Chapter 18

1. The sequences for recognition by a restriction enzyme often occur near one another on the opposite strands of DNA. For example,

   5′. . . .GAATTC. . . .3′

   3′. . . .CTTAAG. . . .5′

   The top and bottom strands have the same 5’-to 3’-sequence. A restriction enzyme recognizes this and cuts between the G and A:

   5′. . . .GAATTC. . . .3′

   3′. . . .CTTAA   G. . . .5′

   Note that the cuts are staggered, not directly opposite each other.

2. DNA from two genetically different strains of bacteria, each carrying a different antibiotic resistance gene, was cut with the restriction enzyme EcoRI and then spliced together using DNA ligase.

1. GFP reporter: all cells alive. Antibiotic resistance: most cells dead and only a selection alive. GFP reporter: need only a UV lamp to see the reporter gene product. Antibiotic resistance: need to alter growth medium and wait until nonselected cells are dead to identify cells with the vector.

2. The sequence would be: d, e, a, b, c.

1. 1. This involves tissue-specific gene expression, so use RT-PCR, which is a “snapshot” of the mRNAs in the cell at a given time and can identify the amylase mRNA.

   2. This calls for genomic analysis, so use a genomic library and identity the amylase gene in the strains.

2. All cellular RNA is isolated and cDNA copies of the RNAs are made using reverse transcriptase. Primers for DNA replication are made for the two ends of the DNA for the specific gene whose expression is to be studied. Then a PCR procedure is carried out using these primers. The degree of amplification from these primers is proportional to the specific cDNA target, which in turn is proportional to the amount of the specific RNA in the original cell extract.

3. Synthetic DNA can be used to make a probe for hybridization to identify a genotype, as in genetic screening for human diseases. Mutations in the DNA can be used to detect mutations in patients.

4. Both tissues express some identical genes that are involved in cellular activities, such as energy metabolism (e.g., genes encoding enzymes in the citric acid cycle) and informational functions (e.g., making rRNA). The tissues will differ in tissue-specific mRNAs. For example, the brain makes proteins needed for electrical activity and communication between cells, while the pancreas makes the hormone insulin. But note that all tissues have the same DNA sequences—all of them.

1. Antisense RNA and siRNA act at to prevent translation by binding to mRNA by base pairing and leading to its breakdown in the cytoplasm.

2. Germinate pea seeds and extract RNA from the embryos at different times. Make cDNA from the mRNA’s and hybridize the cDNA’s to a library of genes from the entire plant genome. The extent of hybridization to the genes will indicate differences in which genes are expressed.

3. CRISPR has the additional advantage of specifically changing the nucleotides in genes, that is, introducing mutations or reversions.

1. Expression vectors have restriction site(s) for insertion of genes, a DNA replication origin, a tissue-specific active promoter for the proposed host cell, and often a sequence for signaling the protein to be sent to the extracellular medium for isolation in the lab.

2. Biotechnology is faster (one instead of many generations of breeding) and specific (only a single gene is introduced, instead of many during selection for a complex characteristic).

3. Because the bacterium E. coli normally lives in the human intestine, there was a danger that lab strains resistant to multiple antibiotics might infect people. Furthermore, there may have been concern that the genes of antibiotic-resistant strains might recombine with the genes of normal strains, resulting in the development of multiple-antibiotic-resistant strains. Another concern was that bacteria could be used to harbor genes encoding toxins as biological warfare agents. To alleviate these concerns, the host E. coli strains were developed with multiple mutations so that they would require a special lab environment to survive. For example, mutations in genes for the synthesis for vital metabolites were introduced so the bacteria would need to be supplied with metabolites that the human intestine does not provide.

1. With no TPA there was no clot dissolution, but with TPA there was dissolution. Lab-made TPA was somewhat superior to natural TPA both in terms of the rate of clot dissolution and the final percentage of dissolution.

   

2. Again, lab-made TPA was somewhat superior to natural TPA in dissolving clots in the rabbits. A comparison could be made between the final percent of dissolution using a t-test.

1. The daughters all are homozygous for the transgene. Under Mendelian expectations, the daughters should be heterozygous.

2. The male offspring inherit their X chromosome from their mother. The female parent in this cross lacked the transgene.

3. Because the F₁ females are homozygous for the transgene, they should produce male offspring that all have the transgene, regardless of their mate’s genetic makeup. Males have only one copy of the X chromosome. These males should then produce daughters that are all homozygous for the transgene. The cycle would continue, yielding more and more flies with the transgene.

4. Additional transgenes that inhibit malaria transmission would be needed. In actual practice, there are genes that cause the mosquitoes to be resistant to Plasmodium. Thus the mosquitoes would still bite humans, but the likelihood that they would transmit malaria would be reduced.

5. Because the technology is very powerful and CRISPR can theoretically be used in just about any organism, there is a legitimate concern about accidental release of the construct. Laboratory safety protocols are vitally important. The researchers have used stringent containment procedures in accordance with approval from their institutions.