NAME: Donald L. Robertson, Ph.D. (E-mail: DONinLA@pacbell.net)

PROFESSIONAL POSITIONS:

• University Professor: Department of Chemistry and Biochemistry; Brigham Young University; Provo, Utah 84602
  
  • Professor of Chemistry and Biochemistry (1993-1995)
  • Associate Professor of Chemistry and Biochemistry (1985-1993)
  • Assistant Professor of Chemistry and Biochemistry (1980-1985)

• Community College Professor: MiraCosta College (8/99-present); Santa Monica College (8/98-6/99); El Camino College (8/98-6/99); LA Pierce College (9/97-12/98); Glendale College (8/97-5/98); Pasadena City College (1/98-5/98)

• Biotechnology Consultant: Stratagene Corporation consultant from September 1993 - August 1995.

• Post-doctoral: University of California San Francisco (1977-1980); Department of Microbiology and Immunology (with Dr. Harold E. Varmus; current Director NIH); School of Medicine; San Francisco, CA 94143

EDUCATION:

• Ph.D. Department of Biological Chemistry (1972-1976); Washington University Medical School (with Dr. Robert E. Thach); 660 So. Euclid Ave.; St. Louis, MO 63110

• B.S. Department of Chemistry (1970-1972); Brigham Young University; Provo, UT 84602

• Department of Chemical Engineering (1966-1968); University of Idaho; Moscow, ID 83843

HONORS:

• Graduated Cum Laude, Brigham Young University (1972)
• USPHS Pre-doctoral fellowship, Washington Univ. (1972-76)
• Junior Research Fellowship, American Cancer Society, California Division (1977-78)
• Senior Research Fellowship, American Cancer Society, California Division (1979-80)
• Summer Faculty Research and Engineering Program, USAMRIID, Ft. Detrick (1984)
• National Research Council, Senior Research Associate, USAMRIID, Ft. Detrick (1986-87); Professional Development Leave (sabbatical)
• U.S. Patent 5,814,493: "Viruses and expression vectors containing LTR size variants"

MEMBERSHIPS:

• American Society for Microbiology
• American Association for the Advancement of Science
• American Chemical Society
• American Society for Biochemistry and Molecular Biology

I. Scientific Research Background.

Research with mouse mammary tumor virus. I have studied mouse mammary tumor virus (MMTV) and the effects of glucocorticoid hormones on MMTV gene expression. We recently discovered (patent approved) that the MMTV glucocorticoid response element (GRE), which is required for hormone-induced transcription, has been duplicated within the large terminal repeat (LTR) isolated from a cell line which was originally infected with the C3H strain of MMTV. This cell line was grown continually in the presence of dexamethasone, a synthetic glucocorticoid hormone, and extremely high levels of MMTV-specific RNA were observed. Some of the proviral DNA isolated from these cells had undergone recombinational events which duplicated part of the GRE. These modified LTRs are highly inducible and produce extremely high levels of viral RNA without an increase in the background level of expression. These MMTV variant promoters exceed 500-fold induction levels and are at least as active as the CMV promoter. In contrast, the normal MMTV LTR usually has a 7-10-fold increase in transcription after hormone addition. Mathematical projections suggest that additional copies of the 78-bp repeat found exclusively in our variant MMTV LTRs could increase the transcriptional activity another 2-5-fold, making it the strongest inducible promoter for mammalian gene expression.

The variant MMTV LTRs can be used for the inducible expression of heterologous genes. We have fused these variant LTRs to the lac operator from LacSwitch^TM (Stratagene, Inc.) for tighter repression using the lac repressor. Pleiotropic effects due to unwanted hormone activation of normal cellular genes are virtually eliminated since our promoter requires at least a 10-fold lower level of hormone to achieve the same amount of transcription as the normal MMTV promoter and other hormonally responsive cellular genes. Viral vectors containing these variant MMTV GREs can be used to study the expression of specific genes, such as those required for cell cycle regulation or neoplastic transformation, and for the selective expression of genes in transgenic animals. A Figure showing the relative activities of these promoters can be viewed.  This gene expression system is covered by U.S. Patent Number 5,814,493, which can be viewed online.

Retrovirus oncogene research. For several years, my laboratory has studied the genetic differences present in normal and cancer cells using retroviruses and their oncogenes. Following this experimental approach, we have studied some of the biochemical and genetic changes that occur in cells transformed by v-src, v-mos, v-myc, v-abl, v-fos, v-fes or v-ras oncogenes. Transformation-specific differences include differential gene expression, altered cellular morphology, increased growth rates and nutrient consumption, the ability to grow in soft agar, etc. When cells containing these oncogenes were treated with cyclic AMP (cAMP), or related analogs (e.g., 8-chloro-cAMP), a complete reversal of the transformed phenotype is observed. cAMP affects the transcription of many regulatory genes, including transcription factors and other growth genes.

In collaboration with Dr. Daniel Simmons (BYU), we also studied the enhanced transcription of gene induced by the viral oncogene v-src After the expression of active pp60^v-src, the mitogen-inducible prostaglandin G/H synthase (PGHS-2; cyclooxygenase-2) gene (pghs-2) is induced and transcribed at high levels. We have observed that pghs-2, or its normal cellular homolog (pghs-1), is expressed at elevated levels in mouse NIH 3T3 cells transformed by v-src, v-mos, v-myc, v-abl, v-fos, v-fes or v-ras genes. These data suggest that the enhanced expression of one, or both, of these cyclooxygenase enzymes is important, if not essential, for cellular transformation by these oncogenes. The JUN and FOS proteins are apparently involved in this process, with the AP-1 transcriptional complex being affected by the addition of cAMP.

It is interesting to note that research conducted by Dr. Simmons has demonstrated that the chemical inhibition of the PGHS-2 (by non-steroidal anti-inflammatory drugs) prevents cellular transformation by the Rous sarcoma virus. We have also observed that cells transformed by v-src, and the other oncogenes listed above, produce less cyclooxygenase-specific mRNA following the addition of cAMP, when there is a loss of the transformed phenotype.

Bacillus anthracis gene expression. We have also used different Bacillus spp., including Bacillus anthracis, to develop a high-level, inducible Bacillus-based gene expression systems for the high-level production and secretion of proteins. We have constructed several plasmid vectors that can be used for the production and secretion of proteins (prokaryotic and eukaryotic) whose genes are placed downstream from the T7 RNA polymerase promoter. Proteins produced in these bacilli are secreted for easier purification. Other modified bacilli are being used for vaccine development against the highly virulent and lethal B. anthracis, which causes anthrax.

II. Experimental Techniques and Procedures.

The following list shows some of the experimental techniques and procedures with which I am familiar and use in my research program. I also teach the recombinant DNA and biochemistry laboratories where many of these techniques are taught to students. I feel comfortable, not only in using these procedures myself but in teaching them in a supervisorial capacity. I have used most procedures conducted in a modern biochemistry and molecular biology laboratory.

• Manual and automated (ABI 373A) dideoxy terminator DNA sequencing.
• HPLC analysis of proteins, nucleic acids and amino acids.
• Polymerase Chain Reaction and cloning of reaction products, including a quantitative analysis of RNA transcripts using PCR.
• Micro-DNA preparation for PCR.
• Animal cell tissue culture and virus growth and characterization.
• Eukaryotic and prokaryotic gene expression and transcription control.
• Eukaryotic and prokaryotic expression vector construction.
• Plasmid, bacteriophage (M13, lambda), and cosmid cloning in E. coli, including standard transformation and electroporation.
• Synthesis of prokaryotic and eukaryotic cDNA and cloning in plasmid and phage vectors.
• Genome DNA cloning.
• Transformation of Bacillus species with plasmids, including integration plasmids for production of foreign genes.
• Analysis of proteins, including purification, polyacrylamide gel electrophoresis, western blotting and immunological detection.
• Analysis of RNA and DNA, including agarose and acrylamide gels, blotting and hybridization.
• Radiolabeling of RNA, DNA and proteins.
• Synthesis and purification of synthetic oligonucleotides (ABI 381A).
• Construction and screening of recombinant DNA libraries, both genomic and cDNA.
• Mutagenesis of cloned DNA in plasmid and phage vectors.
• DNA-protein binding interactions and analysis.
• Protein and enzyme isolation and characterization.

III. Graduate School and Post-doctoral Experience.

As a biochemistry graduate student at the Washington University Medical School working in the laboratory of Dr. Robert E. Thach, I performed a biochemical characterization of the proteins and nucleic acid components of the RNA tumor virus A-type particles found in mouse myeloma (plasmacytoma) tumors. These studies analysis resulted in the publications of several significant manuscripts (including a Cold Spring Harbor Symposium article) about the relatedness of these intracellular virus-like particles and the mouse retroviruses.

As a post-doctoral fellow in the laboratory of Dr. Harold E. Varmus (currently Director of the NIH) and Dr. J. Michael Bishop at the University of California San Francisco, I studied mouse mammary tumor virus (MMTV) gene expression. These studies helped us understand the mechanism by which retroviral genes are regulated and expressed. Included in these studies were biochemical characterizations of the viral proteins and nucleic acids, including the dexamethasone induced expression of the virus-specific RNA and proteins.

After going to BYU in 1980, my research program was externally funded from the outset. Under my direction, five students completed their Ph.D. degrees and six have completed M.S. degrees since 1985.

IV. Graduate and Undergraduate Students Supervised.

While at BYU, I supervised many graduate and undergraduate students. I also supervised the molecular biology-related research for many students from other labs when recombinant DNA techniques were involved. The research of my graduate students was published. For my students who completed their M.S. degrees, all have gone to other graduate schools to work on their Ph.D. or M.D. degree. My Ph.D. students have taken post-doctoral positions, with the exception of Dr. Kent Hill who went to medical school. An undergraduate student performed research entitled "A Study of Antigenes Against HIV" for his senior Honor's Thesis. Listed below are the graduate students who have completed advanced biochemistry degrees in my laboratory. The titles of their theses or dissertations, as well as the date of their graduation, are shown.

• Thesis (M.S.) by Roger L. Kaspar (August, 1986): "Purification and characterization of pXO1 and pXO2 plasmids from Bacillus anthracis"
• Thesis (M.S.) by Roger David Johnson (April, 1990): "Sequence analysis of the poison region of the C3H strain of mouse mammary tumor virus DNA"
• Thesis (M.S.) by Yungang Luh (August, 1991): "Genetic modification and expression in Bacillus subtilis of the Bacillus anthracis edema factor toxin gene which encodes a calmodulin-dependent adenylate cyclase"
• Thesis (M.S.) by Scott C. Simpson (August 1993): "Cloning and construction of a restriction map of Bacillus anthracis pXO1"
• Thesis (M.S.) by Kuhia Loren Fisher (August 1993): "Characterization of multiple glucocorticoid response elements in the long terminal repeat of mouse mammary tumor virus"
• Thesis (M.S.) by Marshall Cornell Mendenhall (December 1993): "Characterization of the pol gene of the C3H strain of mouse mammary tumor virus"
• Thesis (M.S.) by Sheng Chang (January 1995): "Molecular Cloning and Characterization of the c-src Gene from NIH3T3 Cells and Other Mouse Tissues"
• Dissertation (Ph.D.) by Kent R. Hill (December, 1986): "Biochemical characterization of pp60^src in Rous sarcoma virus transformed cells reverted by cyclic-AMP analogs"
• Dissertation (Ph.D.) by M. Todd Tippetts (April, 1987): "Molecular cloning of the chloroplast genome of Carthamus tinctorius L. and of the edema factor gene from Bacillus anthracis"
• Dissertation (Ph.D.) by Thomas S. Bragg (December, 1989): "Restriction map of plasmid pXO2 and characterization of the lethal factor gene from Bacillus anthracis"
• Dissertation (Ph.D.) by Weilin Xie (December, 1991): "Cloning and characterization of a pp60^v-src-inducible prostaglandin G/H synthase"
• Dissertation (Ph.D.) by Lin Xu (December 1992): "Characterization of the liver cytosolic serine hydroxymethyl transferase gene from human and rabbit"

V. Research Grants and Contracts.

I have had grant or contract funding from the National Science Foundation, American Cancer Society, U.S. Army Medical Research and Development Command, Naval Research Office, the Bireley Foundation, and the BYU Cancer Research Center. The source, title, duration and monies associated with these grants are as follows:

• National Science Foundation (6/81-11/83): $75,000. "Mouse mammary tumor virus gene expression"
• American Cancer Society (7/81-6/82): $5,000. "Rous sarcoma virus pp60^src metabolism in reverted transformed cells"
• United States Army Medical Research and Development Command (6/85-7/88): $235,136. "The use of recombinant DNA techniques for the production of a more effective anthrax vaccine"
• U.S. Army Research Office and Office of Naval Research (10/88-9/89): $110,000. "Use of an automated DNA Sequencer for DNA Sequence Determination of Bacillus anthracis Plasmids pXO1 and pXO2"
• The Bireley Foundation (Joint Award with Dr. Daniel Simmons) (3/90-2/93): $108,000. "The use of oncogenes to study cancer development"
• The BYU Cancer Research Center (7/88-7/93): $36,000. "The use of oncogenes to study cancer development"
• United States Army Medical Research and Development Command (1/93-12/94): $269,366. "The use of the Bacillus species to express the Bacillus anthracis toxin genes for vaccine studies"

VI. Refereed Publications:

1. Cannon, J.F., D.L. Robertson, and H.T. Hall. 1972. The synthesis of lanthanide-iron Laves phases at high pressures and temperatures. Materials Res. Bull. 7:5-11.
2. Robertson, D.L., J.F. Cannon, and H.T. Hall. 1972. High pressure and high-temperature synthesis of LaCo₂. Materials Res. Bull. 7:977-982.
3. Cannon, J.F., D.L. Robertson, and H.T. Hall. 1972. The effect of high pressure on the formation of LRu₂ and LOs₂ (L=lanthanide) compounds. J. Less-Common Metals 29:141- 146.
4. Cannon, J.F., D.L. Robertson, H.T. Hall, and A.C. Lawson. 1973. The effect of high pressure on the crystal structure of LaOs₂ and CeOs₂. J. Less-Common Metals 31:174- 176.
5. Lawson, A.C., J.F. Cannon, D.L. Robertson, and H.T. Hall. 1973. Superconductivity of lanthanum-osmium (LaOs₂). J. Less-Common Metals 32:173-174.
6. Cannon, J.F., D.L. Robertson, H.T. Hall, and A.C. Lawson. 1974. High pressure of -tungsten-type triniobium telluride. J. Phys. Chem. Solids. 35:1181-1182.
7. Thach, R.E., D.L. Robertson, N.L. Baenziger, and D. Dobbertin. 1974. Reverse transcriptase associated with A-type particles from murine myeloma cells. Cold Spring Harbor Symposium on Quantitative Biology 39:963-968.
8. Robertson, D.L., N.L. Baenziger, D. Dobbertin, and R.E. Thach. 1975. Characterization of DNA polymerase and RNA associated with A-type particles from murine myeloma cells. J. Virol. 15:407-415.
9. Robertson, D.L., P. Yau, D. Dobbertin, T.K. Sweeney, S. Thach, and R.E. Thach. 1976. Relationships between intracisternal type A and extracellular oncornavirus-like particles in murine MOPC-460 myeloma cells. J. Virol. 18:334-355.
10. Robertson, D.L., and H.E. Varmus. 1979. Structural analysis of the intracellular RNAs of murine mammary tumor virus. J. Virol. 30:576-589.
11. Robertson, D.L., P. Jhabvala, T. Geodfrey-Colburn, and R.E. Thach. 1979. Characterization of the proteins of intracisternal type A and extracellular oncornavirus-like particles produced by MOPC-460 myeloma cells. J. Virol. 32:114-122.
12. Robertson, D.L., and H.E. Varmus. 1981. Dexamethasone induction of the intracellular RNAs of mouse mammary tumor virus. J. Virol. 40:673-682.
13. Robertson, D.L. 1984. Dexamethasone stimulated expression of a proviral copy of the env mRNA of mouse mammary tumor virus. J. Virol. 50:632-635.
14. Robertson, D.L. 1984. S1 nuclease analysis of a deleted mouse mammary tumor virus DNA. Virus Research 1:649-654.
15. Leppla, S.H., D.L. Robertson, S.L. Welkos, L.A. Smith, and M.H. Vodkin. 1986. Cloning and analysis of genes for anthrax toxin components, pp. 275-278. In Bacterial protein toxins, Suppl. 15. Zentralblatt für bakteriologie und hygiene. l. Abteilung. Gustav Fischer, Stuttgart.
16. Robertson, D.L., and S.H. Leppla. 1986. Molecular cloning and expression in Escherichia coli of the lethal factor gene of Bacillus anthracis. Gene 44:71-78.
17. Kaspar, R.L. and D.L. Robertson. 1987. Purification and analysis of Bacillus anthracis plasmids pXO1 and pXO2. Biochem. Biophys. Res. Commun. 149:362-368.
18. Tippetts, M.T. and D.L. Robertson 1988. Molecular cloning and expression of the Bacillus anthracis edema factor toxin gene (cya): A calmodulin-dependent adenylate cyclase. J. Bacteriol. 170:2633-2636.
19. Robertson, D.L., M.T. Tippetts and S.H. Leppla. 1988. Nucleotide sequence of the Bacillus anthracis edema factor (cya) gene: A calmodulin-dependent adenylate cyclase. Gene 73:363- 371.
20. Robertson, D.L. 1988. Relationships between the calmodulin-dependent adenylate cyclases produced by Bacillus anthracis and Bordetella pertussis. Biochem. Biophys. Res. Commun. 157:1027-1032
21. Bragg, T. and D.L. Robertson. 1989. Nucleotide sequence and analysis of the Bacillus anthracis lethal factor gene (lef). Gene 81:45-54.
22. Robertson, D.L., T.S. Bragg, S.C. Simpson, R. Kaspar, W. Xie, and M.T. Tippetts. 1990. Mapping and characterization of the Bacillus anthracis plasmids pXO1 and pXO2. Salisbury Medical Bulletin 68:55-58.
23. Robertson, D.L. and T.S. Bragg. 1990. Nucleotide sequence of the lethal factor (lef) and edema factor (cya) genes from Bacillus anthracis: Elucidation of the EF and LF functional domains. Salisbury Medical Bulletin 68:59.
24. Tippetts, M.T., D.L. Robertson and M.A. Smith. 1991. Complete cloning of the chloroplast genome of safflower in EMBL3 and mapping of the 23S and 16S rRNA genes. Molecular and Cellular Biochemistry 100:61-70.
25. Xie, W., J.G. Chipman, D.L. Robertson, R.L. Erikson, D.L. Simmons. 1991. Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc. Natl. Acad. Sci. USA. 88:2692-2696.
26. Carl, M., R. Hawkins, N. Coulson, J. Lowe, D.L. Robertson, W.M. Nelson, R.W. Titball, and J.N. Woody. 1992. Detection of spores of Bacillus anthracis using the polymerase chain reaction. J. Infectious Diseases 165:1145-48.
27. Xie, W., D.L. Robertson, and D.L. Simmons. 1992. Mitogen-inducible prostaglandin G/H synthase: A new target for nonsteroidal antiinflamatory drugs? Drug Development Research 25:249-265.
28. Evett, G.E., Xie, W., Evett, G., Merrill, J., Robertson, D.L., Bradshaw, W.S. 1993. Drug inhibition and cellular regulation of prostaglandin G/H synthase isoenzyme 2. J. Lipid Med. 6:113-117.
29. Evett, G.E., Xie, W., Chipman, J., Robertson, D.L., and Simmons, D.L. (1993) Prostaglandin G/H synthase isoenzyme 2 expression in fibroblasts: Regulation by dexamethasone, mitogens, and oncogenes. Arch. Biochem. Biophys. 306:169-177.
30. Robertson, D.L. and F. Spangler. 1996. The use of a regulated T7 RNA polymerase-based transcription system for the expression of the anthrax toxin and heterologous genes in Bacillus anthracis. Salisbury Medical Bulletin 87:94-96.
31. K.L. Fisher and D.L. Robertson. 1997. Characterization of multiple glucocorticoid response elements in the long terminal repeat of mouse mammary tumor virus. Manuscript written, waiting for patent progress.
32. Simpson, S.C., and D.L. Robertson. 1997. Complete cloning, restriction mapping and characterization of the Bacillus anthracis toxin plasmid pXO1. Manuscript in preparation, submitted to Medical Microbiology.
33. Bragg, T.S. and D.L. Robertson. 1996. Construction of a restriction map of the Bacillus anthracis capsule plasmid pXO2. Submitted to Plasmid.
    

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