SummarySince a dysregulated synthesis of tumor necrosis factor a (TNF-a) may be involved in the pathogenesis of autoimmune diseases, it was ofinterest to precisely locate the recently reported Ncol restriction fragment length polymorphism (RFLP) of the TNF-a region . However, by mapping of 56.8 kb of overlapping cosmid clones and direct sequencing, we could localize the polymorphic Ncol restriction site within the first intron of the TNF-0 gene and not in the TNF-a gene. To study whether regulatory mechanisms are affected by this polymorphism, we analyzed the TNF-a/TNF-0 production of phytohemagglutinin-stimuated peripheral blood mononuclear cells ofindividuals homozygous for the TNF-0 Ncol RFLP by ELISA and concomitant Northern blot analysis. On days 2-4 after stimulation with mitogen, the TNFB*1 allele corresponding to a 5.3-kb Ncol fragment presented with a significantly higher TNF-0 response. A mRNA analysis demonstrated that higher protein levels of TNF-0 correlate also with increased amounts ofTNF-0 transcripts . No allelic association was found in respect to TNF-a production . To further investigate a possible allelic influence on transcription, we determined the DNA sequence of 2 kb ofthe 5' portion of our cloned TNFB*2 allele and compared it with the available TNF-0 sequences. By computer-aided recognition motif search ofDNA binding factors, we report putative binding sites conserved between mouse and man in the 5' flanking region as well as in intron 1 ofthe TNF-0 gene, found also in other cytokine promoter sequences. In addition, by polymerase chain reaction amplification and sequencing of 740 by of the 5' part of TNF-0 of individuals typed homozygously for the Ncol RFLP, we could show that amino acid position 26 is conserved as asparagine in the TNFB*1 and as threonine in the TNFB* 2 sequence. A previously reported, EcoRI RFLP in the 3' untranslated region of TNF-0 does not segregate with either of the two alleles. Thus, four TNFB alleles can de defined at the DNA level .

Limiting factors in the detection of micrometastatic tumor cells by RT-PCR are (1) the illegitimate transcription of tumor-associated or epithelial-specific genes in hematopoietic cells, and (2) the deficient expression of the marker gene in micrometastatic tumor cells.

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Autoimmune disorders in humans are often associated with particular alleles of major histocompatibility genes. However, the chronic inflammatory liver disease primary biliary cirrhosis (PBC) has not been found to be correlated with certain haplotypes so far. Interestingly, an impaired production of tumour necrosis factor β (TNF‐β) upon mitogen stimulation was observed for PBC patients, especially in the immunologically active stages of the disease. Furthermore, the identification of alleles of the TNF‐β gene which differ in one unique amino acid, and in the production of TNF‐β after phytohaemagglutinin stimulation, has prompted the idea of a possible linkage between the impaired TNF‐β response in PBC and the genetic prevalence of a certain TNF haplotype. We report here a rapid method for typing the TNFB*1 and TNFB*2 genes by a standard polymerase chain reaction, PBC patients(n = 60)as well as randumized healthy controls (n = 179) of the Munich area were studied for the occurrence of the TNF alleles. No deviation was found in the PBC collective (0.7) for the TNFB*2 distribution when compared with the control (0.67).