BIOL 3301 Genetics EXAM 3

BIOL 3301 EXAM 4

Section 07870

GENETICS Thursday April 24, 2003, 5.30-7.00 pm

NOTE: MAKE SURE YOU READ THE QUESTIONS CAREFULLY!!! ________________________________________________________________________ Name:

SSN:

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True-false statements (write True or False in front of the statement) (2 points each, total 20 points). 1. Dideoxy-sequencing was devised by Maxam and Gilbert. F 2. The blue-white screen for recombinant plasmids involves the tetracyclin-resistance gene. F 3. Southern blotting is used for the analysis of total RNA. F 4. DNA fingerprinting in forensic science and in paternity tests makes use of VNTRs. T 5. SNPs enable the most refined mapping of genes on chromosomes. T 6. A forward mutation is any change back to the wildtype allele. F 7. Alkyltransferase is required for direct reversal of photodimers. F 8. Intercalating agents lead to frameshift mutations. T 9. Cyclins act to control the progression of the cell cycle by dephosphorylating Rb protein. F 10. The normal activity of the RB protein is to negatively regulate the progression from G1 to S of the cell cycle. T

Multiple choice (circle the correct answer) (2 points each, total 50 points). 1. The recognition sequence for the restriction site EcoRI is: a. GGATCC b. GAATTC c. GGGCCC d. AGATCT e. TCTAGA 2. Complete the following statement by picking the correct pair of words from the list. 'A critical feature of cloning plasmids is the presence of a …….................. marker such as ..................……………. resistance'. -

visible & blue-white selectable & ampicillin lethal & tetracyclin none of the above

3. The blue-white screen for recombinant plasmids depends on: a. alpha complementation of -galactosidase b. antibiotic resistance c. permease activity d. replica plating e. luck 4. A plasmid vector is cut in one place and a poly(G)tail is added to the 3’ ends. For effective cloning, the donor DNA should have a. poly(G) at the 3’ ends b. poly(G) at the 5’ ends c. poly(C) at the 3’ ends d. poly(C) at the5’ ends e. poly(G) at the 5’ ends and poly(C) at the 3’ends 5. First strand cDNA is synthesized from mRNA by means of: a. DNA polymerase I b. DNA ligase c. S1 nuclease d. Reverse transcriptase e. RNA polymerase III 6. In the dideoxy sequencing method the use of dideoxy thymidine triphosphate stops nucleotide polymerization a. opposite A´s in the template strand b. opposite T´s in the template strand

c. opposite G´s in the template strand d. opposite C´s in the template strand e. opposite any base selected randomly in the template strand

7. In comparison to the inserts in phage  recombinants, inserts in plasmids are typically: a. shorter b. longer c. equally long d. non-existent e. 20-times longer 8. To date, the type of enzyme used in the PCR reaction is: a. DNA polymerase I b. Klenow fragment c. a heat-stable DNA polymerase d. DNA ligase - topoisomerase 9. Which of the following methods is used for assigning loci to specific chromosomes but not for high resolution mapping: a. RFLP b. PFGE c. SSLP d. RAPD e. BAC 10. The largest DNA inserts are contained by: a. phage  vector b. plasmid c. YAC ( but I also count d and e as good answers) d. BAC e. PAC 11. The yeast-two-hybrid system is used to study: a. transcription initiation b. translation c. protein-protein interaction d. mRNA e. none of the above 12. Microarrays are used for the analysis of: a. genomic sequence b. transcriptomes c. proteomes d. posttranslational modifications e. none of the above 13. GC->AT mutation is called a. nonsense

b. missense c. frameshift d. transition e. transversion

14. An individual cell homozygous for a mutant allele of the gene for the human enzyme phenylalanine hydroxylase contains no detectable activity for that enzyme. The mutation is best described as: a. dominant b. recessive c. prototrophic d. gain of function e. null 15. A missense mutation is associated with the replacement of: a. stopcodon by an amino acid codon b. amino acid codon by a stop codon c. an amino acid codon by a codon encoding a different amino acid d. an amino acid codon by a synonymous codon e. …this doesn't make sense 16. In E. coli a region of a gene with repeats of the sequence CTGG will be prone to a. reversion b. missense mutation c. amber mutation d. nonsense mutation e. frameshift mutation 17. The AP repair pathway is a specific excision pathway that removes a. heteroduplex DNA b. photoadducts c. apurinic sites d. damaged bases e. damaged deoxyribose 18. Mismatch repair depends on the recognition of the old DNA strand by virtue of its: a. phosphorylation b. restriction c. methylation d. acetylation e. glycosylation 19. Huntington’s disease is caused by a. trinucleotide repeats b. free radicals c. microdeletions d. 5-bromouracil e. transversions 20. Frameshift mutations can be explained by the model proposed by:

a. b. c. d. e.

Streisinger Benzer Chargaff Einstein Beadle and Tatum

21. Which of the following enzymes plays an important role during apoptosis? a. luciferase b. catalase c. superoxide dismutase d. caspase e. lysozyme 22. Which of the following is not a general characteristic of a tumor suppressor gene? a. Tumor tissue can display a loss of heterozygosity of DNA in the vicinity of the gene b. Mutant versions of tumor-suppressor genes can segregate in families with an apparent autosomal dominant pattern of inheritance c. Tumor-suppressor genes typically display loss-of-function mutations d. Tumor-suppressor genes are typically recessively acting cancer genes e. Cells can become spontaneously transformed when transfected with a mutant version of a tumor-suppressor gene. 23. Which class of proteins is not generally specified by an oncogene? a. Ion channels b. Growth factors c. Transcription factors d. Growth factor receptors e. Signal transduction proteins 24. Which of the following mutations is frequently seen in many different types of cancer? a. Missense mutations of the p53 gene b. Chromosomal translocation involving a transcriptional enhancer and bcl2 gene c. Missense mutations of the breast cancer gene BRCA1 gene d. Chromosomal translocation of the bcr and abl genes (the Philadelphia chromosome) e. Amplification and overexpression of the BRCA1 gene 25. Which of the following does not apply to a growth factor receptor with a deleted ligandbinding domain a. constitutively dimerized b. constitutively activating c. oncogene d. dominant mutation e. tumor suppressor

Fill in (2 points each, total 10 points). 1. Some restriction enzymes create blunt ends, while others lead to sticky ends. 2. Northern blotting is a technique in which RNA is fractionated on a gel, and transferred to a membrane. The RNA attached to the membrane is incubated with a labeled probe, and an autoradiogram is used to identify any band that matches the probe. 3. Ultraviolet light induces the formation of T-T adducts . As a consequence, specific pairing is lost. The defect can be repaired by the enzyme photolyase in combination with white light. 4. Conditional mutations (e.g. temperature- sensitive mutations) cause a phenotype only under restrictive conditions. 5. The Philadelphia chromosome is a leading cause of Chronic Myelogenous Leukemia (CML).

Open question (20 points). Maximum of 2 pages ! Describe in detail how mouse gene knock-outs are generated. The goal of these experiments is to replace a wildtype copy of a gene X by a mutant version. Components for the experiment: 1. Replacement vector.

NeoR

TK

Homologous sequences required for homologous recombination. The small boxes flanking the NeoR gene correspond to parts of an exon of gene X we wish to replace. NeoR: neomycin-resistance gene confers resistance to G418 TK: thymidylate kinase (from Herpes simplex) confers sensitivity to gancyclovir, because gancyclovir is converted into a cell-toxic product by TK 2. ES cells. Embryonic cells have been established in cell culture and were originally derived from a brown mouse. 3. Transfection, selection and verification of ES cells. The targeting vector is introduced into ES cells by transfection experiments and the cells are grown on medium that contains G418 and gancyclovir. We distinguish three possibilities.

A. The ES cells have not taken up any targeting vector or none has been inserted. The cells will not grow on G418 due to the lack of NeoR. B. Random insertion of the targeting vector. The cells are NeoR but will not grow on gancyclovir since TK is also inserted and that converts gancyclovir into a cell-toxic product.

C. Homologous recombination. The cells will grow on G418 because of the NeoR and will also grow in the presence of gancyclovir due to the absence of TK, which was removed during homologous recombination. Subsequently, the ES cells with the gene replacement (due to homologous recombination) are verified for the correct replacement by either PCR or Southern blotting. 4. Generating mice homozygous for the mutant copy of the gene. -

Derive clones of ES cells with gene replacement. Inject cells into blastocysts from a black mouse. Implant blastocysts into a pseudopregnant mother. Allow embryos to develop to term and identify chimeric mice. Breed chimeric mice to establish heterozygotes. Interbreed heterozygotes to obtain homozygotes. Study the mutant phenotypes in the homozygotes. A representation of less than 25% of homozygotes in the progeny is indicative of a lethal or subviable mutation.