Transgenic mice and transhybridomas producing chimeric mouse/human anti-human interleukin-2 receptor recombinant antibodies.

Transgenic mice were developed that secreted chimeric mouse/human anti-human interleukin-2 receptor (IL-2R) antibodies (Ab) into their serum. In addition, hybridomas producing the chimeric Ab in tissue culture were generated from the transgenic mice. The presence of the mouse/human immunoglobulin (Ig) transgene did not appear to affect rearrangement of endogenous murine Ig in the hybridomas. Serum levels of the chimeric Ab correlated with transgene copy number. Although many of the transgenic lineages had serum titers of the chimeric Ab comparable to endogenous mouse IgG, there was no apparent correlation with endogenous mouse IgG levels.

Replication-dependent transactivation of the polyomavirus late promoter.

When a plasmid containing the wild-type polyomavirus intergenic regulatory region fused to the bacterial cat gene was introduced into mouse NIH 3T3 cells along with a plasmid coding for the early viral proteins (T antigens), chloramphenicol transacetylase enzyme activity and mRNA levels were increased about 10-fold over levels observed in the absence of early proteins. To investigate this transactivation phenomenon further, 11 specific deletion mutant derivatives of the wild-type parent plasmid were constructed and studied. One mutant (NAL) with a minimal level of chloramphenicol transacetylase expression in the absence of T antigens was capable of being transactivated more than 40-fold. A number of other mutants, however, had little capacity for transactivation. Each of these mutants had in common a defect in large T-antigen-mediated DNA replication. Interestingly, one of the transactivation-defective mutants showed a basal late promoter activity fivefold higher than that of wild type and replicated in mouse cells in the absence of large T antigen. Subsequently, a small deletion abolishing viral DNA replication was introduced into those mutants capable of transactivation. The effect of the second deletion was to eliminate both replication and transactivation. Finally, wild-type and mutant constructs were transfected into Fisher rat F-111 cells in the presence or absence of early proteins. No transactivation or replication was ever observed in these cells. We concluded from these studies that the observed transactivation of the polyomavirus late promoter by one or more of the viral early proteins was due to either higher template concentration resulting from DNA replication or replication-associated changes in template conformation.

Deletion analysis of the polyomavirus late promoter: evidence for both positive and negative elements in the absence of early proteins.

We have been interested in understanding more about the sequences that constitute the polyomavirus late promoter. Our approach has been to target specific deletions to the viral intergenic region by oligonucleotide-directed mutagenesis. Wild-type and mutant promoter cassettes with defined deletions were then inserted into a promoterless expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene (cat). Plasmids were introduced into mouse NIH 3T3 cells by transfection, and promoter activities were assessed by quantitation of both CAT enzyme and cat mRNA levels. In this report, we present the results of experiments designed to map promoter elements which affect late transcription in the absence of early viral proteins and viral DNA replication. Using this approach, we mapped two major cis-acting elements (a positive and a negative one) which affect transcription in our transient expression system. The first, positive, element coincided with the enhancer A element, which is known to be important for early transcription and viral DNA replication. Removal of this element reduced late transcription by 50- to 100-fold. The second element was a negative one; removal of 89 base pairs that included two high-affinity large-T-antigen-binding sites just to the early side of the inverted repeat structure within the replication origin resulted in a 5- to 10-fold increase in late promoter activity. The implications of these findings for late promoter function and regulation are discussed.