Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions.

Zarrin AA, Fong I, Malkin L, Marsden PA, Berinstein NL

Recombination activating gene 1 (RAG1) and RAG2 are the essential and tissue-specific components of V(D)J recombination. We have characterized the genomic organization of the human RAG locus, mapped the transcriptional initiation sites, and partially sequenced and performed functional reporter assays on the 5' flanking regions of human RAG1 and RAG2. Transcription initiation sites were mapped by rapid amplification of 5' cDNA ends, primer extension, and/or RNase protection in normal thymocytes, three pre-B cell lines, and a mature B cell line. A single promoter region was used for RAG1 transcription. In contrast, transcription of RAG2 initiates at two distinct regions of the genome. The 5'-flanking region of the human RAG2 gene is TATA-less; however, there is a GATAA consensus at position -34 with respect to the major transcriptional initiation site of RAG1. Promoter regions of human RAG1 and RAG2 are active in both lymphoid and nonlymphoid cell lines, suggesting that an outside regulatory element is probably involved in the tissue-specific transcriptional regulation of the RAG genes.

Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines.

Iwasaki A, Stiernholm BJ, Chan AK, Berinstein NL, Barber BH

In the course of examining epitope-specific CTL responses to intramuscular plasmid DNA immunization with influenza nucleoprotein (NP)-expressing vectors, a nonimmunogenic mutant NP (NP(o)) was identified. The coding region of NP(o) differed from the wild-type A/PR/8/34 NP sequence (designated NP(v)) by three amino acid alterations in the carboxyl-terminal portion of the molecule remote from the H-2K(d) epitope (147-155) being monitored. Correction of these mutations restored the immunogenicity of the native sequence, indicating that sequence alterations remote from the CTL epitope in question can profoundly influence its immunogenicity. In an effort to identify general, nonstructural means of enhancing the CTL response to weak plasmid DNA immunogens, vectors were constructed expressing NP(o) in tandem with the costimulatory molecules B7-1 or B7-2. Co-linear expression of NP(o) with B7-2, but not B7-1, significantly increased the NP epitope-specific CTL response. In addition, coinjection of these NP(o) plasmids with granulocyte-macrophage CSF- and/or IL-12-expressing vectors also restored near native NP-specific CTL responses. Thus, the coexpression of certain costimulatory molecules and/or cytokines, in concert with a non-self gene delivered as an intramuscular plasmid DNA immunogen, can significantly enhance Ag-specific CTL responses.