NAME: Donald L. Robertson, Ph.D. (E-mail: DONinLA@pacbell.net)
PROFESSIONAL POSITIONS:
EDUCATION:
HONORS:
I. Scientific Research Background.
Research with mouse mammary tumor virus. I have studied mouse mammary tumor virus (MMTV) gene expression. We recently discovered (patent approved September 1997) that the MMTV glucocorticoid response element (GRE), which is required for hormone-induced transcription, has been modified and duplicated within the large terminal repeat (LTR) of the viral DNA isolated from a particular cell line infected with the C3H strain of MMTV. This cell line was grown constantly in the presence of dexamethasone, a synthetic glucocorticoid hormone, resulting in a cell line producing ultra-high levels of MMTV-specific RNA. When proviral DNA was isolated, alterations in the GRE region were observed. These variant LTRs produce high levels of viral RNA and are at least as active as the CMV promoter, and exceed a 500-fold induction ratio after the addition of hormone. There is no increase in background transcription. In contrast, the normal MMTV LTR has a 7-10-fold increase in transcription after hormone addition. Mathematical projections suggest that additional copies of the duplicated 78-bp repeat (found exclusively in our variant MMTV LTRs) could increase the transcriptional activity another 2-5-fold, making it one of the strongest mammalian promoters available.
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 LacSwitchTM (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. In fact, our data suggest that our modified MMTV promoter is a better gene expression system than the Invitrogen ecdysone promoter or promoters using the tetracycline receptor (unpublished data).
Retrovirus oncogene research. For several years, my laboratory studied genetic differences in normal and cancer cells using retroviruses and their oncogenes. Following this experimental approach, we 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 pp60v-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 Rous sarcoma virus. We have also observed that cells transformed by v-src, and the 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
used in my research. I also taught the recombinant DNA and biochemistry
laboratories at BYU where many of these techniques were taught to students.
III. Graduate School and Post-doctoral Research Experience.
As a biochemistry graduate student at the Washington University Medical School I worked in the laboratory of Dr. Robert E. Thach and 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 resulted in the publications of several significant manuscripts (including a Cold Spring Harbor Symposium article) about the relatedness of these intracellular virus-like particles with 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 joining the faculty at BYU in 1980, my research program was externally funded from the outset. Under my direction, five students completed their Ph.D. degrees and six completed M.S. degrees since 1985.
IV. Selected Scientific Publications:
This short scientific CV can be viewed online at http://home.pacbell.net/doninla/cv2.htm. Additional professional information is available at http://home.pacbell.net/doninla/cv.htm.
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