Detection of mitochondrial DNA mutations in pancreatic cancer offers a "mass"-ive advantage over detection of nuclear DNA mutations.

We sequenced the complete 16.5-kb mitochondrial genome (mtDNA) in 15 pancreatic cancer cell lines and xenografts. Homoplasmic mtDNA somatic mutations and novel variants were identified in nearly all samples. Southern blot analysis and direct sequencing of mutation sites showed that the intracellular mass of mtDNA was greatly (6-8-fold) increased in pancreatic cancer cells in relation to corresponding normal cells; this property accounted for and greatly facilitated the identification of these mutations among the dense desmoplastic host reaction characteristic of primary pancreatic cancers. Structural characteristics and mathematical modeling of the evolution of mtDNA mutations suggested that many of the mutations identified might represent a random evolution of homoplasmic variants, rather than necessarily being a product of selective pressures. Complete sequencing of the nuclear MnSOD gene, which protects cells from the mitogenic and toxic effects of oxygen radicals, did not reveal any mutations. Nevertheless, the nearly ubiquitous prevalence and high copy number of mtDNA mutations suggest that they be considered of promising clinical utility in diagnostic applications.

Transcriptional regulation of the human polymeric immunoglobulin receptor gene by interferon-gamma.

IgA is transported into external secretions by the polymeric Ig receptor (pIgR). Interferon-gamma (IFN-gamma), a major regulator of pIgR expression, has been shown to increase pIgR mRNA levels in HT-29 human colon carcinoma cells. To determine the molecular mechanisms of pIgR regulation, genomic DNA containing the 5'-flanking region of the human pIgR gene was isolated and a single start site of transcription in human intestinal epithelial cells was identified. Using chimeric reporter plasmids containing flanking regions of the pIgR gene, a segment of the pIgR promoter which is necessary and sufficient for induction of transcription by IFN-gamma in HT-29 cells was identified. Significantly, the pIgR promoter contains three motifs homologous to the interferon-stimulated response element (ISRE), two in the 5'-flanking region and one in exon 1 of the pIgR gene. The upstream ISREs bind nuclear protein(s) which are constitutively expressed by HT-29 cells, while the exon 1 ISRE binds interferon regulatory factor-1 (IRF-1), following stimulation with IFN-gamma. Furthermore, induction of the IRF-1 promoter by IFN-gamma correlates with induction of the pIgR promoter by IFN-gamma. It has previously been demonstrated that induction of pIgR mRNA by IFN-gamma, requires de novo protein synthesis. It is now shown that IRF-1 is not detected in nuclear extracts from HT-29 cells stimulated with IFN-gamma in the presence of cycloheximide, suggesting that de novo synthesis of IRF-1 is required for induction of pIgR transcription by IFN-gamma.

Alternate junctions and microheterogeneity of Tlr1, a developmentally regulated DNA rearrangement in Tetrahymena thermophila.

A large number of developmentally regulated DNA rearrangements occur during the development of the macronucleus in Tetrahymena thermophila. Tlr1 is a deletion element which has large inverted repeats near the rearrangement junctions and deletes more than 13 kbp of internal DNA. Previous analysis of caryonidal lines revealed alternate left junctions for the Tlr1 rearrangement in B strain cells. We show here that C2 strain Tetrahymena also use alternate rearrangement junctions. We have mapped and sequenced two additional rearrangement variants and find that both the left and right junctions can vary over a range of approximately 200 bp. We also demonstrate the presence of sequence microheterogeneity in the most commonly found Tlr1 rearrangement product.