MBB 141 Questions 1-17

1. A piece of DNA 900 bp long is cloned and then cut out of the vector for analysis. Digestion of this linear piece of DNA with three different restriction enzymes singly and in all possible pairs of enzymes gave the following restriction fragment size data

Enzymes	Restriction fragment sizes (bp)
EcoRI	200, 700
HindIII	300, 600
BamHI	50, 350, 500
EcoRI + HindIII	100, 200, 600
EcoRI + BamHI	50, 150, 200, 500
HindIII + BamHI	50, 100, 250, 500

Construct a restriction map from these data

2. The ability of complementary nucleotides to base pair using hydrogen bonding, and the ability to selectively disrupt or retain accurate base pairing by treatment with chemicals (e.g., alkaline conditions) and/or heat is critical to many methods used to produce and analyze recombinant DNA. Give three examples of methods that rely on complementary base pairing, and explain what role complementary base pairing plays in each of these methods.
3. A new restriction endonuclease is isolated from a bacterium. This enzyme cuts DNA into fragments that average 4,096 base pairs long. Like many other known restriction enzymes, the new one recognizes a sequence in DNA that has twofold rotational symmetry. From the information given, how many base pairs of DNA constitute the recognition sequence for the new enzyme?
4. E. coli, like all bacterial cells, has its own restriction endonucleases that could interfere with the propagation of foreign DNA in plasmid vectors. For example, wild-type E. coli has a gene, hsdR, that encodes a restriction endonuclease that cleaves DNA that is not methylated at certain A residues. Why is it important to inactivate this enzyme by mutating the hsdR gene in strains of E. coli that will be used to propagate plasmids containing recombinant DNA?
5. Suppose you have cloned a eukaryotic cDNA and want to express the protein it encodes in E. coli. What type of vector would you use, and what features must this vector have? How would this vector need to be modified to express the protein in a mammalian tissue culture cell?
6. Suppose you wanted to produce human insulin (a peptide hormone) by cloning. Assume that you could do this by inserting the human insulin gene into a bacterial host where, give the appropriate conditions, the human gene would be transcribed and then translated into human insulin. Which would be better to use as your source of the gene: human genomic insulin DNA or a cDNA copy of this gene? Explain your choice.
7. You have inserted human insulin cDNA in the cloning vector pUC19 and transformed the clone into E. coli, but insulin was not expressed. Propose several hypotheses to explain why not.
8. Genomic libraries are important resources for isolating genes and for studying the functional organization of chromosomes. List the steps you would use to make a genomic library of yeast in a plasmid vector. In what fundamental way would you modify this procedure if you were making the library in a BAC vector?
9. The human genome contains about 3 x 10^9 bp of DNA. How many 20 kb fragment would you have to clone in a BAC library to have a 90% probability of including a particular sequence?
10. Suppose a researcher wants to clone the genomic sequences that include the human gene for which a cDNA has already been obtained. She has available a variety of genomic libraries that can be screened with a probe made from the cDNA.
1. Assuming that each library has an equally good representation of the 3 x 10^9 base pairs in a haploid human genome, about how many clones should be screened if the researcher wants to be 95% sure of obtaining at least one hybridizing clone and

i. The library is a plasmid library with inserts that are, on average, 7 kb?

ii. The library is a YAC library with inserts that are, on average, 1 Mb?

2. What advantages and disadvantages are there to screening these different libraries?
3. What kinds of information might be gathered from the analysis of genomic DNA clones that could not be gathered from the analysis of cDNA clones?
10. A researcher interested in the control of the cell cycle identifies three different yeast mutants whose rate of cell division is temperature-sensitive. At low, permissive temperatures, the mutant strains grow normally and produce yeast colonies having a normal size. However, at elevated, restrictive temperatures, the mutant strains are unable to divide and produce no colonies. She has a yeast genomic library made in a plasmid shuttle vector, and wants to clone the genes affected by the mutants. What steps should she take to accomplish this objective?
11. It’s 3 am. Your best friend has awakened you with yet another grandiose scheme. He has spent the last 2 years purifying a tiny amount of a potent modulator of the immune response. He believes that this protein, by stimulating the immune system, could be the ultimate cure for the common cold. Tonight, he has finally been able to obtain the sequence of the first seven amino acids at the N-terminus of the protein: Met-Phe-Tyr-Trp-Met-Ile-Gly-Tyr. He wants your help in cloning a cDNA for the gene so that he can express large amounts of the protein and undertake further testing of its properties. After you drag yourself out of bed and ponder the sequences for a while, what steps do you propose to take to obtain a cDNA for this gene?
12. A piece of DNA 5,000 bp long is digested with restriction enzymes A and B, singly and together. The DNA fragments produced are separated by DNA electrophoresis and their sizes are calculated, with the following results:

Digested with

A	B	A+B
2,100 bp	2,500	1,900
1,400	1,300	1,000
1,000	1,200	800
500		600
		500
		200

Each A fragment is extracted from the gel and digested with enzyme B, and each B fragment is extracted from the gel and digested with enzyme A. The sizes of the resulting DNA fragments are determined by gel electrophoresis, with the following results

A fragment	Fragments produced by digestion with B	B fragment	Fragment produced by digestion with A
2,100 bp	1,900; 200	2,500	1,900; 600
1,400	800; 600	1,300	800, 500
1,000	1,000;	1,200	1,000; 200
500	500

Construct a restriction map of the 5,000 bp DNA fragment.

14. A 10-kb genomic DNA EcoRI fragment from a newly discovered insect is ligated into the EcoRI site of the pUC19 plasmid vector and transformed into E. coli. Plasmid DNA and genomic DNA from the insect are prepared and each DNA sample is digested completely with the restriction enzyme EcoRI. The two digests are loaded into separate wells of an agarose gel, and electrophoresis is used to separate the products by size.
1. What will be seen in the lanes of the gel after it is stained to visualize the size-separated DNAs?
2. What will be seen if the gel is transferred to a membrane to make a Southern blot, and the blot is probed with the 10-kb EcoRI fragment? (Assume that the fragment does not contain any repetitive DNA sequence.)
15. During Southern blot analysis, DNA is separated by size using gel electrophoresis, and then transferred to a membrane filter. Before it is transferred, the gel is soaked in an alkaline solution to denature the double-stranded DNA, and then neutralized. Why is it important to denature the double-stranded DNA? (Hint: Consider how the membrane will be probed.)
16. A researcher digests genomic DNA with the restriction enzyme EcoRI, separates it by size on an agarose gel, and transfers the DNA fragments in the gel to a membrane filter using the Southern blot procedure. What result would she expect to see if the source of the DNA and the probe for the blot is as described as follows?
1. The genomic DNA is from a normal human. The probe is a 2.0-kb DNA fragment obtained by excision with the enzyme EcoRI from a plasmid containing single-copy genomic DNA.
2. The genomic DNA is from a normal human. The probe is a 5.0-kb DNA fragment that is a copy of a LINE (long interspersed elements) sequence with an internal EcoRI site.
3. The genomic DNA is from a normal human. The probe is a 5.0-kb DNA fragment that is a copy of a LINE sequence that lacks an internal EcoRI site.
4. The genomic DNA is from a human heterozygous for a translocation (exchange of chromosome parts) between chromosomes 14 and 21. The probe is a 3.0-kb DNA fragment that is obtained by excision with the enzyme EcoRI from a plasmid containing single-copy genomic DNA from a normal chromosome 14. The translocation breakpoint on chromosome 14 lies within the 3.0-kb genomic DNA fragment.
5. The genomic DNA is from a normal female. The probe is a 5.0-kb DNA fragment containing part of the testis determining factor gene, a gene located on the Y chromosome.
17. A molecular genetics research laboratory is working to develop a mouse model for bovine spongiform encephalopathy (BSE, “mad cow”) disease, which is caused by misfolding of the prion protein. As part of their investigation, they want to investigate the structure of the gene for the prion protein in mice. They have a mouse genomic DNA library made in a BAC vector and a 2.1-kb long cDNA for the gene. List the steps they should take to screen the BAC library with the cDNA probe.