Bio 423 Sample Problems

Problems 1. From in situ hybridizations, five different YACs containing genomic fragments were known to hybridize to one specific chromosome band of the human genome. Genomic DNA was digested with a long-cutter restriction enzyme, and radioactively labeled YACs were each hybridized to blots of the digest. The autoradiogram was as follows:

a. Use these results to order the three hybridized restriction fragments. b. Show the locations of the YACs in relation to the three genomic restriction fragments in part a.

Unpacking the Problem 1. State two types of hybridization used in genetics. What types of hybridizations are used in this problem, and what is the molecular basis for such hybridizations? (Draw a rough sketch of what happens at the molecular level during hybridization.) 2. How are in situ hybridizations done in general? How would the specific in situ hybridizations in this problem be done (as in the first sentence)? 3. What is a YAC? 4. What are chromosome bands, and what procedure is used to produce them? Sketch a chromosome with some bands and show how the in situ hybridizations would look. 5. How would five different YACs have been shown to hybridize to one band? 6. What is a genomic fragment? Would you expect the five YACs to contain the same genomic fragment or different ones? How do you think these genomic fragments were produced (what are some general ways of fragmenting DNA)? Does it matter how the DNA was fragmented? 7. What is a restriction enzyme? 8. What is a long cutter? If you do not know what a long cutter is, what do you think it might be, and does your guess make sense of this part of the problem? If not, refer to discussions of long cutters in the chapter. 9. Why were the YACs radioactively labeled? (What does it mean to radioactively label

something?) 10. What is an autoradiogram? 11. Write a sentence that uses the words DNA, digestion, restriction enzyme, blot, and autoradiogram. 12. Explain exactly how the pattern of dark bands shown in the problem was obtained. 13. Approximately how many kilobases of DNA are in a human genome? 14. If human genomic DNA were digested with a restriction enzyme, roughly how many fragments would be produced? Tens? Hundreds? Thousands? Tens of thousands? Hundreds of thousands? 15. Would all these DNA fragments be different? Would most of them be different? 16. If these fragments were separated on an electrophoretic gel, what would you see if you added a DNA stain to the gel? 17. How does your answer to question 16 compare with the number of autoradiogram bands in the diagram? 18. Part a of the problem mentions "three hybridized restriction fragments." Point to them in the diagram. 19. Would there actually be any restriction fragments on an autoradiogram? 20. Which YACs hybridize to one restriction fragment and which YACs hybridize to two DNA fragments? 21. How is it possible for a YAC to hybridize to two DNA fragments? Suggest two explanations, and decide which makes more sense in this problem. Does the fact that all the YACs in this problem bind to one chromosome band (and apparently nothing else) help you in deciding? Could a YAC hybridize to more than two fragments? 22. Distinguish the use of the word band by cytogeneticists (chromosome microscopists) from the use of the word band by molecular geneticists. In what way do these uses come together in this problem?

2. Three genes, leu2, ade3, and mata, were cloned in yeast. A Neurospora geneticist wanted to find out if Neurospora had these three genes and, if so, wanted to clone the Neurospora equivalents. As a first step to this analysis, he hybridized the clones as radioactive probes to PFGE preparations of Neurospora chromosomes, with the following results. Which genes are present in Neurospora and what chromosomes are they on?

3. A Neurospora geneticist wanted to clone the gene cys-1, which was believed to be near the centromere on chromosome 5. Two RFLP markers (RFLP 1 and RFLP 2) were available in that vicinity, so he made the following cross:

Then 100 ascospores were tested for RFLP and cys-1 genotypes, and the results were:

a. Is cys-1 in this region of the chromosome? b. If so, draw a map of the loci in this region, labeled with map units. c. What would be a suitable next step in cloning the cys-1 gene? 4. In a certain haploid fungus, there had been extensive genetic analysis, including genetic mapping, and four linkage groups had been developed, suggesting four chromosomes. However, the chromosomes were very small and difficult to see under the microscope, so it was not known if there really were four chromosomes. The advent of PFGE technology showed that there are four chromosomes. However, the linkage groups still needed to be assigned to these chromosomes. To begin this process, a cloned gene P was used in a Southern analysis with a PFGE preparation from wild type and from two translocations that were known from genetic studies to be between linkage groups 1 and 4 in one case and between 2 and 4 in the other. The results follow:

a. From these data, determine which of the four real chromosomes (bands) the gene is on. b. Determine which chromosome (band) corresponds to each of the four linkage groups. 5. A cloned gene from Arabidopsis is used as a radioactive probe against DNA samples from cabbage (in the same plant family) digested by three different restriction enzymes. For enzyme 1, there were three radioactive bands on the autoradiogram; for enzyme 2, there was one band; and, for enzyme 3, there were two bands. How can these results be explained? 6. Five YAC clones of human DNA (YAC-A through YAC-E) were tested for sequence-tagged sites STS 1 through STS 7. The results are shown in the following table, in which a plus sign shows that the YAC contains that STS.

a. Draw a physical map showing the STS order. b. Align the YACs into a contig.

7. Seven human rodent radiation hybrids were obtained and tested for six different human genome molecular markers A through F. The results are shown here, where a plus sign

indicates the presence of a marker.

a. What marker linkages are suggested by these results? b. Is there any evidence of markers being on separate chromosomes? 8. A RAPD primer amplified two bands in Aspergillus nidulans haploid strain 1 and no bands in A. nidulans strain 2 (which was from a different country). These strains were crossed, and seven progeny were analyzed. The results were as follows:

a. Draw diagrams that explain the difference between the parents. b. Explain the origin of the progeny and their relative frequencies. c. Draw an example of a single tetrad from this cross, showing RAPD bands.

9. A Caenorhabditis contig for one region of chromosome 2 is as follows, where A through H are cosmids:

a. A cloned gene pBR322-x hybridized to cosmids C, D, and E. What is the approximate location of this gene x on the chromosome? b. A cloned gene pUC18-y hybridized only to cosmids E and F. What is its location? c. Explain exactly how it is possible for both probes to hybridize to cosmid E? 10. A certain disease is inherited as an autosomal dominant N. It was noted that some patients carry reciprocal translocations in which one of the chromosomes is always chromosome 3 and the break is always in band 3q3.1. Four molecular probes (a through d) are known to hybridize in situ to this band, but their order is not known. In the translocations, only probe c hybridizes to chromosome 3 carrying a part of another chromosome, and probes a, b, and d always hybridize to the translocated fragment of chromosome 3. a. Draw diagrams that illustrate the meaning of these findings. b. How would you use this information for positional cloning of the normal allele n? c. Once n is cloned, how would you use this clone to investigate the nature of the mutations in disease patients who do not have translocations?

11. The gene for the autosomal dominant disease shown in this pedigree is thought to be on chromosome 4, so five RFLPs (1 5) mapped on chromosome 4 were tested in all family members. The results are shown in the diagram; the superscripts represent different alleles

of the RFLP loci.

a. Explain how this experiment was carried out. b. Decide which RFLP locus is closest to the disease gene (explain your logic). c. How would you use this information to clone the disease gene? 12. A couple has three children with cystic fibrosis (CF). Their oldest son has recently married his second cousin. He has molecular testing done to determine if there is a chance that he may have children with CF. Three probes detecting RFLPs known to be very closely linked to the CF gene were used to assess the genotypes in this family. Answer the following questions, explaining your reasoning. a. Is this man homozygous normal or a carrier? b. Are his three normal siblings homozygous normal or carriers? c. From which parent did each carrier inherit the disease allele?