The human homologue of the RNA polymerase II-associated factor 1 (hPaf1), localized on the 19q13 amplicon, is associated with tumorigenesis.

The 19q13 amplicon in pancreatic cancer cells contains a novel pancreatic differentiation 2 (PD2) gene (accession number AJ401156), which was identified by differential screening analysis. PD2 is the human homologue of the RNA polymerase II-associated factor 1 (hPaf1). In yeast, Paf1 is part of the transcription machinery, acting as a docking protein in between the complexes Rad6-Bre1, COMPASS-Dot1p, and the phosphorylated carboxyl terminal domain of the RNA polymerase II. As such, Paf1 is directly involved in transcription elongation via histone H2B ubiquitination and histone H3 methylation. The PD2 sequence is highly conserved from Drosophila to humans with up to 98% identity between rodent and human, suggesting the functional importance of PD2/hPaf1 to maintain cellular homeostasis. PD2 is a modular protein composed of RNA recognition motif, DEAD-boxes, an aspartic/serine (DS)-domain, a regulator of the chromosome condensation domain and myc-type helix-loop-helix domains. Our results further showed that PD2 is a nuclear 80 kDa protein, which interacts with RNA polymerase II. In addition, we have demonstrated that the overexpression of PD2 in the NIH 3T3 cells result in enhanced growth rates in vitro and tumor formation in vivo. Altogether, this paper presents strong evidence that the overexpression of PD2/hPaf1 is involved in cancer development.

Systemic RNAi in Caenorhabditis elegans.

RNA interference (RNAi) in Caenorhabditis elegans induced by ingestion or injection of double-stranded RNA (dsRNA) spreads throughout the organism and is even transmitted to the progeny. We have identified two proteins required for spreading of RNAi, SID-1 and SID-2, whose structure, subcellular localization, and expression pattern have been informative for how dsRNA can be transported into and between cells. SID-1 is a transmembrane protein that functions as a pore or channel that transports dsRNA into and out of cells. Proteins homologous to SID-1 are present in a wide range of invertebrate and vertebrate animals but are absent from plants. SID-2 is a small transmembrane protein that is expressed in the gut and localizes strongly to the luminal membrane where it appears to act as a receptor for uptake of dsRNA from the environment. Characterization of SID-2 activity in a variety of Caenorhabditis nematodes indicates that C. elegans SID-2 may have a novel activity.

Phylogenetic relationships of north American field crickets inferred from mitochondrial DNA data.

A well-supported molecular phylogeny for North American Gryllus species based on a combined data set of mitochondrial (mt) DNA is presented. A total of 26 individuals representing 13 populations of 11 species of the genus Gryllus and 4 individuals of two outgroup species, Teleogryllus oceanicus and Acheta domestica, were sampled in this study. The complete cytochrome b gene (1036 bp) and a 500-bp fragment of the 16S rRNA gene were sequenced for each individual. Since results from separate analyses of the cytochrome b and 16S data sets, as well as a previously published mtDNA restriction-site data set, were not conflicting, all data were combined for phylogenetic analyses. The clade of European Gryllus was clearly separated from the North American clade. The amount of sequence divergence between these clades was significantly greater than within the clades, suggesting a basal drift-vicariant event in the genus. This is the first phylogenetic analysis of North American Gryllus that includes western species. Four well-supported groups were identified but their relationships showed no clear east-west structure. Our phylogeny supports the recent reassignment of G. integer Scudder 1901 from Texas to G. texensis Cade and Otte 2000. The evolution of cricket song and life cycle is discussed using the new phylogenetic framework.

Expression of three UDP-N-acetyl-alpha-D-galactosamine:polypeptide GalNAc N-acetylgalactosaminyltransferases in adenocarcinoma cell lines.

The levels of mRNA expression of three UDP-N-acetyl-alpha-D-galactosamine:polypeptide GalNAc N-acetylgalactosaminyltransferases (GalNAc-transferases) were quantified for human adenocarcinoma cell lines from pancreas, colon, stomach, and breast. Two of the GalNAc-transferases, GalNAc-T1 and GalNAc-T2, were expressed constitutively and at low levels in most or all cell lines examined. A third GalNAc-transferase, GalNAc-T3, was differentially expressed. Well-differentiated adenocarcinoma cell lines expressed high levels and moderately differentiated cell lines expressed lower levels of GalNAc-T3. Cell lines classified as poorly differentiated failed to express GalNAc-T3 mRNA at levels that could be detected by Northern blot analysis. Differential expression of the GalNAc-T3 protein was confirmed in these cell lines by Western blotting. We propose that glycosylation in tumor cell lines may be regulated in part by differential expression of GalNAc-transferases, and we suggest that GalNAc-T3 gene expression may be a molecular indicator of differentiated adenocarcinoma.

Duct epithelial cells cultured from human pancreas processed for transplantation retain differentiated ductal characteristics.

A procedure is described for the isolation and growth in vitro of epithelial cells from the duct network of human pancreas, referred to as DEC. A significant advantage of our procedure over previously published procedures is that it enables the isolation of DEC from small pieces of pancreas tissue (< 5 g) and, also, from the digest remaining after the isolation of islet cells from human pancreas, material that would normally be discarded. These were the only reliable sources for pancreas tissue available to us. This procedure shows that some of the techniques that have been successfully used for the isolation of rodent DEC are also valuable in the isolation of human DEC. In particular, the use of cholera toxin to prevent fibroblast growth and contamination obviates the need for the time-consuming procedure of physically removing fibroblasts or the use of expensive fibroblast-specific monoclonal antibodies. The use of sieving to separate the digest immediately achieves a partial purification, which, coupled with that of allowing duct cysts to form, adds to the purity of the final preparation. The ductal system of the intact pancreas tissue and the DEC derived from it expressed cytokeratins 7, 8/18, and 19 and markers for the presence of MUC1, CFTR, and carbonic anhydrase II, which are specific for ductal epithelial cells or for pancreatic ductal functions. This study showed that it is possible to obtain selectively viable DEC from small ducts in otherwise waste pieces of human pancreas. It showed that these cells retained all of the epithelial characteristics that were examined and, in combination with data from an earlier study, showed that the cultured DEC retain the metabolic functions of duct epithelial cells in vivo.

Selective lineage specification by mab-19 during Caenorhabditis elegans male peripheral sense organ development.

The action of the gene mab-19 is required for specification of a subset of Caenorhabditis elegans male peripheral sense organ (ray) lineages. Two mab-19 alleles, isolated in screens for ray developmental mutations, resulted in males that lacked the three most posterior rays. Cell lineage alterations of male-specific divisions of the most posterior lateral hypodermal (seam) blast cell, T, resulted in the ray loss phenotype in mab-19 mutant animals. Postembryonic seam lineage defects were limited to male-specific T descendent cell divisions. Embryonic lethality resulted when either mab-19 mutation was placed over a chromosomal deficiency encompassing the mab-19 locus. The earliest detectable defect was aberrant hypodermal cell movements during morphogenesis. From these data, it is inferred that both mab-19 alleles described are hypomorphs, and further reduction of mab-19 function results in embryos that are unable to complete morphogenesis. Thus, mab-19 may play a larger role in developmental regulation of hypodermal cell fate, including sensory ray development in males. Body morphology mutations, passage through the dauer stage, and heat or CdCl2 treatment suppressed mab-19 male phenotypes. A model is presented in which all three types of suppression result in a physiological stress response, which in turn leads to correction of the mab-19 defect.

Two distinct yolk lipoprotein complexes from Caenorhabditis elegans.

The four yolk polypeptides of the nematode Caenorhabditis elegans are found in two types of lipoprotein particle: 12 S particles with Mr estimated at 450,000 and 8 S particles with Mr estimated at 250,000. Both types of particle contain approximately 8% phospholipids, 3% triglycerides, and 3% other lipids by mass. All four C. elegans yolk polypeptides can be found in either 12 or 8 S particles, depending upon the conditions of isolation. While the properties of the 12 and 8 S lipoprotein particles are consistent with a dimermonomer relationship, the asymmetric distribution of the yolk polypeptides between 12 and 8 S fractions suggests that at least two different oligomeric lipoprotein complexes are present in C. elegans embryos. In order to clarify the subunit composition of the C. elegans yolk lipoproteins, the patterns of polypeptides retained in immunoaffinity binding procedures by immunoglobulins of different antigenic specificities have been compared. When immunoaffinity binding is performed in the absence of sodium dodecyl sulfate, three C. elegans yolk proteins (yp170A, yp115, and yp88) are retained together by polyclonal immunoglobulins directed against either yp115 or yp88. A monoclonal immunoglobulin also retains these three proteins together. In contrast, a second monoclonal immunoglobulin retains only the fourth yolk protein (yp170B). Aggregate species, evidently reflecting the spontaneous formation of interchain disulfide bonds, indicate that yp170A and yp88 are physically associated, whereas yp170B self-associates in dimers. It is concluded that there are two distinct lipoprotein complexes in C. elegans: the A complex, which consists of yp170A, yp115, and yp88 and is essentially heterodimeric and the B dimer, a simple dimer of yp170B.