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5 POINT QUESTIONS 1. A. Give the anticodon sequences (with 5' 3' direction being indicated) of all possible Aspartic Acid (Asp) tRNAs. 3’ CUA 5’ AND 3’ CUG 5’ B. Give the anticodon sequences of a minimum set of Aspartic Acid (Asp) tRNAs. 3’ CUU 5’ 2. For a UUU Phenylalanine (Phe) codon, how many of the transversion base pair substitution mutations would be in each category below? Silent 3. __0___ Missense __6___ Nonsense ___0__ Briefly describe the structure of the primary DNA lesion produced by exposure to ultravioltet (UV) light. Two adjacent thymines in the same strand are crosslinked by two covalent bonds to produce a “cyclobutane pyrimidine dimer”. See diagram in Fig. 18.15. 4. A. Identify the primary DNA lesion that results from the deamination of Cytosine. A GU m mismatch. B. Identify the primary DNA lesion that results from the deamination of 5-Methyl Cytosine? A GT mismatch. 5. What is a "two-way" mutagen? Two way mutagens are capable of inducing several types of mutation that will reverse each other’s effects. i.e. BOTH AT - GC AND GC - AT. 6. Give one specific example each of constitutive heterochromatin: centromeres; Y chromosomes of Drosophila and humans facultative heterochromatin: Barr Bodies (inactive X chromosomes); mentioning Bar Eye in Drosophila or "position effect variegation" 7. 8. For each human karyotype shown below state the biological gender and the # of Barr bodies. Karyotype Gender # Barr Bodies XYY MALE 0 XXY MALE 1 XXXX FEMALE 3 Describe the molecular structure of a nucleosome. A nucleosome consistes of about 2 turns of double helix wound around a core consisting of 2 molecules of each histones H2A, H2B, H3 and H4. One molecule of histone H1 attaches outside the core. See text Fig. 12.5. 9. In a certain eukaryotic species, the DNA sequence found at the right-hand ends of chromosomal DNAs is: - - - A T A G G C G A T A G G C G A T A 3' - - - T A T C C G C 5' Predict (below) the sequence of the telomerase-associated RNA: 5' - U A U C G C C U A U - 3' 10. Which two of the processes of bacterial gene transfer would NOT be inhibited by the presence of DNA degrading enzymes (DNases) in the exterior environment? 1: CONJUGATION 2: TRANSDUCTION 11. The diagram shows the genetic map of a bacterial genome with the locations of genes A, B, and C indicated. B C Bacteriophage X can transduce any of the 3 genes to recipient cell. a Bacteriophage Y can transduce only gene A to a recipient cell. Explain fully the basis for the difference between bacteriophage X and Y. A Bacteriophage Y carries out GENERALIZED TRANSDUCTION. Bacteriophage X is a temeperate phage and carries out SPECIALIZED transduction. Its chromosomal insertion site is near genes B and C. 12. An F+ E. coli cell is not able to transfer bacterial genes to a recipient by conjugation. Describe what must happen in an F+ cell to convert it to an Hfr strain that would be capable of transferring bacterial genes. The F DNA must undergo homologous recombination with a complementary sequence (IS element or transposon) in the chromosome so that the 2 DNA molecules fuse. 13. The DNA sequences recognized by regulatory proteins such as the Lac Repressor protein often share a certain feature with the DNA sequences recognized by restriction enzymes. What common feature do they share, and why is this the case? The sequences are often inverted repeats because the proteins that bind to them are dimers of 2 identical subunits that are oriented in opposite directions. 14. Assume that there is a single base pair mismatch (SNP) between a 40 base nucleotide hybridization probe and its complementary target DNA sequence. What factor will primarily determine the thermal stability of the hybrid between the probe and its complementary sequence? The position of the mismatch along the length of the double-stranded hybrid ("effective length") determines the thermal stability. 15. A. InDel polymorphisms are detected by two different molecular methods. What are they? 1. ASO hybridization 2. PCR of the InDel region followed by gel electrophoresis B. What factor determines which of these two methods of detection (above) would be appropriate for a given InDel? Length or size of the InDel. Smaller InDels (1-2 bp) are detected by ASO hybridization. Larger InDels are detected by PCR amplification of the InDel region followed by gel electrophoresis. 10 POINT QUESTIONS 1. A woman is born with Turner Syndrome (an aneuploid condition) and red-green color blindness; associated with expression of an X-linked allele. Both her parents had normal vision. Explain as fully as possible. The woman inherited the X-linked recessive allele from her mother, who was heterozygous for the normal allele. The father’s sperm did not contain either an X or a Y chromosome as the result of meiotic non-disjunction. It is not possible to determine whether the non-disjunction occurred at the first or second meiotic division. 2. The genomes of three species D, E, and F all have the same basic number of chromosomes, x=7. These genomes were originally derived from an ancestral plant species, and they diverged from each other sufficiently long ago so that the chromosomes from one genome no longer pair with chromosomes from the other species at meisosis. For plants with the following chromosome complements state (a.) the number of chromosomes, (b.) the appropriate term to describe the plant's genetic makeup, (c.) whether or not the plant is likely to be fertile, and (d.) the # chromosomes in the gametes. Genotype EEE DEF FFFF DD FF DD E FF a. # Chromosomes 21 21 28 28 35 b. autotriploid allotriploid autotetraploid allotetraploid allopentaploid c. Fertile? NO NO FERTILE FERTILE NO b. # Gametic Chromosomes 14 14 3. A. For each of the 2 diploid genotypes below for the Lac Operon, determine whether the gene products will be made in the presence of and absence of, the inducer (lactose). Give your answers by filling in the 4 boxes with "+" or "-" (See example.) B. Give the overall cell phenotype. The possibilities would include Wild-type (Lac+ regulated = Lac+ inducible) Lac+ unregulated (constitutive) LacEXAMPLE: GENOTYPE I+ P+ O + Z + Y+ I+ P+ O + Z + Y+ No Inducer I- P+ O + Z + Y+ Inducer Present No Inducer Inducer Present DIPLOID GENOTYPE I- P+ O + Z + Y+ I- P+ O c Z + Y- Permease [LacY] - - Cell Phenotype Wild-Type DIPLOID GENOTYPE I- P+ O + Z - Y- b-Galactosidase [LacZ] No Inducer Inducer Present + + b-Galactosidase [LacZ] Permease [LacY] + + + + b-Galactosidase [LacZ] Permease [LacY] + + + + Cell Phenotype Lac+ unregulated (constitutive) Cell Phenotype Lac+ unregulated (constitutive) 4. The genome of the bacterial virus Lambda is 50 kilobases of linear double-stranded DNA. The overall base composition is 50%GC:50% AT. Predict the number and the size of restriction fragments obtained by digestion of Lambda DNA with the restriction enzyme BssHI (5' GCGCGC 3'). A. SIZE of fragments = 46 = 4,096 base pairs B. NUMBER of fragments = 50kb / 4.096 kb = 12 fragments C. You isolate the double-stranded DNA genome of a different, unrelated bacterial virus that is also 50 kb in length. When you digest this DNA with BssHI you several times more restriction fragments, with much smaller sizes, than you get from Lambda DNA. Propose a simple explanation for why 2 genomic DNAs of the same size would give very different numbers of fragments when they are cut with the same restriction enzyme. CREDIT FOR EITHER ANSWER: The base composition of the second viral genome is lower %GC. Some potential BssHI restriction sites in the Lambda DNA were blocked by methylation. 5. A particular PCR reaction uses the 3 temperatures listed in Column A in the cycle. The temperatures are not necessarily given in the correct order. In Column B, number the steps 1, 2, and 3 in their proper sequence assuming that you have just mixed the reaction components together and started the thermocycler. In Column C, indicate what is actually happening in the reaction at each temperature by using the appropriate choice from Column D. A Cycle Temperature 55 °C B Sequence # C D Choices for Column C 2 b. 72 °C 3 c. 95 °C 1 a. a. Denaturation (separation) of double-stranded template DNA b. Hybridization of primers with complementary sequences in the template DNA c. Synthesis of DNA by Taq polymerase 6. The diagram below is a restriction map of a gene containing 2 introns. The vertical arrows indicate the location of restriction enzyme cut sites for a certain restriction enzyme. The number give the distance between the cut site in nucleotide base pairs. 220 120 115 110 80 200 160 130 The dark horizontal arrows show the binding sites for 2 PCR primers used to amplify a portion of the gene. A. Predict the size of the PCR product in kb. __________800 kb__________kb B. The PCR product is digested with the restriction enzyme whose cut sites are shown, and the fragments are separated by agarose gel electrophoresis. Sketch in the appropriate # of DNA bands you expect to see, in the appropriate locations in the gel, relative to the MW size standards. C. You transfer the restriction fragments from the gel to a membrane and perform a Southern Blot. The hybridization probe is a synthetic oligonucleotide whose sequence determined by "reverse translation" of the first 20 amino acids in the protein sequence. Label the bands that would contain DNA sequences that would hybridize with this probe. 250 200 200 160 150 130 120 100 100 110 80 Label 7. Transformation C. A. Conjugation H. B. Telomerase E. C. GATC J. D. Intercalating Agent D. E. Minisatellite I. F. 300 Angstrom Fiber B. G. Wobble A. H. Operator K. I. cAMP G. J. Microsatellite F. K. A special set of base pairing rules that apply to the interaction of the 3' base of a codon with an anticodon. The DNA sequence to which a trtanscriptional repressor protein binds. A process of gene transfer involving the uptake of fee DNA from solution. Mutagenic chemical that inserts between DNA base pairs to cause InDel mutations. DNA sequence that is used in E. coli to determine the relative age of DNA strands. Genetic variant that is hypervariable due to high frequency of slipped mis-pairing during DNA replication. Molecule involved in positive regulation of the E. coli Lac operon in response to glucose levels. A process of DNA transfer involving a DNA element known as a "plasmid". The copy number variant used for forensic DNA fingerprinting. An enzyme that is found in germ-line cells and some cancer cells, but not ordinarily in somatic cells of humans. Formed by the association of nuclear DNA with histones.