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88 Cards in this Set
- Front
- Back
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What is the main difference between interspersed repeats/mobile DNA elements and other types of DNA? |
Repeated (unlike spacer DNA) and are transposable, not created from simple slippage (unlike satellite DNA) |
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What are the two classes of transposons? |
DNA transposons and retrotransposons |
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How do the two classes of transposons differ? |
DNA transposons use a cut and paste method of insertion, in which the DNA is excised from its original place and then inserted at another place.
Retrotransposons are a copy and paste method, where the DNA is transcribed and then the RNA is reverse-transcribed back to DNA and inserted. |
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How do DNA transposons expand their copy number in eukaryotic genomes? |
Expansion can occur if the cut and paste occurs in the S phase and the cut comes from previously-replicated DNA and the paste is into not-yet-replicated DNA |
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What are the two main types of retrotransposons? |
LTR (long terminal repeat) containing and non-LTR containing |
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What proteins are encoded for by LTR retrotransposons |
Reverse transcriptase and integrase |
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What function do the LTR regions serve? |
Leftward: promoter Rightward: directs host-cell processing enzymes to add polyA tail |
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What are the two classes of non-LTR? How do they differ? |
LINEs, which encode proteins, and SINEs, which do not. |
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What proteins are encoded by non-LTR retrotransposons? |
ORF1, which is an RNA-binding protein, and ORF2, which acts as a reverse transcriptase and a DNA endonuclease |
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What enzyme catalyzed the coupling of an amino acid with a tRNA? |
Aminoacyl-tRNA synthetase |
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How does the # of amino acids compare with the number of aminoacyl-tRNA synthetases in the cell? |
Same. Each aminoacyl-tRNA synthetase adds a unique amino acid. |
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How does the # of tRNAs in the cell compare to the number of amino acids? |
More tRNAs than amino acids because each tRNA recognizes a specific code, but more than one code corresponds with an amino acid. |
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How does the # of tRNAs compare to the number of codons? |
Less tRNAs than codons in bacteria, but may be more in plants and animal cells |
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What molecule proves energy for coupling a tRNA to an amino acid? |
ATP |
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How do aminoacyl-tRNA syntheatases help reduce the number of errors in translation? |
They contain proofreading capabilities to double-check the amino acid and catalyze the removal of wrong AAs |
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What is the wobble position? |
1st NT (5' end) in the anticodon; 3rd in a codon (3' end). |
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What codons can be read by a tRNA with the anticodon sequence GCA? |
CGU or UGU (because of wobble) |
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What are the main differences between prokaryotic and eukaryotic ribosomes? |
Different size rRNAs: 28 and 18s in vertebrates, 23 and 16s in prokaryotes. Different ribosomal proteins In vertebrates, a 5.8s rRNA is basepaired to the 28S rRNA |
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How many types of tRNAs "carry" methionine? |
Two: metthionyl-tRNAi-MET (for recognizing intial start codon) and tRNA-MET adds to a growing chain |
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Which initiation factors keep the large and small ribosomal subunits apart? |
eIF3 and eIF6 |
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Which GTP-bound eIF interacts with pre-initiation complex, and what is it's function? |
eIF2-GTP: it binds to tRNAi-Met, and which is hydrolyzed in positioning the initiation complex at the start codon |
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What IF complex associates with the 5' cap of an mRNA and functions in formation of the initiation complex? |
eIF4 |
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Once the large and small subunits combine, what ensures they don't dissociate? |
eIF5-GTP is hydrolyzed |
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What 3 sites form in the ribosome when the sununits combine, and what happens at each site? |
A: Aminoacyl-tRNAs enter here P: The growing polypeptide chain rests here (except when it is transferred to the A site when forming a polypeptide bind) E: The exit site, where tRNAs leave the complex. |
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What role do the GTP-coupled EFs have in translation elongation? |
Conformational changes: EF1alpha: positions the amino acids close to each other to facilitate bonding EF2: monitors translocation |
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How does eRF1 binding facilitate translation termination? |
Similar in shape to a tRNA, it binds to the A site to directly recognize the stop codon. |
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What does eRF3 do? |
Coupled with GTP, it acts with ERF2 to promote cleavage of the peptidyl-tRNA to release the polypeptide. |
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What are the two main ways translation efficiency is increased? |
Rapid recycling of ribosomes and formation of polysomes. |
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What are the two different types of viruses, and how do they differ? |
DNA viruses, which have a DNA genome, and RNA viruses, which have an RNA genome |
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Where do the two different types of viruses replicate? |
RNA viruses replicate in the cytoplasm; DNA viruses replicate in the nucleus |
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What to functions does the capsid serve? |
Protect the viral genome and interact with the host cell for infection |
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What is a virus's host range? |
The range of host cells it can infect, determined by the types of proteins in the capsid (the viruses are restricted to cells with certain receptors) |
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What are the two capsid structures of viruses? |
Helical (rod shaped) and icosahedron (20 equilateral triangular faces). |
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What does a virus assay determine? |
The number of infections particles in a sample. |
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How does a virus assay work? |
Culture a sample and count the plaques that develop. Viruses replicate and then lyse the cell, which then makes it infect more. When enough cells lyse in an area, a plaque forms. |
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What are the two types of viral growth cycles, and how do they differ? |
Lytic (which causes the cells to lyse or break open) and non lytic (in which the virus's genome is integrated into the host genome) |
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What are the five stages of the lytic cycle, and what occures in each? |
1. Adsorption: virus attaches to host cell 2. Penetration: virus genome is inserted into host cell 3: Replication: genome is translated into proteins and replicated. 4: Assembly: Capsid proteins assemble around genomes to create virions 5: Release: host cell dies and virions are released to infect more cells |
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How does the lytic cycle differ amongst RNA and DNA viruses? |
In DNA viruses, the host cell's transcriptional machinery is used. RNA viruses use viral enzymes to product mRNAs. DNA viruses need to be transported into the nucleus. |
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What are the 3 main questions asked when studying a novel protein? |
What is the function? What is the structure? Where is it located? |
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Genomic regions of LINE insertions are rich in what nucleotides? Why? |
Rich in As and Ts. Functions in retrotransposition. |
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How are SINES transposed in the genome? |
Using LINE proteins (ORF1 and ORF2). |
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How have transposons played a role in the evolution and reorganization of our genomic material? |
Exon shuffling, gene duplication, and formation of complex regulatory regions. |
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What are the three types of RNA involved in translation, and what are their functions? |
mRNA: template for protein synthesis. tRNA: carries amino acids. rRNA: forms the ribosome, the site of synthesis |
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How many different codons are there? How many code for an amino acid, and how many are stop codons? |
64 codons; 61 code for an amino acid and 3 code for a stop codon. |
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Why is an mRNA's reading frame important? |
Each 3-nucleotide codon is a "block." It reads by 3s, so it reads 1-3, and then 4-6, etc. Where the mRNA's frame starts determines the whole sequence of codons and thus polypeptides. |
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How does a forward genetic screen differ from a reverse screen? |
Forward screens start with a phenotype and then look to a gene; reverse screens involve manipulating a gene first and then observing the phenotype. |
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What substance is commonly used to induce point mutations in cell lines and organisms? |
EMS, ethyl methanesulfonate |
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What causes a frameshift mutation? |
An insertion or deletion |
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If a true-breeding wild type yeast cell was mated with a true-breeding yeast cell with a dominant mutation, what fraction of the F2 progeny will exhibit the mutant phenotype? What if it were recessive? |
In both cases, equal amounts because yeast cells sporulate into four, each with only one allele. |
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What characteristic do all temperature sensitive mutations have in common? |
Protein denaturation at a lower temperature than the wild-type protein. |
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What is the goal of a complementation assay? |
Determine whether different recessive mutations are in the same gene |
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What is the main difference between a genomic and cDNA library? |
Genomic libraries contain all of an organism's DNA; a cDNA library only contains protein-coding DNA |
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Which type of DNA library is more useful for studies of higher organisms and why? |
cDNA libraries are more useful because they do not contain non-coding regions |
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What determines how often a restriction enzyme will digest an organism's genome? |
How many specific restriction sites there are in the genome, which is usually determined by the probability of the particular sequence showing up randomly in the genome. This is often determined by length of the restriction site. |
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How do bacteria protect their genomic DNA from digestion by the restriction enzymes they produce? |
Using a modification enzyme (for each RE),they methylate one or two bases to prevent the RE from cutting. |
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What function does DNA ligase serve during cloning? |
Ligates the inserted cDNA to the vector. |
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What are the three plasmid components that are essential for cloning? |
ORI (initatiates replication) Amp-R: Makes resistant to ampicillin Polylinker: contains restriction sites so exogenous DNA can be inserted without disturbing its ability to replicate. |
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What is the process by which bacteria take up recombinant DNA called? |
Transformation |
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What are the key steps of cDNA library synthesis? |
1: Isolate mRNA 2: Synthesis cDNA 3: Remove RNA 4+5: Use oligo-dC to create dsDNA 6: Methylate to protect from REs 7: Prepare for cloning by ligating cDNA to restriction site linkers 8: Digest! 9: Ligate 10: Transform into e. coli
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What are the basic steps of e. coli DNA library screening? |
1: Attach to nitrocellulose membrane 2: Denature and incubate with probe 3: Detect with autoradiography |
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What property of DNA contributes to its ability to be manipulated by gel electrophoresis? |
The negative charge. |
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What determines the pore size of an agarose gel? |
Concentration |
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What property of ddNTPs lead to termination of daughter strand synthesis? |
They lack the 3' OH used in forming phosphodiester bonds. |
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What is the targeted difference in length between daughter strands of a Sanger sequencing reaction? |
1 nucleotide. |
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How does a sequencing machine detect the terminal nucleotide of the daughter segments? |
The terminal nucleotide is a fluorescently-labelled ddNTP. Each base is a different color. The machine detects the fluorescent color. |
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What are the three stages of PCR and what occurs during each stage? |
1. Denaturation (94 C): melts the nucleic acid into single-strands. 2. Annealing (50-60 C): Primer attaches to the single-strand nucleic acid. 3. Polymerization (72 C): DNA is synthesized on the new strand |
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What must you include in your PCR reaction in order to generate probes used for hybridization? |
32P ("heavy" phosphorous) labelled dNTPs. |
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Which hybridization technique would you use to detect the expression of a gene in HEK298 cells? What about for mice? |
Use a northern blot for HEK293 and in-situ for mice. |
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How would you run the northern blot assay? |
Denature the RNA sample, then separate using gel electrophoresis and transfer to nitrocellulose membrance. Incubate with a probe specific towards the mRNA desires, and then subject to autoradiography. |
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How would you go about detecting whether a particular transcirption control region is present in the genome of alien species? |
Run a southern blot, using DNA, and probe for the particular region to see if the sequence is there. You can also test by attempting to grow sample cells in permissive and non-permissive environments. |
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How do the two methods of microarray synthesis differ from one another? |
In one, 1kb DNA is attached to glass directly. In the other, a specific small sequence that starts as ~20 nucleotides is programmed in. |
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How are samples processed for microarray analysis? |
Isolate mRNA, RT to cDNA with a colored dye, and then mix and hybridize to a DNA mircoarray |
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What E. Coli expression system is commonly used to produce large quantities of protein and how does this system work? |
Using expression vectors with operons, protein synthesis is coupled to specific promoters, such as the LacZ gene, that can be induced. When coupled, proteins are synthesized rapidly. When using LacZ, IPTG is the promoter, and proteins are synthesized rapidly. |
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What is the introduction of recombinant DNA into animal cells called? |
Transfection. |
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What are three main methods of animal cell transfection? |
1. Expose cells to lipid prepration 2. Subject to brief shock 3. Viral infection
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What is the difference between transient and stable transfection? |
Transient transfection does not integrate the recombinate DNA into the genome, so it is not replicated and dies out over generations. Stable transfection does integrate the DNA, so it is replicated and becomes a part of the genome. |
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What is the function of the LTR sequences in the vector plasmid for virus production? |
Directs the synthesis of a viral RNA molecule. |
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What other two plasmids are required for the production of virus partciles |
Packaging and viral coat plasmid vectors. |
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What does protein tagging allow you to do? |
Couple expression of a protein with the expression of a marker, which allows you to visualize expression of a protein. |
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How does protein tagging work? |
Hybrid genes are created by connected expression of a reporter protein to the protein of interest to link the proteins together. |
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What are the three approaches researchers use to inactivate genes in an organism? |
Replace a normal gene with another sequence, introduce an allele that codes for an inhibitory product, and promote destruction of the mRNA. |
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What is the name of the construct in yeast cells that is used to replace a gene in the genome? How is it generated, and what does it code for? |
Disruption construct, generated through PCR, and codes for a marker that confers resistance to G-418 |
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How are disruption constructs integrated into the yeast genome? |
Homologous recombination at primer sites. |
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Why is it important to transform diploid yeast cells with disruption constructs instead of haploid cells? |
If gene is important for growth, integrated cells will not survive if haploid because they will not carry a second, correct copy of the gene. |
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How do researchers go about studying yeast cells with lethal knockouts? |
After recombination in a dipolid cell, the cell sporulates to produce half recombinant and half wild type haploid cells. If lethal, the recombinant cells will die. |
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What cells are sued for targeted recombination when making a knockout mouse? |
Embryonic stem cells. |
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Which two genes are present in the disruption construct used for generating a knockout mouse? |
neo-r (confer resistance to G-418) tk-HSV (confer sensitivity to ganciclovir and inserted outside of target sequence) |
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How does expression of these genes allow for the selection of ES cells containing the properly recombined disruption construct? |
Properly combined genes will grow in presence G-418 and ganciclovir, because neo-r integrates in but tk-HSV should not if targeted properly. |
