Ada Rosen

In the NOD mouse, the onset of beta-cell destruction is associated with spontaneous development of T-lymphocytes reactive to members of the 60 kDa heat shock protein (hsp60) family, including the Mycobacterial (MT) and the human (H) hsp60 molecules. Diabetes in the NOD mouse is a spontaneous tissue-specific autoimmune disease occurring without prior immunization. Therefore, it has been suggested that the anti-hsp60 T cells involved in the autoimmune diabetes of NOD mice might reflect molecular mimicry between MT-hsp60 and a beta-cell tissue specific molecule sharing similar T cell epitopes, the p277 peptide of hsp60 in particular. We cloned and expressed the mouse hsp60 cDNA from a beta-cell tumour. This mouse beta-cell hsp60 cDNA was found to be identical in sequence to the hsp60 of mouse fibroblasts. We further report that NOD spleen cells and an NOD diabetogenic T cell clone C9 responded to the recombinant mouse hsp60 and to its peptide M-p277 to the same extent as to H-hsp60 and H-p277. Splenocytes of mice of other strains did not respond to p277. Moreover, treatment of 3 month old NOD mice with the non-modified self M-p277 peptide was as efficient as H-p277, from which it differs in one amino acid, in halting progression of the disease. Thus, anti-H-p277 T cells modulating diabetes in the NOD mouse are autoreactive, and are targeted at the mouse beta-cell hsp60, which is not tissue specific. These findings raise the question of how a non-tissue specific molecule may be a target of a tissue-specific autoimmune disease.

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on multiple DNA sequence elements located in the 5' flanking region of the gene. Of particular importance in the rat insulin I gene are two closely similar 9 bp sequences (IEB1 and IEB2): mutation of either of these leads to 5-10 fold reduction in transcriptional activity. We have screened an expression cDNA library derived from mouse pancreatic endocrine beta cells with a radioactive DNA probe containing multiple copies of the IEB1 sequence. A cDNA clone (A1) isolated by this procedure encodes a protein which shows efficient binding to the IEB1 probe, but much weaker binding to either an unrelated DNA probe or to a probe bearing a single base pair insertion within the recognition sequence. DNA sequence analysis indicates a protein belonging to the helix-loop-helix family of DNA-binding proteins. The ability of the protein encoded by clone A1 to recognize a number of wild type and mutant DNA sequences correlates closely with the ability of each sequence element to support transcription in vivo in the context of the insulin 5' flanking DNA. We conclude that the isolated cDNA may encode a transcription factor that participates in control of insulin gene expression.

A 13.4 kb rat genomic DNA fragment containing two related tRNA gene clusters was isolated from a rat lambda recombinant and analyzed for gene arrangement and nucleotide sequence. One cluster was found to contain a tRNALeuCUG gene while the second contained a tRNALeuCUA pseudogene with multiple base substitutions. The tRNALeu gene was found to possess an intact coding region and a functional transcription termination signal at the 3' end as demonstrated by in vitro transcription and processing of precursors to mature size tRNA. The first tRNA gene cluster was found to contain in addition to tRNALeu, three other transcribable genes coding for tRNAAspGAC(U), tRNAGlyGGA(G) and tRNAGluGAG; the second cluster contained in addition to tRNALeu pseudogene, the tRNAAsp tRNAGly and tRNAGlu genes. Examination of flanking sequences of the corresponding tRNA genes in the two clusters shows no homology at the 5' ends and partial conservation of sequences at the 3'-end region. Genomic rat DNA blot hybridizations show that the tRNALeu gene is distributed together with the tRNAAsp, tRNAGly and tRNAGlu on a 10 fold repeat of 3.2 kb EcoRI fragment.

The E2A gene encodes transcription factors of the helix-loop-helix family that are implicated in cell-specific gene expression as part of dimeric complexes that interact with E box enhancer elements. It has previously been shown that transcripts of the E2A gene can be detected in a wide range of cell types. We have now examined expression of the mouse E2A gene at the protein level using polyclonal antisera directed against distinct portions of the E2A protein to probe blots of cellular extracts. A 73 kDa protein was identified by this analysis: this protein is highly enriched in cell lines of B lymphoid origin as compared to pancreatic beta-cells and fibroblast cells. The detection of this protein selectively in extracts of lymphoid cells correlates with the presence of the E box-binding activity LEF1/BCF1 in these cells; this binding activity was previously shown to be efficiently recognized by antiserum directed against E2A gene products. Transfection of cells with full length E2A cDNA leads to appearance of protein co-migrating with the 73 kDa protein on SDS gel electrophoresis and co-migrating with LEF1/BCF1 on mobility shift analysis. Our results are consistent with the view that the DNA-binding activity LEF1/BCF1 is a homodimer of E2A proteins; the selective appearance of this putative cell-specific transcription factor in B lymphoid cells seems to be attributable, at least in part, to the elevated E2A protein concentrations in these cells.

A rat genomic library was screened for tRNA genes with an unfractionated rat liver tRNA probe. About 70 clones containing tRNA genes were detected per rat genome. The organization of tRNA genes in five clones was analyzed by restriction endonuclease digestion, RNA-DNA hybridization and in vitro transcription with nuclear extracts from Xenopus oocytes. Evidence is presented suggesting that tRNA genes are distributed in the rat genome in small clusters spanning 1 to 2 kb and interspersed with large regions (minimum 8 to 20 kb) of non tRNA-coding DNA. The tRNA gene clusters were found to contain the sequences for a variety of tRNA species. Genes for a single isoacceptor, were found in more than one clone. The detailed study of one clone shows the repetition of a cluster of four tRNA sequences at a distance of about 8 kb. The arrangement of tRNA genes in rat appears to follow the irregular pattern of tRNA gene organization previously reported in Drosophila and Xenopus.