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1) What can both transposition and conservative site-specific recombination do?
a. They can alter gene order along a chromosome, and cause unusual types of mutations that add new information to genomes. They move a variety of specialized DNA segments
1) What is the cut-and-paste method of DNA-only transposons?
- a. First, each of the special short DNA sequences that mark the two ends of the element binds a molecule of transposase, which come together to form a multimeric “transposome” that produces a DNA loop juxtaposing the two ends of the element.
- b. The transposase then introduces cuts at the base of the loop and removes the element completely from its original chromosome, forming the central intermediate in the transposition process.
- c. To complete DNA movement, the transposase catalyzes a direct attack of the elements two DAN ends on a target molecule and creating two new ones as it joins the element and target DNAs together
- d. This can occur without the imput of energy
- e. The product DNA ends with two short, single-strand gaps at each of the inserted transposon due to staggered breaking.
- f. DNA polymerase and ligase fill in and seal the gaps to finish the process.
1) What happens when a DNA-only transposon is removed?
- a. It leaves a hole in the chromosome and can be perfectly healed by recombinational double-strand beak repair, provided that the chromosome has been replicated and an identical copy of the damaged host sequence is available; the transposon is restored.
- b. It can also be recombinationally repaired using the chromosomal homolog, which does not restore the transposon but causes a loss of heterozygosity.
- c. Third, a nonhomologous end-joining reaction can reseal the break; the DNA sequence that flanked the transposon is altered, producing a mutation at the chromosomal site from which the transposon was excised.
1) What are the steps in transposition of retroviral-like retrotransposons?
a. First step in their transposition is the transcription of the entire transposon, prodcing an RNA copy of the element that is typically several thousand nucleotides long. This transcript encodes a reverse transcriptse enzyme. This enzyme makes a double-stranded DNA copy of the RNA molecule via an RNA/DNA hybrid intermediate, mirroring retroviral sages. The dsDNA then integrates into the chromosome by using integrase
1) Compare and contrast transposition and conservative site-specific recombination.
- a. First, conservative site-specific recombination requires specialized DNA sequences on both the donor and recipient DNA. These sequences have recognition sites for the particular recombinase that will catalyze the rearrangement
- b. Transposition requires only that the transposon have a specialized sequence; for most transposons, the recipient DNA can be of any sequence
- c. Second, the reaction mechanisms are different
- i. Recombinases that catalyze conservative site-specific recombination resemble topoisomerases in the sense that they form transient high-energy covalent bonds with the DNA and use this energy to complete the DNA rearrangmenets; all phosphate bonds that are broken during recombination are restored upon completion
- ii. Transposition uses a transesterification reaction that does not proceed through a covalently joined protein-DNA intermediate. This process leaves gaps in the DNA that must be resealed by DNA polymerase and ligase, both of which require the input of energy in the form of nucleotide hydrolysis.
1) What is the recombination process for bacteriophage lambda?
- a. The process begins when several molecules of the integrase protein bind tightly to a specific DNA sequence on the circular bacteriophage chromosome, along with several host protiens
- b. The DNA-protein comple then binds to an attachment-site DNA sequence on the bacterial chromosome, bringing the bacterial and bacteriophage chromosomes together
- c. Integrase then catalyzes the required cutting and resealing reactions that result in recombination.
- d. A tiny heteroduplex joint is formed at the point of exchange
- e. Bacteriophage lambda DNA can also exit from the integration site. Excision is catalyzed by integrase enzyme and host factors with a second bacteriophage protein, excisionase, which is produced by the virus only when its host cell is stressed
1) When the special sites recognized by a conservative site-specific recombination enzyme are inverted n their orientation, what happens?
a. The DNA sequence between them is inverted rather than excised; and, this inversion can control gene expression, which is advantageous in heredity, since the new DNA arrangement is transferred to daughter chromosomes automatically when a cell divides.
How can a conservative site-specific recombination enzyme from bacteria be used to delete specific genes from particular mouse tissues?
a. This approach requires insertion of two specially engineered DNA molecules into the animal’s germ line. The first contains the gene for a recombinase (ex: Cre recombinase from bacteriophage P1) under the control of a tissue-specific promoter, which ensures that the recombinase is expressed only in that tissue. The second DNA molecule contains the gene of interest flanked by recognition sites (loxP sites) for the recombinase. If the recombinase is expressed only in the liver, the gene of interest will be deleted there and only there.