18. Molecular Genetic Tools

Genetic Cloning

Open Question
What roles do restriction enzymes, vectors, and host cells play in recombinant DNA studies? What role does DNA ligase perform in a DNA cloning experiment? How does the action of DNA ligase differ from the function of restriction enzymes?
Open Question
Why are most recombinant human proteins produced in animal or plant hosts instead of bacterial host cells?
Open Question
One of the major causes of sickness, death, and economic loss in the cattle industry is Mannheimia haemolytica, which causes bovine pasteurellosis, or shipping fever. Noninvasive delivery of a vaccine using transgenic plants expressing immunogens would reduce labor costs and trauma to livestock. An early step toward developing an edible vaccine is to determine whether an injected version of an antigen (usually a derivative of the pathogen) is capable of stimulating the development of antibodies in a test organism. The following table assesses the ability of a transgenic portion of a toxin (Lkt) of M. haemolytica to stimulate development of specific antibodies in rabbits.Immunogen Injected Antibody Production in SerumLkt50*—saline extract +Lkt50*—column extract +Mock injection -*Lkt50 is a smaller derivative of Lkt that lacks all hydrophobic regions. indicates at least 50 percent neutralization of toxicity of Lkt; indicates no neutralization activity.Source: Modified from Lee et al. (2001). Infect. and Immunity 69:5786–5793.With regards to development of a usable edible vaccine, what work remains to be done?
Open Question
One of the major causes of sickness, death, and economic loss in the cattle industry is Mannheimia haemolytica, which causes bovine pasteurellosis, or shipping fever. Noninvasive delivery of a vaccine using transgenic plants expressing immunogens would reduce labor costs and trauma to livestock. An early step toward developing an edible vaccine is to determine whether an injected version of an antigen (usually a derivative of the pathogen) is capable of stimulating the development of antibodies in a test organism. The following table assesses the ability of a transgenic portion of a toxin (Lkt) of M. haemolytica to stimulate development of specific antibodies in rabbits.Immunogen Injected Antibody Production in SerumLkt50*—saline extract +Lkt50*—column extract +Mock injection -*Lkt50 is a smaller derivative of Lkt that lacks all hydrophobic regions. indicates at least 50 percent neutralization of toxicity of Lkt; indicates no neutralization activity.Source: Modified from Lee et al. (2001). Infect. and Immunity 69:5786–5793.What general conclusion can you draw from the data?
Open Question
The human insulin gene contains a number of sequences that are removed in the processing of the mRNA transcript. In spite of the fact that bacterial cells cannot excise these sequences from mRNA transcripts, explain how a gene like this can be cloned into a bacterial cell and produce insulin.
Open Question
Although many cloning applications involve introducing recombinant DNA into bacterial host cells, many other cell types are also used as hosts for recombinant DNA. Why?
Open Question
The human genome is 3×10⁹bp. You wish to design a primer to amplify a specific gene in the genome. In general, what length of oligonucleotide would be sufficient to amplify a single unique sequence? To simplify your calculation, assume that all bases occur with an equal frequency.
Open Question
Using DNA sequencing on a cloned DNA segment, you recover the nucleotide sequence shown below. Does this segment contain a palindromic recognition sequence for a restriction enzyme? If so, what is the double-stranded sequence of the palindrome, and what enzyme would cut at this sequence? (Consult Figure 20.1 for a list of restriction sites.)

CAGTATGGATCCCAT
Open Question
Using animal models of human diseases can lead to insights into the cellular and genetic bases of the diseases. Duchenne muscular dystrophy (DMD) is the consequence of an X-linked recessive allele.How would you make a Drosophila model of DMD?
Open Question
Restriction sites are palindromic; that is, they read the same in the 5' to 3' direction on each strand of DNA. What is the advantage of having restriction sites organized this way?
Open Question
Using animal models of human diseases can lead to insights into the cellular and genetic bases of the diseases. Duchenne muscular dystrophy (DMD) is the consequence of an X-linked recessive allele.How would you make a mouse model of DMD?
Open Question
List the advantages and disadvantages of using plasmids as cloning vectors. What advantages do BACs and YACs provide over plasmids as cloning vectors?
Open Question
Compare methods for constructing homologous recombinant transgenic mice and yeast.
Open Question
What are the advantages of using a restriction enzyme whose recognition site is relatively rare? When would you use such enzymes?
Open Question
Chimeric gene-fusion products can be used for medical or industrial purposes. One idea is to produce biological therapeutics for human medical use in animals from which the products can be easily harvested—in the milk of sheep or cattle, for example. Outline how you would produce human insulin in the milk of sheep.