_________ ____Key Last name Circle TA name
_________________ First name Paula Wade John
_________________ Student number Andy Stacey
3rd Midterm Exam LS 4 Lecture 2 March 4, 2002
1. Replica plates with 4 different media with colonies from the same master were compared to determine the genotype of the colonies.
Fill out the table with the genotype for each colony with + for wild type and for mutant. Arg 1 2 strain 3 4 + + + + Genotypes (+ or -) bio + + gal + -
2.
A Hfr strain with resistance to the drugs streptomycin, kanamycin, penicillin and ampicillin but sensitive to the drug chloramphenicol is crossed to an F- strain resistant of geneotype strep- kan-, pen-, amp- chlor+. After 30 minutes of conjugation, mating is disrupted and diluted bacterial samples are plated on master plates containing only chloramphenicol, and then replica plated onto plates containing the indicated antibiotic combinations and the number of resistant colonies is counted: Antibiotics added number resistant colonies chlor 3,008 chlor, strep 677 chlor, kan 180 chlor, pen 0 chlor, amp 483 a. What is the order of gene transfer? Strep before amp before kan before pen What is the purpose of adding chloramphenicol to the master plate? Remove Hfr parents Why were no penicillin resistant recombinants observed? Transfer after 30 min - not enough time
b.
c.
3.
Interrupted mating experiments using four Hfr strains derived from the same F+ strain are shown to transfer the following genetic markers with the time of transfer indicated in parenthesis next to each marker: Hfr Strain
1 2 3 4 arg+(10) suc+(8) tyr+(3) arg+(6) mal+(13) ton+(19) uvr+(11) pur+(22)
Order of transfer
lys +(27) uvr+(21) ton+(13) leu+(26) phe+(33) tyr+(29) suc+(24) pro+(31) tyr+(47) phe+(43) pro+(31) suc+(38) uvr+(55) lys +(49) leu+(36) ton+(45)
Using this data, draw a map of the bacterial chromosome including the distance between each gene pair in minutes. Use arrowheads to indicate the point of F factor insertion and direction of transfer for each Hfr strain.
4.
The first gene transferred by an Hfr strain in the figure is indicated by the arrowhead. Based on what is shown on the map, choose Hfr and F- genotypes for an experiment to map arg and thi (locations not indicated on the map). Circle the best F- strain for this mapping cross Met+ val+ pro+ (arg- thi-) Met+ val+ pro- (arg+ thi+) Met val- pro+ (arg- thi-) Met- val- pro- (arg+ thi+)
Circle the best Hfr strain for this mapping cross Met+ val+ pro+ (arg- thi-) Met+ val+ pro- (arg+ thi+) Met- val- pro+ (arg+ thi+) Met- val- pro- (arg- thi-)
5.
DNA transduction experiments using phage grown on a pur+ pro+ his+ donor and a pur- prohis- recipient strain give the following results when replica plated: Plate 1 2 3 4 5 Supplements pur pro his + + + + + + + # of colonies 378 16 312 401 29 Genotypes pur+ pur+ pro+ pur+ his+ pro+ pro+ his+
(a) What are the cotransduction frequencies for each gene pair? Pur-his Pur-pro Pro-his 16/378 312/378 29/401
Draw a map of the DNA fragment containing these genes showing gene order and the relative distances between the genes. Pur his pro
6. In the plant Arabidopsis mutants A, B, C, D, E, F, and G all fail to synthesize the green pigment chlorophyll and are albino. Pairwise combinations of the mutants give the following complementation results, where a + = complementation and indicates failure to complement: A B C D E F A B + C + + D + + + E + + F + + + + + + 3 G + +
E a. How many genes are involved in chlorophyll biosynthesis? b. Indicate which mutations are allelic. A, D, F B, G C, E
7. Several auxotrophic fungal mutants are isolated, all of which require the compound Z for growth. Compounds S, T, W, X and Y are also know to components of the same biosynthetic pathway, although their order in the pathway is not yet determined. Each fungal mutant is grown on minimal media supplimented with each pathway intermediate and the following data are obtained, where a + indicates growth and a indicates failure to grow: Compound tested S T W X Y Z Mutant 1 + + 2 + + + 3 + 4 + + + + 5 + + + + + Draw the biosynthetic pathway for formation of compound Z showing the order of the pathway intermediates. Indicate at which step each mutant 1-5 is blocked.
8.
Recessive mutations a,b,c,d,e and f are known to map to a chromosome region covered by a series of deletions. Using the complementation data between each point mutant and the deletion map, assign each mutant to the appropriate interval. (Solid line indicates deleted region.) a b c d e f Deletion 1 + + + + = growth Deletion 2 + + + + Deletion 3 + + + - = no growth Deletion 4 + + + Deletion 5 + + + -
___ = missing sequences
b
a
c
e
d
f
9.
The table below gives partial results comparing the amino acid sequences and the mRNA sequences (codons) of a wild type gene and the same gene with a double frameshift mutant that restores function to the polypeptide chain. Fill in the missing amino acids or codons in the table. Use all the information, and the code table on the last page of the exam to be as specific as possible in choosing which codon is present. Thu_ ACA Tyr_ UAU Leu_ CUG Arg AGG Ala GCA
Amino acids Wild type Codons
Amino acids Double mutant Codons
Thr ACA
Phe UUC
Cys UGA
Gly
U
Ala GCA
A G
GGc
10. A section of the rIIB gene codes for the polypeptide sequence Met His Ile (FC88) Val (FC9) Lys
FC88 and FC9 are proflavin induced mutants that map to the sections coding for the His and Val amino acids, respectively. The FC88 FC9 double mutant has an r phenotype when grown on E. coli strain B. It has a wild type phenotype when grown on the E. coli strain Suppressor (tRNA), a derivative of the B strain. Write out a wild type RNA sequence and show where mutant insertions or deletions could explain these results.
(need to create a stop codon in the interval between FC88 and FC9) Alternative: