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Genetics 380, Spring 2004 Exam III

NAME___ANSWER PAGE 1 2 3 4 5 TOTAL TOTAL

KEY__________
Points Received Grader Initials 25 20 20 20 15 100 100

Possible Pts.

Genetics 380, Spring 2003, Name__________________________ Page 1 1. E. coli possesses a mechanism called the "SOS response" to cope with various types of DNA damage. More than 20 SOS genes, which are known to promote the survival of DNA-damaged cells, are induced by treatments that damage DNA (e.g., UV irradiaton). All the SOS genes share similar sequences in the regulatory regions called the "SOS box," to which LexA repressor binds to repress the transcription in the absence of DNA damage. Upon the occurrence of DNA damage, singlestranded DNA produced in the vicinity of DNA damage acts as a signal. A protein called RecA binds to the single-stranded DNA, and is activated by it. The activated RecA then promotes cleavage of LexA protein. Once cleaved, LexA no longer represses transcription. Hence, the SOS genes are allowed to be transcribed. The induced products of the SOS genes then repair the DNA lesions by various mechanisms, including recombination, excision repair and error-prone repair. Once repaired, DNA damage signal in the cell decreases and the repression of the SOS genes is restored. a. (3 pts) Draw out a linear genetic pathway for the SOS response. Be sure to include the following components: single stranded DNA, LexA, the SOS box, RecA, the SOS genes, and DNA damage. Be sure to indicate whether each genetic relationship is stimulatory ( ) or inhibitory ( ). DNA Damage b. single stranded DNA RecA LexA SOS Box SOS genes

(22 pts) You obtain mutants for various components in the SOS response. You test each mutant for its ability to respond (i.e., turn on the SOS genes) to DNA damage by UV radiation. In the following table, fill in whether you would expect high activity (+) or low activity(-) for the regulated SOS gene for each of the following genotypes in the presence or absence of UV radiation. For each gene, please note that a + indicates the wild-type allele, whereas a - indicates a loss-of-function allele. The first genotype has been done for you as an example. SOS Gene Activity UV treatment GENOTYPE No UV
recA+ lexA+ recA- lexA+ recA+ lexArecA+ lexA+ recA+ lexA+ recA+ lexA+ recA- lexA+ recA- lexArecA+ lexA+ /FrecA+ lexA+ recA+ lexA+ /FrecA+ lexA+ recA+ lexA+ /FrecA+ lexArecA+ lexA/FrecA+ lexA+ SOS box+ SOS box+ SOS box+ SOS boxSOS box+ SOS boxSOS boxSOS box+ SOS box+ SOS boxSOS boxSOS box+ SOS box+ SOS box+ SOS box+ SOS box+ SOS gene+ SOS gene+ SOS gene+ SOS gene+ SOS geneSOS geneSOS gene+ SOS gene+ SOS gene+ SOS geneSOS gene+ SOS geneSOS gene+ SOS geneSOS gene+ SOS gene-

+ + + + + -

+ + + + + + + + +

Genetics 380, Spring 2003, Name__________________________ 2.

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You are characterizing a pattern formation mutant called nanos. You decide to clone the nanos gene. You map it to a region of the Drosophila third chromosome between the genes pink and radius. pink nanos radius

a.

(15 pts) You have two different cosmid clones, one that contains genomic DNA sequences for the pink gene, and another that contains the DNA sequences for the radius gene. How could you isolate a genomic DNA clone for the nanos gene? Be sure to describe the reagents that you will need and the techniques that you will use.

You will need a genomic DNA library (4 points). Make a probe using the cosmids for one of the cloned genes, and probe the library for overlapping cosmids (4 points). Use each identified cosmid as a probe to pull adjacent cosmids as the next step (3 points). With each new cosmid, hybridize it to the cosmid of the other gene to see if you have walked the entire interval. (4 points)

b.

(5 pts) How would you confirm that the DNA clone that you identify contains the nanos gene?

The nanos gene should be in your contig- could sequence candidate genes or inject cosmids to test for phenotypic rescue.

Genetics 380, Spring 2003, Name__________________________

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3. A circular DNA molecule carries the -gal gene. You perform single and double digests with the indicated restriction enzymes, and you run the DNA out on two agarose gels. You stain one of the gels with ethidium bromide so that you can see all of the DNA bands (panel on the left). You transfer the DNA out of the other gel onto a filter and perform a Southern blot using sequences for the -gal gene as your hybridization probe (the resulting autorad is the panel on the right). The sizes, in kilobases, are indicated for each band. Ethidium Bromide EcoRI 4.1 PstI 1.9 1.7 1.6 1.7 1.4 1.0 0.6 0.5 EcoRI PstI Southern Blot EcoRI 4.1 PstI 1.9 1.6 1.1 EcoRI PstI

1.4 0.6 0.5

1.1

a.

(4 pts) For each restriction enzyme, indicate how many sites there are on the circular DNA molecule. 2 EcoRI__________ 3 PstI____________

b.

(4 pts) What would the number of sites be if the molecule were linear DNA? 1 EcoRI__________ 2 PstI____________

c.

(12 pts) Using all of the information from both the ethidium-stained gel and the Southern blot, draw a map of the circular DNA. Indicate the position of the EcoRI and PstI sites on the circle below. Indicate the distances between the sites in kilobases (kb). Use a box to indicate the position of the -gal gene. Use the additional space and the back of this sheet for scratch work if needed. They might do either of these orientations. 12 points for a flawless answer. 8 points if there is 1 mistake. 4 points if there is more than 1 mistake. PstI EcoRI 0.5 0.6 EcoRI Plasmid Map EcoRI Plasmid Map 0.6 PstI 0.5 EcoRI

1.4

1.0

1.0

1.4

PstI 1.7

PstI

PstI 1.7

PstI

Genetics 380, Spring 2003, Name__________________________

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4. The hypothetical Grape Fruit Urine Disease (GFUD) causes affected individuals to have pink colored urine, but is otherwise harmless. GFUD is mapped to chromosome 22, where there are 5 different RFLPs (named a-e), each with 3 different alleles (named 1-3). The RFLPs are arranged on the chromosome as follows: a b c d e

Below is a pedigree of a family affected by GFUD. Affected individuals are indicated by the black squares and circles. All 6 individuals give tissue samples. DNA is extracted, cut with restriction enzymes for the different RFLPs, and Southern blotted to detect the different RFLP alleles for each RFLP. The following RFLP allelic data is obtained for each individual (the lines indicate the two different homologs for chromosome 22 with their corresponding RFLPs):

1
a1 b2 c2 d3 e2 a2 b3 c1 d2 e1

2
a2 b3 c2 d2 e1 a3 b1 c2 d1 e3

3
a1 b2 c2 d3 e2 a2 b3 c2 d2 e3

4
a1 b2 c2 d3 e2 a3 b1 c2 d2 e1

5
a2 b3 c1 d2 e1 a3 b3 c2 d2 e1

6
a1 b2 c2 d3 e2 a3 b1 c2 d1 e3

a. (5 pts) Is GFUD in this family inherited as a sex-linked, autosomal recessive, or autosomal dominant trait? autosomal dominant b. (5 pts) Did any of the progeny inherit a recombinant chromosome? If so, then which ones (numbers 3, 4, 5, and/or 6)? Yes (2 points) 3 (1point) 4 (1point) 5 (1point) c. (5 pts) Which RFLP (a, b, c, d, or e) is most closely linked to the GFUD gene? e Partial credit: 2 points if they say d-e. d. (5 pts) Which allele (1, 2, or 3) predicts inheritance of the GFUD gene? e1 (1 would be sufficient)

Genetics 380, Spring 2003, Name__________________________

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e. (12 pts) You decide to clone the GFUD gene, so you build a BAC genomic DNA library and isolate a set of clones that span this region of chromosome 22. You determine the presence (+) or absence (-) of each RFLP in each of the 7 BAC clones. Using the map below, put the BAC clones in their correct order and indicate the location of the markers within them. BAC R has been done for you as an example. RFLP Markers a R T BACs V W X Y Z a R V W Y X Z b + + b + + + c c + + d + + + d T e + e

2 points for each BAC.

f. (3 pts) Which BAC is likely to contain the GFUD gene? T