Final Exam Biotechnology 395

Name_____________________________

General Instructions: This examination is worth 200 points, 40% of the grade for this course. It has two parts: short answer questions worth 10 points each and essay questions each worth 10 points each. The authors of the questions will be identified both for your information and for grading purposes. Read instructions, budget your time, skip over questions you cannot answer promptly and return to them after you have answered all the others you can. Good luck! K.L. Campbell, Course Director.

Multiple Choice: 10 points per question, 2 points per response. Circle all answers that are correct; this may be as many as 5 and as few as 0. Answer 1 through 9 (90 pts).

Dr. Beck:

• Adoptive transfer of acquired immune responsiveness involves the transfer of:



• antibody.
• lymphocytes.
• serum.
• genes.
• legal documents.



• HAT medium is used to:



• immortalize B-lymphocytes.
• culture B-lymphocytes.
• select for hybrids in the hybridoma technique.
• kill B-cell hybridomas.
• fuse B-lymphocytes to myeloma cells.





• The paratopes of immunoglobulins:

• are restricted to light chains.

• are in the constant part of the Ig molecule.
• bind to Fc receptors.

• are involved in hapten recognition.
• are complementary to isotopes on antigens.

Dr. Campbell:

• Embyonic lethality is a common problem during production of knock-out animals. Under what condition(s) would this problem be most pronounced?

• When a single copy gene is targeted.
• When a multicopy gene is targeted.
• When the normal gene is expressed late in embryogenesis.
• When the normal gene is expressed early in embryogenesis.
• In first generation animals heterozygous for the knocked-out gene.





• Postive-negative selection



• May be used as an alternative to intensive DNA screening by Southern blotting or PCR as a means of assuring incorporation of a transgene into an engineered animal.
• Involves checking that the first generation offspring of an animal infected with an engineered retrovirus carries the transgene while the second generation of such animals do not.
• Is designed to allow random gene insertion into target cell DNA of embryonic stem cells with elimination of the non-homologously incorporated transgenes following exposure of the transfected cells to two drugs.
• Increases the efficiency of generation of transgenic animals versus that found with simple infection of early stage embryoes with engineered retroviruses.
• Assures that engineered DNA will be incorporated into the DNA of the host cells and will allow production of transgenic offspring.





• To date



• all gene therapies have been total failures.
• all gene therapies have been restricted to applications to somatic tissues.
• in vivo gene therapies have been preferred to ex vivo approaches.
• therapies have only worked temporarily, if at all, when given in vivo.
• therapies have utilized naked DNA, liposomes, replication-deficient adenoviruses, modified RNA retroviruses, and Herpes simplex virus mutants as vehicles for delivering therapeutic DNA sequences.

• Clones of all the following species have been generated in the laboratory and grown to adulthood, or at least well past infancy, as of Spring 1999:



• Rabbit
• Mouse
• Rhesus macaque
• Human
• Cow





• Clonal animals



• are often incapable of transmitting their modified genes to the next generation but are still valuable as pharmaceutical bioreactors.
• are always capable of transmitting their modified genes to the next generation but may be worth little as pharmaceutical bioreactors due to unforeseen phenotypic effects.
• that are created using adult cell nuclei as the source of the zygote nucleus are less identical to their adult cell progenitors than are identical twins formed by spontaneous separation of the blastomeres of early stage embryos.
• that are created using adult cell nuclei as the source of the zygote nucleus are just as identical to their adult cell progenitors as are identical twins formed by spontaneous separation of the blastomeres of early stage embryos.
• are a technology of choice over transgenic animals in production of disease models because of their lower cost of production.

• Regulation of DNA-based technologies



• was originally a result of self-governance by molecular biologists in the 1980's and stemmed from James Watson's publication of a letter in The New York Times.
• generated a review body at NIH called the RAC dedicated to reviewing proposals to conduct DNA modification research.
• now centers on questions of the propriety of producing transgenic proteins for drug use.
• has become so obsolete that no restrictions apply unless the genes are to be introduced directly into humans.
• now revolves primarily around questions of environmental modification(s) by transgenic organisms and of effects of gene therapies on patient populations.

Short Response: Briefly answer any 3 of 10 through 13 (30 pts).

Dr. Sugumaran:

• What are the biochemical steps that lead to the activation and toxicity of parasporal crystals from Bacillus thuringenesis?

• How did scientists establish which plasmid codes for the crystal protein of Bacillus thuringenesis spores?

















• How can Baculovirus in combination with juvenile hormone esterase gene be used as a biological control agent?

















• How can transition state analogs be useful for developing new drugs?

Dr. Davis

Short Response: Briefly answer 14 through 16 (30 pts).

• What is the main difference between batch, fed batch, and continuous culture?























• What are three genetic engineering approaches to decreasing the need for nitrogen fertilizers for crop plants?





















• List what happens when the Ti plasmid of Agrobacterium tumefasciens transforms plant cells.

Essays: Answer 17, 18 or 19, 20, 21, and 22. Total = 50 pts, 10 points per question.

Dr. Beck:

• While working for a small start-up biotechnology company you design and prepare an immunotoxin by conjugating diphtheria toxin with a monoclonal antibody specific for a tumor cell antigen. At a meeting with the marketing vice-president and the company legal staff they inquire as to the safety of the product. They have a series of questions they need answered by you.



• If this immunotoxin is injected into an animal, will any normal cells be killed? Explain.

























• If the antibody part of the immunotoxin is degraded so that the toxin is released, will normal cells be killed? Explain.

Dr. Sugumaran

Answer 1 of the following 2 essays:

• Write an essay on various techniques used in purification of proteins. Make sure you discuss at least 5 of them.

Dr. Sugumaran

• Illustrate with a diagram how the portioning and mixing (also known as split-mix) technique works in making a tripeptide combinatorial library.

Dr. Campbell

Answer 20 and 21.

• Many people and animals become diabetic as they age. Often they are found to produce normal to above-normal levels of biologically active insulin. The problem in this condition, a form of Type II diabetes mellitus, proves to be the lack or loss of insulin receptor in the major target tissues liver, muscle, and fat. One possible approach to treating the condition would be introduction of additional species-specific receptors into normal targets.

• Describe at least two approaches for introducing these receptors into the affected organisms.

























• Discuss the problems and limitations of these approaches in humans.

• To quote Glick and Pasternak, 2^nd Edition, Molecular Biotechnology, 1998, p 609: "Discuss the premise 'Genetic engineering ... is a radical new technology that violates the fundamental laws of nature.'"

Dr. Davis

• Read the attached paper, which describes a whole transgenic plant kind of bioreactor for producing recombinant proteins, with the aim of answering the following questions.

[Vocabulary you may need:

root exudate: material secreted by root tips

hydroponic: growth with roots in liquid nutrient medium (see Fig. 2D)

heterologous: from a different source

apoplastic space: the cell walls and intercellular liquid in plant tissues

in planta: in intact plants

rhizosphere: the space around the roots]

• Underline important points as you read.







• In one paragraph of your own words, briefly summarize their studies.

• Make a table comparing the productivity (grams recombinant protein per day per gram dry weight of root or seed) of root secretion of GFP, root secretion of SEAP, and maize seed production of avidin. Add at least two other criteria that should also be compared (even if all data are not present).

• In one paragraph, discuss what might account for these productivity differences, and suggest what conditions might be changed to optimize the root secretion method (based on the general characteristics of industrial scale cell cultures).