BSCI 410-Liu Homework#1 Key Spring 05

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1 1. (8 points) The following is a list of mutational changes. For each of the specific mutations indicate which type of mutation best describes the change. Sometimes, more than one term could be used to describe each of the following statement (such as transition, transversion, translocation, insertion, deletion etc.). (1) An A-T base pair is changed to a T-A pair. 1 transversion (2) An A-T base pair in the wild type gene is changed to a G-C pair. 1 transition (3) The sequence of AAGCTTATCG is changed to AAGCTATCG. 1 deletion (4) The sequence AACGTTATCG is changed to AATGTTATCG. 1 transition (5) The sequence AAGCTTATCG is changed to AAGCTTTATCG. 1 insertion (6) The sequence AACGTCACACACACATCG is changed to AACGTCACATCG. 1 deletion (7) The gene map in a given chromosome arm is changed from bog-rad-fox1- fox2-try-duf to bog-rad-fox2- fox1-try-duf. 1 Inversion (7) The gene map in a chromosome is changed from bog-rad-fox1-fox2-try-duf to bog-rad-fox1-met1-qui-txu-sqm. 1 Translocation 2. (10 points) Design a mutagenesis screen to isolate mutations involved in the biosynthesis of cytosine in yeast? Note: such a mutant is expected to be lethal. See fig Mutagenize (and then induce meiosis) 2 Grow in complete media 2 Transfer (replica-plating) to minimal media 2 Those that fail to grow in minimal media will be tested in the next step 2 Test for growth in minimal media supplemented with cytosine to confirm. Those that fail to grow in minimal media (MM) but can now grow in MM supplemented with cytosine will be the mutants we want. 3. (20 points) Briefly compare and contrast following pairs of terms: 1. Auxotroph and Prototroph 2 Auxotroph: Strain requires growth factors not normally needed by Wt cells for viability 2 Prototroph: Strain which does not require any special growth factors (usually WT) 1

2 2. Complete medium and Minimal medium 2 Complete medium: Contains all nutrients necessary for growth 2 Minimal medium: Contains only a carbon source (usually glucose), inorganic salts and water. 3. Mutagenesis screen and Mutagenesis selection 2 Selection: Conditions set up so only the mutant of interest will survive (use death vs. growth to select) 2 Screen: Both Wt and mutant organisms are present (ie. survive) and require one to look through all of them to identify the mutant of interests. 4. Haploinsufficient and dominant negative mutations 2 Haploinsufficiency is very rare and occurs when a phenotype is observed in individuals heterozygous for a loss-of-function allele (or null) and its phenotype is similar to homozygous loss-of-function mutants. 2 Dominant negative mutations are those in which a mutant protein product interferes (poisons) with the wild type product. Often this occurs in cases in which there are multiple subunits of a protein, and a mutation in one subunit interferes with the activity of the entire protein complex. An individual heterozygous for the dominant gainof-function mutation exhibits a phenotype similar to homozygous lossof-function. 5. Retrotransposons and Transposons 2 Retrotransposons: Use a reverse transcribed RNA intermediate to transpose to a new location, and has a polya tail at one or LTR at both ends. Still maintain the original (starting) retrotransposon (ie. increase in copy number rapidly). 2 Transposons: Requires no RNA intermediate, short inverted repeats at end. It may excise itself and insert itself into a new location (no net gain of the transposon). 4. (8 points) Wild type mouse with normal pigmentation is grey in color. Albino means white (no pigment at all). The following mutations (m1, m2, m3, and m4) affecting pigment synthesis in mouse are identified. They all affect the activity of the same gene. Given the following genotype as well as corresponding phenotype, could you describe the type of mutations using following terms: "recessive-null", "dominant-gainoffunction", "dominant-negative", "hypomorphic", "hypermorphic", and "neomorphic". Each situation listed below could sometimes be described by more than one terms (1) m1/+ grey mouse m1/m1 albino mouse 2 recessive-null, hypomorph, or loss of function (2) m2/+ albino mouse m2/m2 albino 2 dominant negative or haploid-insufficient 2

3 (3) m3/+ Black (darker than normal) m3/m3 Black 2 hypermorph or dominant gain-of-function (4) m4/+ Redish brown (unusual color) m4/m4 Redish brown 2 neomorph 5. (10 points) You have isolated five recessive maize mutants (d1-d5) which are defective in pigment biosynthesis. While wild type kernels are Red (R) in color; these d1- d5 mutant kernels are Yellow (Y) in color. To test if any of these mutations are allelic to each other, you did pairwise crosses among all five mutations and observed F1 phenotypes, which are summarized in the table below. How many genes are represented by these five mutations? How are these five mutations grouped into different genes? d1 d2 d3 d4 d5 d1 Y R R Y R d2 Y R R R d3 Y R Y d4 Y R d5 Y (R: red; Y: Yellow) 2.5 d1=d4 2.5 d3=d5 2.5 d2 different from rest genes 6. (14 points) The intermediates A, B, C, D, E, and F all occur in the same biochemical pathway. G is the product of the pathway and mutants 1 through 7 are all G- (ie. fail to produce G). The following table shows which intermediates will promote growth in each of the mutants. Arrange the intermediates in order of their occurrence in the pathway, and indicate the step in the pathway at which each mutant strain is blocked. + in the table indicates that the strain will grow if given that substance, an O means lack of growth. Supplement Mutant A B C D E F G O + 2 O O O O O O + 3 O + + O + O + 4 O + O O + O O + O O O O O + O + 3

4 F D A C B E G pnt for each correct letter and number 7. (10 points) Ethylene is a plant hormone that facilitate fruit ripening. Two types of tomato mutants were isolated that either could not ripen even when exposed to ethylene treatment or could ripen even without ethylene treatment. To determine the epistatic relationship among these mutations, double mutants were constructed and their phenotypes were observed and summarized below. Gene names Single mutant phenotype: never red (nrd): Form green tomatos even after ethylene treatment never ripen (nrp): Form green tomatos even after ethylene treatment always ripen (ar): Form red tomatos even without ethylene treatment (nrd, and nrp are defective in different genes). Gene names Double mutant phenotype: nrd ar: Form green tomatos even after ethylene treatment nrp ar: Form red tomatos even without ethylene treatment What is the regulatory relationship among these three genes? (Draw the regulatory pathway starting with Ethylene and ending with tomato ripening. Use arrows to indicate positive regulations and bars ( )to indicate negative regulations. Ethylene (arrow) nrp (bar) ar (bar) nrd (arrow) red tomatoes 2pnts for each correct gene placement in pathway 1pnt for each correct arrow or bar placed in pathway (and ethylene and red tomatoes being in the correct places in the pathway) 8. (8 points) Name and describe what type of transposable element each of the following is: (1).Ty1 transposon from yeast. 2 Retrotransposon, uses RNA intermediate (2). P-element from drosophila, 2 Transposon, does not require an RNA intermediate (3). Ac/Ds from maize 2 Transposon Ac: activator, autonomous Dc: dissociation, non autonomous Inverted repeats at end (4). T-DNA from agrobacteria, 4

5 2 Inserts into host DNA upon agrobacteria infection, can cause transformation 9. (12 points) What kinds of effect on DNA each of following mutagens could cause? (1) UV irradiation 2 thymine thymine dimmer (2) X-ray 2 double stranded DNA break, causing inversions, deletions or other rearrangements (3) EMS 2 Chemical change (alkylating agents) causing transitions (4) Proflavin 2 Intercalation causes insertions and deletions (frameshifts) (5) Nitrous acid 2 Deaminating agent causes transitions (6) Transposon insertion 2 inserts different nucleic acids into a gene and can disrupt frame, change encoding, introduce stops, alter splicng etc. 5