitate binding to the negatively charged DNA.
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What are the major Histone types?
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H1, H2A, H2B, H3, and H4
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What is the structural unit of chromatin?
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the nucleosome.
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How were nucleosome core particles discovered?
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extensive digestion of chromatin with micrococcal nuclease
fig. 5.11 |
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What is the structure of a chromatosome?
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147 base pairs are wrapped around DNA 1.67 times areound a histone core consisting of 2 molecules each of H2A, H2B, H3 and H4 (fig. 5.12). One molecule, H1 is bound around the DNA as it enters each nucleosome core particle. this forms a chromatin subunit, the chromatosome, which consists of 166bp of DNA wrapped around the histone core and held in place by H1.
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Interphase chromatin
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chromatin is randomly scattered and decondensed throughout the nucleus. called euchromatin
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How much of the interphase chromatin is heterochromatin?
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10% and is very highly condensed that resembles the chromatin of cells undergoing mitosis.
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What is the function of the centromeres?
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centromere is a highly specialized region of the chromosome that plays a role in the correct distribution of duplicated chromosomes to daughter cells during mitosis.
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How are sister chromatids held together during mitosis?
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they are held together by the centromere and microtubules attach to the centromere and the sister chromatids migrate to opposite ends.
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What does the kinetochore consist of?
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the structure that binds the microtubules and the centromeres.
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Where are telomeres located?
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the are at the ends of eukaryotic chromosomes
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What is the purpose of telomeres?
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they play critical roles in chromosome replication and maintenance. They are highly unstable
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What is the role of telomeres in replication?
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DNA polymerase is able to extend a growing linear DNA molecule, but cannot initiate synthesis of a new chain at the terminus of a linera DNA molecule, and so the ends cannot be replicated. This problem is solved by telomerase, which uses reverse transcriptase to replicate telomeric DNA
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Why are telomeres important?
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are important factor in determining the life span and reproductive capacity of cells
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What are open-reading frames?
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they are long stretches of nucleotide sequence that can encode polypeptides because they contain none of the tree chain terminating codons
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What are the chain terminating codons?
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UAA, UAG, and UGA
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What is FISH?
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Fluorescence in situ hybridization
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How is FISH used?
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it is used to localize genesin situ hybridization of probes labeled with fluorescent dyes to chromosomes
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Does in situe hybridization to human metaphase chromosomes provide mapping information?
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no, but prometaphase ir interphase can be used.
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what were the strategies for genome sequencing?
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the "shot-gun" approach was used by Venter and the HGP. HGP used BACs for sequencing and reassembled the sequence using overlapping pieces. (p.188)
Venter did shot-gun on the whole genome and then attempted to put it all back together. |
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What is bioinformantics?
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biology and computer science, focused on developing the computational methods needed to analyze and extract useful biological information from DNA bases.
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How do you screen a gene for function?
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you can "knock-out" the gene in a genome by homologous recombination with an inactive mutant allele.
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What is the use of RNAi?
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uses double-stranded RNAs to induce degradation of the homologous mRNA cells. can be used for a wide RNAi genome analysis.
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Genome-wide RNAi screen:
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used for drosphila, individual double stranded RNAs from the library are tested in microwells in a high thoughput format to identify those that interfere with cell growth
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How is gene expression regulated?
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most are regulatory sequences of DNA spanning about 10bp. The occur frequently in genomic DNA.
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How is gene expression being studied?
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they employ microarrays in which gene expression is represented by an oligonucleotide. They also use fluorescent cDNA copies of mRNA.
There is also comparative analysis. fig. 5.34 |
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How is this useful in Medicine?
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it can be useful in identifying abnormalities in individuals, especially diseases.
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What is the most of the variation in the bases?
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mostly single nucleotide polymorphisms (SNPs)
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How were alleles found?
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Gregor Mendel's observations and experiments with pea plants
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What is the genetic material?
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DNA
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What molecule did scientists think was the genetic material?
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Protein because there was more of it and they understood protein better
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Chromosome segregation is similar to?
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This type of segregation is very similar to Mendel's
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Where are genes located?
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Chromosomes
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How was it discovered that DNA was the genetic material?
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by experimenting with pathogenic strain (Pneumococcus) surrounded by a capsule and forms smooth colonies (S). Addition of S to R colonies results in the transformation of the R colonies to S colonies.
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How did Watson and Crick come up with the model of DNA?
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Help of x-ray crystallography (Rosalin Franklin) and they also knew that amount of G= amount of C, and amount of A= amount of T.
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How many bonds does GC make?
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3 H bonds, they are heavier by one dalton
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How many bonds does AT make?
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2 H bonds
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How does DNA replicate?
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Through the semi-conservative method.
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How was the semi-conservative replication demonstrated?
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Grew E.coli in 14N and another in radioactive 15N. The 15 N was transferred to the 14N media and what resulted was a medium density molecule
fig. 4.7 |
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What is the central dogma of Biology?
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DNA --> RNA --> Protein
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What is the one gene-one enzyme hypothesis?
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the current accepted state ment of this hypothesis is that each gene specifies the structure of a single poly-peptide chain
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What is colinearity of genes and proteins?
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A series of mutations were mapped in E.coli gene encoding tryptophan synthase. the amino acid resulting from each mutations were determined by sequence analysis of the proteins of mutant bacteria. This revealed that the order of mutations on DNA was the same as the order of amino acid substitutions in the encoded protein
fig. 4.8 |
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How is RNA synthesized from DNA?
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The two strands of DNA unwind and one is used as a template for synthesis of a complementary strand of RNA, which is called transcription
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How are proteins put together?
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the tRNA serves as an adaptor during protein synthesis. Each amino acid is attached to the 3' end of a specific tRNA by an enzyme, The tRNA aligns on the mRNA template by complementary base-pairing
fig. 4.10 |
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How do we know that the codons come in triplets?
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there is genetic evidence, a series of mutations were studied in rII gene of bacteriophage T4. Additions of one or 2 nucleotides altered the reading frame. As a result, an in active protein is produced giving a mutant phage. The addition of 3 nucleotides, only alters a single amino acid, the reading frame of the gene is normal and an active protein, giving rise to wildtype phage.
fig. 4.11 |
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What are RNA Viruses?
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they are retroviruses, like HIV that inject their RNA into a cell and through reverse transcription, they produce a DNA molecule.
fig. 4.13 |
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What is a restriction endonuclease?
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they are enzymes that cleave DNA at specific sequences. They were identified in bacteria.
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How can restriction endonucleases be used?
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the can cleave a DNA molecule at unique sites, such as EcoRI. These fragments can then be separated by gel electrophresis
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How can molecular recombination occur?
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Put DNA into a plasmid vector, the plasmid replicates in the bacteria. Cut the plasmid with restriction enzymes insert and deal with DNA ligase, replicate and purify.
fig. 4.16 |
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What is a BAC?
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Bacterial artificial chromosome, these vectors are derived from a naturally occurring plasmid of E.coli
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What is required for cloning in plasmid vectors?
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DNA fragment, origin of replication and a region that is resistant, in order to isolate a colony that contains the recombinant DNA.
fig. 4.19 |
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How does sequencing by polymerization/termination method work?
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DNA synthesis in the presence of fluorsecent-labeled chain terminating dideoxynucleotides are used, the reaction contains 4 dideoxynucleotides. the products are sent through gel-electrophoresis, as they pass though a laser beam that excites the fluorescent labels, the light is detected by a photomultiplier, which is connected to a computer.
fig. 4.20 |
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What is PCR?
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Polymerase chain reaction
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How does polymerase chain reaction work?
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PCR can achieve amplification of DNA via reactions in vitro. DNA polymerase is used for repeated replication of a defined segment of DNA
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What is the process of PCR?
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DNA is heated and denatured, 2 primers are then used to initiate DNA synthesis using DNA polymerase in opposite directions from complementary DNA strands.
fig. 4.23 |
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What is Nucleic Acid Hybridization?
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The key to detection of specific nucleic acid sequences is base pairing between complementary strands of DNA or RNA. When DNA is denatured, and incubated under specific conditions, they then renature to form double-stranded molecules. Nucleic acid hybrids can be formed between 2 strands of DNA and 2 of RNA or one of each.
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How else are RNA or DNA sequences detected?
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The cloned DNA is labeled with a radioactive nucleotide or modified nucleotides and this is used as a probe for hybridization to complementary DNA or RNA sequences, which are detected by virtue of the radioactivity, resulting in double stranded hybrids.
Southern Blotting and Northern Blotting are techniques that are used. |
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What is Southern Blotting?
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it is used for detection of specific genes in a cellular DNA.
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How is the DNA in Southern Blotting analyzed?
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the DNA is digested with restriction endonucleases and then the fragments are separated by gel electrophoresis. The gel is then overlaid with a nitrocellulose filter to which the DNA fragments are transferred to yield a replica of the gel. the filter is incubated and labeled with a probe, which hybridizes the fragments.
Fig. 4.25 |
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What is Northern Blotting?
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it detects RNA instead of DNA and works the same way as Southern Blotting
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What is the advantage of using DNA microarrays as opposed to blotting?
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Hybridization to Microarrays allows tens of thousands of genes to be analyzed simultaneously as opposed to blotting, which only allows for analyzing one gene at a time.
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What do DNA microarrays consist of?
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they consist of a glass slide or membrane filter onto which oligonucleotides or fragments of cDNA are printed.
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How can nucleic acid hybridization be used to detect homologous DNA or RNA sequences not only in cell extracts?
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they can be detected in intact cells, in a procedure called in situ hybridization.
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What is Western Blotting?
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it is immunoblotting is immunoprecipitation. Antibodies can be used to detect proteins in cell extracts. Proteins are first separated according to size by electrophoresis. They are separated by SDS-polyacrylamide gel electrophoresis, proteins are dissolved in a solution of negatively charged detergent. This detergent denatures the protein and gives it a negative charge. The proteins are then transferred to a filter and allowed to react with antibodies and the protein can be detected by chemilumiescence.
fig. 4.30 |
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What is gene transfer?
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genetic manipulations that can still be assayed by the introduction of cloned DNA into plant and animal cells.
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What is transfection?
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introduction of DNA into animal cells initially developed for infectious viral DNAs.
(transformation + infection) |
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How can DNA be introduced into animal cells in culture?
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direct micro-injection into the cell, coprecipitation of DNA with calcium phosphate.
Fig. 4.33 |
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What is transient expression?
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cells are taken up by a high fraction of cells and is transported to the nucleus, where it can be transcribed for several days.
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How are retroviruses used?
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they are efficient and can beused to introduce cloned genes into a wide variety of cells.
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How else can cloned genes be introduced into the genome?
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Allowing them to be in an animal rather than cultured cells, such as transgenic mice.
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What is an alternative way of introducing cloned genes into mice?
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Embryonic Stem cells, they can be established in culture from early mouse embryos and participate in normal development.
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What is reverse genetics?
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mutation is introduced into a cell first and then the sequence is found out
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What is the ability to introduce specific mutations into cloned DNA?
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in vitro mutagenesis
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What occurs in in vitro mutagenesis?
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using synthetic oligonucleotides to generate nucleotide changes in a DNA sequence. This synthetic bears a desired mutation is used as a primer for DNA synthesis. The newly synthesized DNA molecules can be isolated and characterized.
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Mutating chromosomal genes is based on what?
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on the ability of a cloned gene introduced into a cell to undergo homologous recombination with its chromosomal copy.
fig. 4.38 |
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What occurs in homologous recombination?
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the cloned gene replaces the normal allele, so the cloned gene in vitro becomes incorporated into the chomosomal copy of the gene.
It occurs frequently in yeast, but is rare in mammals. fig. 4.39 |
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How has homologous recombination been used?
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it has been used to systematically inactivate (knockout) every gene in yeast
in mice about 10% of genes has been knocked out--there is a large scale project underway |
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What has been used to inhibit gene expression?
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antisense nucleic acids into cultured cells RNA or single-stranded DNA complementary to RNA of the gene of interest hybridizes with the mRNA and blocks translation of a protein.
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About how long is antisense DNA?
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about 20 bp long
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What is RNAi?
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interference RNA, used for interfering with gene expression at the mRNA level.
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What is direct inhibition of protein function?
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microinjected anitbodies can bind to proteins within cells, inhibiting their normal function. Sometimes competing with normal protein for interaction with targets.
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What is a beta-barrel?
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it is a protein structure known to span lipid bilayers, formed by folding beta sheets into a barrel like structure, which is found in some membrane proteins (bacteria, chloroplasts and mitochondria)
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What is proteomics and what is its goal?
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it is the analysis of proteins and it has the goal of identifying and quantifying all of the proteins expressed in a given cell and establishing the localization of these proteins to different subcelluar organelles and elucidating the networks of interactions between proteins that govern cell activities.
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What is the function of an enzyme?
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they increase the rate of chemical reactions w/o being consumed and they increase reaction rates without altering the chemical equilibrium between reactants and products.
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What are coenzymes and what is their function?
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they are low-molecular-weight organic molecules participate in specific enzymatic reactions. They work together with enzymes to enhance reaction rates.
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what does feedback inhibition do?
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the product of a metabolic pathway inhibits the activity of an enzyme involved in its synthesis.
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