1. 20.1 – 1 Look at the illustration of “Cloning a Human Gene in a Bacterial Plasmid” (Figure 20.4 in the orange book). If the medium used for plating cells in step 5 did not contain ampicillin, cells containing no plasmid would be allowed to grow into colonies. What color would those colonies be, and why?

2. Cloning a human gene in a bacterial plasmid

3. Cloning a human gene in a bacterial plasmid Bacterial cell Human cell

4. Isolate DNA Bacterial plasmid Human chromosomes lacZ gene amp r gene

5. Restriction enzymes Bacterial plasmid gene of interest amp r gene broken lacZ gene

6. Mix together

8. Add bacteria

10. Grow on ampicillin

12. Add lactose mimic

14. 20.1 – 1 Look at the illustration of “Cloning a Human Gene in a Bacterial Plasmid” (Figure 20.4 in the orange book). If the medium used for plating cells in step 5 did not contain ampicillin, cells containing no plasmid would be allowed to grow into colonies. What color would those colonies be, and why?

15. 20.1 – 1 White (No functional lacZ gene is present.)

16. 20.1 – 2 Imagine you want to study human β-globin, a protein found in red blood cells. To obtain sufficient amounts of the protein, you decide to clone the β-globin gene. Would you construct a genomic library or a cDNA library? What material would you use as a source of DNA or RNA?

17. 20.1 – 2 A cDNA library, made using mRNA from developing red blood cells, which would be expected to contain many copies of β-globin mRNA’s.

18. 20.1 – 3 What are two potential difficulties in using plasmid vectors of human proteins from cloned genes?

19. 20.1 – 3 Some human genes are too large to be incorporated into bacterial plasmids. Bacterial cells lack the means to process RNA transcripts, and even if the need for RNA processing is avoided by using cDNA, bacteria lack enzymes to catalyze the post-translational processing that many human proteins undergo.

20. 20.2 – 1 Suppose you carry out electrophoresis on a sample of genomic DNA isolated from an individual and treated with a restriction enzyme. After staining the gel with a DNA-binding dye, what would you see? Explain.

21. 20.2 – 1 Any restriction enzyme will cut DNA in many places, generating such a large number of fragments that they would appear as a smear rather than distinct bands when the gel is stained after electrophoresis.

22. 20.2 – 2 Explain why restriction fragment length polymorphisms (RFLPs) can serve as genetic markers even though they produce no visible phenotypic differences.

23. 20.2 – 2 RFLPs are inherited in a Mendelian fashion, and variations in RFLPs among individuals can be detected by Southern blotting.

24. 20.3 – 1 What is a major difference between a genetic (linkage) map and a physical map of a chromosome?

25. 20.3 – 1 In a genetic linkage map, genes and other markers are ordered with respect to each other, but only the relative distances between them are known. In a physical map, the actual distances between markers, expressed in base pairs, are known.

26. 20.3 – 2 In general, how does the approach to genome mapping used in the Human Genomic Project differ from the shotgun approach?

27. 20.3 – 2 The three-stage approach employed in the Human Genome Project involves genetic mapping, physical mapping, and then sequencing of short, overlapping fragments that previously have been ordered relative to each other. continued…

28. 20.3 – 2 The shotgun approach eliminates the genetic mapping and physical mapping stages; instead, short fragments generated by multiple restriction enzymes are sequenced and then subsequently ordered by computer programs that identify overlapping regions.

29. 20.4 – 1 Current estimates are that the human genome contains about 25,000 genes, but there is evidence for many more different polypeptides. What process might explain this discrepancy?

30. 20.4 – 1 Alternative splicing of RNA transcripts from a gene and post- translational processing of polypeptides.

31. 20.4 – 2 What is the major value of DNA microarray analysis for studying gene expression?

32. 20.4 – 2 It allows the expression of thousands of genes to be expressed simultaneously, thus providing a genome-wide view of which genes are expressed in different tissues, under particular conditions, or at different stages of development.

33. 20.4 – 3 Why is the genetic variation among people so much less than it is among individuals of many other species?

34. 20.4 – 3 Because the human species arose more recently than many other species, there has been less time for genetic variations in coding and noncoding DNA to accumulate.

35. 20.5 – 1 What is the advantage of using stem cells for gene therapy?

36. 20.5 – 1 Stem cells continue to reproduce themselves.

37. 20.5 – 2 List at least three different properties that have been acquired by crop plants via genetic engineering.

38. 20.5 – 2 Herbicide resistance Pest resistance Disease resistance Delayed ripening Improved nutritional value