Chaim O. Jacob

Molecular characterization of MHC class II antigens (β1 domain) in the BB diabetes-prone and-resistant rat

The BB or BB/Worcester (BB/W) rat is widely recognized as a model for human insulin-dependent diabetes mellitus (IDDM). Of at least three genes implicated in genetic susceptibility to IDDM in this strain, one is clearly linked to the major histocompatibility complex (MHC). In an attempt to define the diabetogenic gene(s) linked to the MHC of the BB rat, cDNA clones encoding the class II MHC gene products of the BB diabetes-prone and diabetes-resistant sublines have been isolated and sequenced. For comparison, theβ1 domain of class II genes of the Lewis rat (RTlL) were sequenced. Analysis of the sequence data reveals that the first domain of RT1.Dβ and RT1.Bβ chain of the BB rat are different from other rat or mouse class 11 sequences. However, these sequences were identical in both the BB diabetes-prone and BB diabetes-resistant sublines. The significance of these findings is discussed in relation to MHC class II sequence data in IDDM patients and in the nonobese diabetic (NOD) mouse strain.

DNA polymorphism in cytokine genes based on length variation in simple-sequence tandem repeats

The possibility of the involvement of cytokines in the genetic predisposition to various diseases has been suggested by a large variety of studies. However, the study of potential disease linkage of cytokine genes has been hampered by a lack of sufficiently polymorphic markers at the restriction fragment length polymorphism (RFLP) level. We have investigated the distribution, the length polymorphism, the informativeness, and the efficiency of analysis, of simple-sequence tandem repeats in the mouse cytokine genes. Highly polymorphic sequences have been identified in the IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-7, and IFN-γ genes. The utility and the value of these sequences as gene markers is exemplified by mapping the IL-7 gene to mouse chromosome 3 close to pgk-1ps3 and Car-2 loci and the IFN-γ gene to chrrmosome 10 near the pg locus. Advantages of short tandemly repeated sequences as genetic markers are discussed in comparison with RFLPs.

Definition of microsatellite size variants forTnfa andHsp70 in autoimmune and nonautoimmune mouse strains

We have cloned and sequenced the upstream regulatory region of tumor necrosis factor α (Tnfa) gene in 12 different mouse strains and identified an allelic polymorphism in the upstream regulatory region of the mouseTnfa gene. TheTNF allele found in the NZW strain is distinct from those of all otherH-2 haplotypes, supporting our previous suggestion that this allele may be associated with a regulatory or structural defect. In addition, simple tandem repeat sequences (microsatellites) within the promoter region of theTnfa gene and the 3′ untranslated region of one of the members of the HSP70 family (Hsp68c clone) were utilized as genetic markers. Ten TNF size variants and twelve HSP70 variants were identified in over forty mouse strains. Using these markers in a set of congenic mice, we mapped this member of the HSP70 family to the central portion of theH-2 complex, centromeric to theTnfa gene. The NOD and NZW strains carry uniqueHSP70 alleles based on the variability in the length of this marker. These findings raise the possibility that this protein may play a role in the association of the major histocompatibility complex with these autoimmune diseases.

Novel DNA polymorphism in the mouse tumor necrosis factor receptors type 1 and type 2.

The introduction of the polymerase chain reaction (PCR) provides an entirely new means of analyzing DNA polymorphism and makes practical the analysis of length variation in simple-sequence tandem repeats of dinucleotides. In the process of cloning and sequencing the mouse genomic DNA for tumor necrosis factor (TNF) receptors type 1 and type 2, we identified two simple dinucleotide repeats within the noncoding regions of TNF receptor type 1 and three such sequences within TNF receptor type 2. PCR analysis of these sequences, using genomic DNA from 21 different inbred and wild mouse strains, as demonstrated by running the amplified products on sequencing gels, showed that the repeats are highly polymorphic. We identified seven alleles of TNF receptor type 2 and five alleles of TNF receptor type 1. Using these polymorphic markers in two sets of recombinant inbred strains of mice, the chromosomal localization of Tnfr-1 was mapped to mouse chromosome 6 and Tnfr-2 was located to the distal portion of mouse chromosome 4.

Mapping of the interleukin 5 receptor gene to human chromosome 3 p25-p26 and to mouse chromosome 6 close to the Raf-1 locus with polymorphic tandem repeat sequences.

Simple-sequence tandem repeat sequences in the 3′ UTR of interleukin 5 (IL5)-receptor gene of human and mouse are polymorphic in their length among humans and different strains of mice. In 20 different human Epstein-Barr virus (EBV)-transformed cell lines, six alleles of IL5R could be distinguished. In the mouse, three different alleles are found. With the human-specific IL5R tandem repeat marker in human-rodent somatic cell hybrids, the IL5R gene was mapped to human Chromosome (Chr) 3 p25–p26. With the mouse-specific IL5R tandem repeat sequence in recombinant inbred strains of mice, the Il5r gene was mapped to the distal part of mouse Chr 6 close to the Raf-1 locus.

Prospects and problems in synthetic vaccine development: what did we learn from the cholera toxin system?

The cholera toxin is a 84-kilodalton protein composed of two noncovalently bound subunits, A and B. The A subunit is the proenzyme form of an ADP-ribosyltransferase that is responsible for the biological activity of the toxin through its ability to activate target cell adenylate cyclase. The B subunit is the immunologically dominant region consisting of five identical, noncovalently associated B chains composed of 103 amino acids that are responsible for binding the toxin to target cell membrane receptors containing the oligosaccharide of the GM1 ganglioside. Neutralizing antibodies raised against the holotoxin react mainly with the B subunit, and antibodies against the B subunit are capable of neutralizing the biological activity of the intact toxin.

The Effect of Tumor Necrosis Factor (TNF) on (NZB × NZW)F1 Lupus Nephritis

The (NZB × NZW)F1 hybrid mouse develops severe autoimmune disease characterized by fatal glomerulonephritis, antibody to nuclear antigens, and female predominance, remarkably similar to systemic lupus erythematosus in humans. (NZB × NZW)F1 × NZB backcross studies by Kotzin and Palmer have shown that the major genetic contribution from NZW mice maps within the H-2 region but may be separate from the class II loci (1). Since the I-Az β chain from the NZW parental strain has an identical sequence to the normal I-Au haplotype (Acha-Orbea and McDevitt) and it was postulated that levels of Ia expression in affected tissues may be involved in autoimmune pathogenesis (2), the regulatory role of cytokines was tested in vivo. We present evidence suggesting the immunoregulation by interferon gamma (IFN-γ) and tumor necrosis factor (TNF) may be involved in the pathogenesis of lupus nephritis in the (NZB × NZW) model system.