Daniel U Rabin

Cloning and Expression of IDDM-Specific Human Autoantigens

A DNA cloning approach was taken to identify islet cell protein antigens that are recognized specifically by insulin-dependent diabetes mellitus (IDDM) sera. A human islet cDNA library was generated and screened with diabetic sera. In this article, identification of two clones is described. Proteins expressed by these λ phages appeared to react specifically with newly diagnosed diabetic sera. Islet cell antibody 12 (ICA12) was tested by Western blotting. ICA512 was not reactive with sera in the Western format but was specifically immunoprecipitated by diabetic sera from an Escherichia coli extract.

An ELISA sandwich capture assay for recombinant fusion proteins containing glutathione-S-transferase

A sandwich capture ELISA technique is presented for detection of recombinant proteins sharing a common affinity domain or reagents such as antibodies that bind to these proteins. An activated carrier protein (BSA) is modified with reduced glutathione (GT), forming an affinity capture reagent for glutathione-S-transferase (GST) and recombinant fusion proteins bearing the GST moiety. GT-BSA is immobilized on microtiter plates, and a sandwich is formed consisting of the recombinant fusion protein, reactive antibodies, and detection antibodies. An example is given that demonstrates that this format yields equivalent results to a conventional ELISA test with a panel of newly diagnosed diabetic sera reacting with an islet cell autoantigen.

Characterization of the LIM/Homeodomain Gene Islet-1 and Single Nucleotide Screening in NIDDM

Islet-1 (Isl-1) is a unique transcription factor that binds to the enhancer region of the insulin gene. To evaluate this gene in non-insulin-dependent diabetes mellitus (NIDDM), a full-length human Isl-1 cDNA was isolated and the genomic structure was characterized. The cDNA [2,395 bp plus additional poly(A) residues] contained an open reading frame from an initiator methionine at nucleotide 240 to an opal stop codon at nucleotide 1,286 (GenBank accession number UO7559), encoding a predicted protein of 349 amino acids (39 kDa). From their ends, 23 additional clones were sequenced, revealing 15 incomplete cDNAs and 8 intron-containing partially processed precursors. As determined by Northern blotting and reverse transcriptase–polymerase chain reaction analysis, Isl-1 was most abundantly expressed as a 2.4-kb mRNA in human islets, with a restricted pattern of expression in other adult human tissues. Analysis of genomic clones revealed that Isl-1 is encoded by six exons, varying in size from 168 bp (exon 5) to 1,230 bp (exon 6). Exons 2 and 3 each encode a LIM domain, while the homeodomain is completely contained within exon 4. The sequence of the proximal promoter region, including 426 bp upstream of the 5′ -end of the cDNA, revealed two potential regulatory elements, GCCAGCCGG (–414 to –406) and GCCACAGG (–357 to – 350), each differing by one base form a homologous sequence in the insulin gene (GCCACCGG) that has been shown to be a major positive regulatory element (Boam DSW, Clark AR, Docherty L: Positive and negative regulation of the human insulin gene by multiple trans-acting factors. J Biol Chem 265:8285–8296, 1990). A search by single-strand conformational polymorphism analysis for variants in the Isl-1 gene was undertaken in NIDDM patients. Three variants were identified in the cDNA, none of which alter the predicted amino acid sequence. No variants were found in the promoter. The results of these studies thus indicate that the Isl-1 gene is not a major contributor to NIDDM susceptibility in this Northern European Caucasian population.

A simple DNA diagnostic method for human genetic disorders

SummaryA fast, reliable, and simple technique for detecting point mutations in unfractionated human DNA has been developed. Oligonucleotide probes complementary to either sickle- or normal β-globin DNA are labeled by primer extension and hybridized to DNA applied to nitrocellulose paper in a dot-blot format. A short hybridization time (about 1 h) and low probe concentration (about 1 nM) yield low background and high specificity. Double-blind trials show 100% agreement with restriction fragment length polymorphism (RFLP) analysis of DNA from normal, sickle, and heterozygous subjects.