THE CONTROL OF CELLULAR GENE EXPRESSION BY A UNIQUE TUMOR VIRAL SEQUENCE: CONSTRUCTION OF MODEL REGULATORY SYSTEM BY RECOMBINANT-DNA METHODOLOGY AND THE EFFECT OF INSERTED ENHANCER-PROMOTER SEQUENCES ON HOST CELLULAR GENES (PLTR, PAAP)

The DNA recombinant technology methods employed in this study, allowed the probing of the human genome for the presence of retroviral sequences. This dissertation describes a technique using a mini-plasmid, (pi)VX, as a probe to detect retroviral homologous sequences in a manner than had been successfully utilized to detect hemoglobin genes in a (lamda) phase library containing the cloned human genomic information. To accomplish this procedure, a method was developed to prepare (pi)VX in a purified form. A novel method for augmenting the yields of the mini-plasmid, (pi)VX, is also described. A human Alu-repeated-ubiquitous sequence was isolated and cloned into the mini-plasmid (pi)VX to make a unique recombinant construct, (pi)BLUR, which was a successful probe to detect Alu homologous regions in a human genome cloned into the lambda library. The detection was, in human genome cloned into the lambda library. The detection was, in fact, quite species specific and did not recognize mouse homologous Alu sequences in a mouse-human hybrid library. Having successfully detected reiterated repeat sequences using the (pi)BLUR as a model system indicated the feasibility of using this procedure to probe for LTR-retroviral-like sequences in the human genome. The LTR is notable for its enhancer activity and its necessity to induce malignant transformation. The Murine Sarcoma Virus (MSV) LTR region was isolated and subcloned into a plasmid and further cloned into the (pi)VX mini-plasmid and used to probe the human genome contained in a phage library. This mini-plasmid construct ((pi)LTR) was able to detect a comparable homologous sequence not only in a human placental DNA containing library, but also in human genomic libraries derived from pancreatic and colonic tumors as well. This unique finding of a human LTR-retroviral element in the human DNA is consistent with it being introduced by either (a) an exogenous human retrovirus or (b) an endogenous route through the heritable course of evolution, since such sequences are commonly known to occur in other mammalian genomic species.