Review Questions Flashcards
DNA transposable elements utilize three alternate pathways to move from place to place in the genome. Describe these three pathways. Use diagrams if necessary.
- Cut and paste pathway: Transposon excised by tranposase enzyme and reinserted in another part of the genome.
- Duplicative: A copy of the transposon is inserted in another part of the genome.
- Retrotranposition: Transposon is transcribed, reverse transcribed into cDNA and converted to double stranded DNA before being transposed to another part of the genome.
Diagram how unequal crossing-over can result in both the deletion and duplication of genes.
Misalignment of homologous chromosomes followed by a recombination event can result in deletion or expansion (duplication) of genes.
Describe and/or illustrate how the renaturation of genomic DNA reveals different classes of DNA sequences in eukaryotic genomes (Cot curve). How does the renaturation of eukaryotic DNA compare with the renaturation of prokaryotic (viral or bacterial) DNA?
When denatured DNA from eukaryotic genomes is reannealed the curve shows three distinct steps that correspond to the three classes of DNA, namely highly repeated, moderately repeated, and nonrepeated. The classes reanneal at different rates because they differ as to the number of times their nucleotide sequence is repeated within the population of fragments. Prokaryotic genomes do not have repeated classes of DNA and renature at a rate correlated with the size of the genome.
Circular plasmid DNA when fractionated on a gel travels faster than a linear fragment of DNA of equivalent length. If treated with topoisomerase II, the plasmid DNA travels at a slower rate, closer to that of the linear DNA. Explain the basis for these observations.
Circular plasmid DNA is supercoiled and more compact than linear DNA. Because of its compact size it travels through the gel faster. Topoisomerase II can relax the
supercoiling of the plasmid DNA which will result in it being less compact leading to slower migration rate through the gel.
Highly repetitive satellite DNA sequences may have functional roles. Describe three putative roles for satellite DNA.
- Formation of the kinetochore: Binding site for kinetochore-specific proteins
- Nucleosome phasing: Uniformity of satellite DNA length (~200 bp) may be related to regular phase of nucleosome cores.
- Satellite DNA transcripts may form ribozymes.
Susumu Ohno predicted that during the evolution of the vertebrate genome, two whole genome duplication events occurred. Describe: (a) how a genome duplication event can occur and (b) how one can determine whether a duplicated gene is the result of a whole genome event or a local duplication.
a) A genome duplication event can occur when two diploid gametes fuse instead of the normal haploid gametes. Diploid gametes can be generated by a meiotic nondisjunction event. Alternatively, if a single cell embryo undergoes chromosome duplication but does not divide mitotically, the embryo can develop into a viable tetraploid embryo.
b) If a whole genome duplication event occurs then the gene synteny (gene order) surrounding the duplicated genes will be conserved between species that diverged from the ancestor in which the whole genome duplication event occurred. If it was a local gene duplication there would not be conserved gene synteny.
Insertion of a transposable element generates flanking “direct repeat” sequences. Explain the basis for the generation of these repeat sequences.
The transposase enzyme makes a staggered cut in the target DNA. After the transposon integrates into the DNA the gaps are filled by DNA polymerase which results in small direct repeat sequences.
The ryanodine receptor gene family shows evidence of gene duplication followed by the process of subfunctionialization. What evidence exists that the RyR1a and RyR1b paralogues are not the result of a whole genome duplication and in what manner do the gene copies demonstrate that they have subfunctionialized?
Comparison of the synteny maps (gene order) for the RyR1a and RyR1b paralogues in different teleost species of fish shows no evidence of conservation of gene order for the genes. If the genes were the result of a whole genome duplication event, one would expect to see similar flanking genes for the RyR1a and RyR1b. The RyR2a/RyR2b and RyR3a/RyR3b genes in contrast do show conservation of flanking genes which suggeststhat they are the product of a whole genome duplication event.
You form a hybrid between hnRNA transcribed from the gene for the enzyme “midtermin” and a DNA fragment from the gene itself. The structure formed is a continuous double-stranded DNA-RNA hybrid. Next, you form a hybrid between the mature mRNA for “midtermin” and the same DNA fragment from the gene mentioned above. What do you see in the electron microscope when you examine this hybrid?Explain the basis for your observation.
When the processed mRNA is hybridized to the genomic (gene) DNA, the genomic DNA corresponding to the intron sequence will loop out since it does not have a complementary sequence in the mRNA to anneal with.
Group II introns in fungal mitochondria and plant chloroplasts are self-splicing. Introns in animal pre-mRNAs are processed much like Group II introns, but are not selfsplicing. Describe the theory of how group spliceosomal introns evolved from group II introns. What replaces the self-splicing activity of Group II introns in spliceosomal introns?
Introns were originally in present in the endosymbiotic organelles in primitive eukaryotic cells. Over time, the introns invaded the nuclear genome. Once in the nuclear DNA the introns transposed to other parts of the genome (duplicative transposition). Over time the catalytic portions of introns moved from the interior of protein coding genes to separate locations in the genome. The RNAs encoded by these new genes evolved into the snRNAs and their catalytic activity became dependent on proteins. Together, the snRNAs and proteins evolved into the snRNP components of the spliceosome
There are two general classes of snoRNAs employed in rRNA processing. Describe the roles for these two classes of snoRNAs.
Small nucleolar RNAs (snoRNAS). One class of snoRNAS (e.g. U3 snoRNA) are responsible for binding to and catalyzing the cleavage of the pre-rRNA transcript. The second class called antisense snoRNAs are responsible for binding to and coordinating the modification of rRNA nucleotides (e.g. methylation and conversion of uridine to pseudouridine).
In an experiment, you purify a cysteine-charged tRNA and chemically alter the amino acid attached to it converting it to alanine. You place this charged tRNA in a cell-free proteinsynthesizing system with all of the other charged tRNAs, mRNA and other substances needed for the synthesis of proteins. What, if anything, will be different from the normal protein coded for by the mRNA in the mixture?
The tRNA would still recognize alanine codons since the anticodon determines the specificity for the mRNA transcript.
You have set up a cell-free transcription system in a test tube. By mixing DNA, RNA polymerase, sigma factors, ribonucleotides and other essential substances, you are able to make mRNAs identical to those made under a variety of conditions in the cells from which these components were isolated. What would happen if you were able to remove the sigma factor first added to the tube and replace it with another sigma factor?
Since sigma factors often can cause transcription from different genes with different promoters, the population of mRNAs made would be likely to change.
What are the roles of general transcription factors (GTFs) in RNA polymerase IImediated transcription, and why are they referred to as “general”?
General transcription factors play several roles in promoting transcription by RNA polymerase II. They help position the RNA polymerase correctly at the promoter, they aid in pulling apart the two strands of DNA to allow transcription to begin, and they release RNA polymerase from the promoter once transcription has begun. They are called ‘general’ because they assemble on all promoters used by RNA polymerase II; they are identified by names beginning with TFII (transcription factor for RNA polymerase II). Labeling them ‘general’ transcription factors also serves to distinguish them from more specialized gene regulatory proteins that enhance transcription at selected promoters in certain cell types.
You are experimenting with two strains of bacteria. The normal strain usually produces extremely low levels of b-galactosidase. In the mutant strain, the operator site is altered, resulting in the constitutive synthesis of high levels of b-galactosidase in the presence andabsence of lactose. Why does this happen?
The alteration in the operator site results in a lessening of the ability of the active repressor to bind to the operator. This allows RNA polymerase to transcribe the operon unimpeded, when no lactose is present. If lactose is present, the repressor cannot bind to the operator because of the mutation at that site either; furthermore, the repressor bound to lactose would not bind to the operator since lactose would render the repressor inactive. In the presence of lactose, b-galactosidase would also be synthesized, if the operator were normal. Synthesis of b -galactosidase in the presence and absence of the lactose inducer without the normal regulation is said to be constitutive synthesis.
