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_____is a pigment product produced by the activity of catechol oxidase

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10.10 Restriction endonucleases are used to construct restriction maps of linear or circular pieces of DNA. The DNA usually is produced in large amounts by recombinantDNA techniques. Generating restriction maps is like putting the pieces of a jigsaw puzzle together. Suppose we have a circular piece of double-stranded DNA that has a length of 5,000 bp. If this DNA is digested completely with restriction enzyme I, four DNA fragments are generated: fragment a is 2,000 bp, b is 1,400 bp, c is 900 bp, and d is 700 bp. If, instead, the DNA is incubated with the enzyme for a short time, the result is partial digestion of the DNA: not every restriction enzyme site in every DNA molecule will be cut by the enzyme, and all possiblecombinations of adjacent fragments can be produced. From a partial digestion experiment of this type, fragments of DNA were produced from the circular piece ofDNA that contained the following combinations of the above fragments: a-d-b, d-a-c, c-b-d, a-c, d-a, d-b, and b-c. Lastly, after digesting the original circular DNA to completion with restriction enzyme I, the DNA fragments are treated with restriction enzyme II under conditions conducive to complete digestion. The resultingfragments are 1,400, 1,200, 900, 800, 400, and 300 bp. Analyze all the data to locate the restriction enzyme sites as accurately as possible.

*10.18 It is 3 a.m. Your best friend has awakened you with yet another grandiose scheme. He has spent the last two years purifying a tiny amount of a potent modulatorof the immune response. He believes that this protein, bystimulating 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 sequence fora while, what steps do you propose to take to obtain a cDNA for this gene?

10.25 A cDNA library is made with mRNA isolated from liver tissue and the vector shown in Figure 10.4. When a cloned cDNA insert from that library is digested with the enzymes EcoRI (E), HindIII (H), and BamHI (B) (described in Table 8.1, p. 174), the restriction map shown in the following figure, part (a), is obtained. When this cDNA isused to screen a cDNA library made with mRNA frombrain tissue and the vector shown in Figure 10.4, three identical cDNAs with the restriction map shown in the following figure, part (b), are obtained. When a uniformlylabeled, 32P-labeled riboprobe made using T7 RNA polymeraseis prepared using either cDNA and the probe is allowed to hybridize to a Southern blot prepared fromgenomic DNA digested singly with the enzymes EcoRI, HindIII, and BamHI, an autoradiograph shows the pattern of bands in the following figure, part (c). When any of the -32P labeled riboprobes are used to probe a northern blotprepared with poly(A)+ mRNA isolated from liver and brain tissues, no signal is seen. However, when the samenorthern blot is probed with a uniformly labeled, -32P labeled probe is prepared using the random primer method (described in Box 10.1), the pattern of bands in part (d) of the figure is seen. Fully analyze these data and then answer the following questions:a. Do these cDNAs derive from the same gene?b. Why are different-sized bands seen on the northern blot?c. Why could hybridization signal be detected on the Southern blot but not on the northern blot when riboprobes were used? Why could hybridization signal be detected on the northern blot when a random-primed probe was used? (Hint: consider how these probes aremade and which nucleic acid strands become labeled.)d. Why do the cDNAs have different restriction maps?e. Why are some of the bands seen on the whole genome Southern blot different sizes than some of the restriction fragments in the cDNAs?

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