Problem Set 12, Fall 2017 Name: 8 points total



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Problem Set 12, Fall 2017 Name:

8 points total



1. A rare autosomal recessive disorder causes pink hair. A non-carrier male and an affected female marry. Consider the following pedigree and RFLP analysis prepared using HindIII, Southern blot, and a probe with a human sequence known to hybridize to sequences closely linked to the gene responsible for this disorder.

Note several arrangements are possible. This shows one of those.

a. Diagram each of the different haplotypes (group of markers that tend to be transmitted together because of linkage) of I-1 and I-2 showing the restriction map and fragment lengths. Show all HindIII restriction sites on each map.




Note that I-1 is heterozygous for the pattern shown whereas I-2 is homozygous for a pattern of sites as shown.




Probe is somewhere in the region shared by these three fragments.

b. On the restriction map, indicate the area to which the radioactive probe must hybridize in order to detect the fragments seen by Southern analysis.

Shown above

c. To which haplotypes (essentially pattern of each chromosome) does the mutant allele of the gene seem to be linked in this family.



The mutation appears linked to the 2.5 kb polymorphism

d. Why does individual III-1 only show one band in the RFLP analysis?



She’s homozygous for a pattern of HindIII sites that produces the 2.5 kb fragment.

e. Which individuals are probably carriers? II-1, II-2, II-3, II-4, III-2



4.0kb


3.5kb
2.5kb
2.0kb
1.0kb

0.2kb


III-1 II-1 II-2 I-1 II-3 I-2 II-4 II-5 III-2 III-3 III-4 III-5

2. Your friend just got a new job in a research lab in Brazil studying tropical butterflies. She has recently isolated a new mutant butterfly that produces fluorescent blue wings. She calls the mutation flb. While attempting to establish a line of these butterflies for her lab, she crosses an flb female with a wild type male and all of the progeny have fluorescent wings. When she performs a reciprocal cross between a wild type female and an flb male the progeny are all wild type. Suggest three plausible explanations for these results. How would you distinguish between these possibilities?



Maternal effect, Extranuclear inheritance, or Genomic Imprinting

A cross of mutant female with wild type male followed by a cross of F1s would distinguish between these possibilities. If maternal affect, all the F2 will be phenotypically wild type. If extranuclear all the F2 will be phenotypically mutant. If imprinting, half the F2 will be phenotypically mutant and half will be wild type.
3A. In general as we have seen this semester, reciprocal crosses produce the same results. However, we have encountered several exceptions to this general rule. Provide 4 examples where reciprocal crosses produce different results. Please include an example of such a cross and an explanation for the reason for the difference.

X linkage

genomic imprinting

extranuclear inheritance

maternal effect

Drosophila linked genes (because males suppress recombination)

You’ve seen many examples of the above this semester so I won’t bother writing any here.
B. In your studies of the poison dart frog C. drakeae you discover that there are two true breeding populations in the wild. The first population has bright red skin and the second has brilliant blue skin. You wish to understand the genetic basis of skin color in C. drakeae, so you cross frogs from population 1 with frogs from population 2 (i.e. red frogs X blue frogs). The F1 progeny all have purple skin. You cross purple F1’s to produce F2’s, and obtain the results shown below.

You see from the numbers that we have a 9:3:3:1 ratio, which says that the results are due to segregation of two alleles of two genes.
307 red frogs  rrB–  

298 blue frogs  R–bb  

883 purple frogs  R–B–  

92 white frogs  rrbb   

1580
i. What are the genotypes of the F2 frogs?

ii. If you cross red F2 frogs X white F2 frogs, what is the probability of getting white offspring?

1/3 are rrBB X rrbb => rrBb

2/3 are rrBb X rrbb => ½ rrBb and ½ rrbb

Therefore the probability of white offspring is 2/3(1/2) = 1/3

4. In your travels through the tropics you isolate a strain of poison dart frogs that has the following unusual features. Every generation you are able to identify a high frequency of mutations. Unfortunately, these mutations are not stable and often revert in the next generation (even thought kept under identical conditions) making them difficult to map genetically. Provide a plausible molecular explanation for the high frequency of reversion seen in this population.



Instability is a characteristic of transposable elements. The high frequency of reversion is due to the transposable element excising from the gene, restoring gene function and therefore the wild-type phenotype.
5. You are studying a rare mutant phenotype and perform PCR to analyze the suspected gene that is associated with the phenotype. You notice that the allele found in mutants is much longer than the wild type allele. You also notice a high frequency of reversion among mutants.

a. What caused the mutant allele?



As in #4, a high frequency of reversion is suggestive of a transposable element.
b. What are two mechanisms by which it could have moved into the gene (depending upon what type of transposable element it is)?

It could have moved via replicative transposition, which is utilized by retrotransposons, or by conservative transposition, utilized by some DNA elements.

6. You wish to study development, the process by which an embryo develops to produce a mature animal. Which organism would you choose? Justify your answer.



Many choices are possible. You might utilize nematodes, Drosophila or zebra fish. Others are possible but perhaps more difficult to justify. Justification would include some of the advantages offered by each. For example, if you chose zebra fish you might point out that they are genetically tractable, easy to store long term as frozen sperm, easy to house, have a relatively short generation time, have transparent embryos and are vertebrate.
7. You study a rare, but evolutionarily interesting species of tunicate. You want to write a grant in which you propose to sequence this animal’s genome. Please describe the general approach that you will use to sequence the genome. Assume that the genome contains many repetitive DNA sequences.

The strategy that’s been used for animals with much repetitive DNA involves preparing a YAC library of large genomic pieces and then figuring out where in the tunicate genome each large fragment came from. You then use the YAC library to order the clones made in a cosmid library, which have much smaller fragments of the genome (~50,000 base pairs vs. hundreds of thousands or more base pairs in YAC). You then determine the sequence of ~1,000 base pair bits of the cosmid clone and, once finished with each cosmid, assemble the sequence. Sequence all of the DNA in each of the cosmids and assemble to determine the final DNA sequence of the tunicate.
8. Reggaephilia, which causes a great fondness for Reggae music, is inherited as an autosomal dominant. Below is the pedigree analysis of reggaephilia in a single family. You perform a Southern blot analysis of DNA from family members, using a probe that recognizes a specific polymorphism.


It’s difficult to say for sure because of the small sample size but it looks like the 4.5 kb band is cosegregating with the disorder and therefore likely to be linked to the mutation that produces it.

a. Based on the blot shown above, are any of the polymorphisms likely to be linked to reggaephilia? If so, which one(s)? Numbers on the left show the sizes in kilobase pairs of bands detected.


Note several arrangements are possible. This shows one of those.

Probe is somewhere in the region shared by these four fragments.

b. Draw a map showing the bands in the original parents. Please indicate the approximate location of the nucleic acid probe that was used to detect the bands that were seen on the blot.


Note that I-1 and I-2 are both heterozygous for a pattern of restriction endonuclease recognition sequences that produces the bands shown.

c. How would linked polymorphisms help you to clone the gene?



You could use a positional cloning strategy using the hybridization probe shown above as your starting probe. This would be viable as long as you could show tight linkage between the polymorphism and the mutation that produces the disease.



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