New retinoid X receptor subtypes in zebra fish (Danio rerio) differentially modulate transcription and do not bind 9-cis retinoic acid.

Retinoid X receptors (RXRs), along with retinoic acid (RA) receptors (RARs), mediate the effects of RA on gene expression. Three subtypes of RXRs (alpha, beta, and gamma) which bind to and are activated by the 9-cis stereoisomer of RA have been characterized. They activate gene transcription by binding to specific sites on DNA as homodimers or as heterodimers with RARs and other related nuclear receptors, including the vitamin D receptor, thyroid hormone receptors (TRs), and peroxisome proliferator-activated receptors. Two additional RXR subtypes (delta and epsilon) isolated from zebra fish cDNA libraries are described here; although both subtypes form DNA-binding heterodimers with RARs and TR, neither binds 9-cis RA, and both are transcriptionally inactive on RXR response elements. In cotransfection studies with TR, the delta subtype was found to function in a dominant negative manner, while the epsilon subtype had a slight stimulatory effect on thyroid hormone (T3)-dependent transcriptional activity. The discovery of these two novel receptors in zebra fish expands the functional repertoire of RXRs to include ligand-independent and dominant negative modulation of type II receptor function.

Vitamin D receptor phosphorylation in transfected ROS 17/2.8 cells is localized to the N-terminal region of the hormone-binding domain.

The human 1,25-dihydroxyvitamin D3 receptor (hVDR) has been recently shown to be phosphorylated in vitro by casein kinase-II. Most of the residues phosphorylated by this enzyme were shown to reside between Asn160 and Asp232, a region near the N-terminal boundary of the hormone-binding domain. We report here that the hVDR is also phosphorylated in vivo after transfection into ROS 17/2.8 cells. In addition to testing full-length hVDR, we analyzed several internally deleted hVDR mutants. The expression and phosphorylation of full-length and mutated hVDRs were monitored in transfected cells by metabolic labeling with either [35S]methionine or [32P]orthophosphate, followed by immunopurification using monoclonal anti-VDR antibody linked to agarose beads. Transfected hVDR is distinguishable from the endogenous rat VDR when the immunoprecipitated proteins are resolved on sodium dodecyl sulfate-polyacrylamide gels. Significant phosphorylation of transfected full-length hVDR was observed in ROS 17/2.8 cells, and it was less dependent on the presence of 1,25-dihydroxyvitamin D3 than that of the endogenous rat receptor. Most importantly, the region of in vivo phosphorylation, as defined by internal deletion mutants, resides between Met197 and Val234. Therefore, we have localized the major site of phosphorylation of hVDR to residues in the N-terminal region of the hormone-binding domain. The boundaries of this region fall within the amino acid segment defined for phosphorylation of hVDR by casein kinase-II in vitro, suggesting that VDR is an in vivo substrate for casein kinase-II or a related protein kinase.

Signal transducers and activators of transcription (Stat) are detectable in mouse trigeminal ganglion neurons.

We studied signal transducers and activators of transcription (Stat) expression in mouse trigeminal ganglia (TG) to gain an understanding of herpes simplex virus (HSV) infection and reactivation. Mouse TG were harvested and were either frozen for Western blot analysis or preserved in 4% paraformaldehyde for subsequent immunohistochemistry study. The thawed specimens were homogenized, and nuclear/cytoplasmic extractions were performed for Western blots and immunoprecipitation. Immunohistochemistry showed that Stat1, Stat3, Stat4, Stat5b, and phosphotyrosine Stat3 localized to TG neurons, not surrounding satellite cells. Western blot of TG nuclear and cytoplasmic extracts confirmed the presence of these Stat at the appropriate molecular weights. Stat2 was undetectable in TG by these methods. Immunoprecipitation of TG nuclear extracts did not confirm the presence of Stat-Stat dimers in these specimens. These studies show that several Stat, including phosphotyrosine Stat3, are present in TG neurons, the site of HSV latency, where they could act upon latent viral DNA to effect reactivation.

Isolation of specialized transducing bacteriophages carrying deletions of the regulatory region of the Escherichia coli K-12 tryptophan operon.

A lysogen of a wild-type strain of Escherichia coli K-12 carrying a heat-inducible lambda-phi80 hybrid prophage was induced to yield transducing phages carrying all of the structural genes of the tryptophan operon. The presence or absence of elements of the trp regulatory region was determined by examining the effects of lambda genes N and cI on trp gene expression. The phages were further characterized by transduction studies and by examining anthranilate synthetase (EC 4.1.3.27) (TRYPE+D) synthesis in the presence of the lambda cI product. A number of phages deleted for the trp promoter were found. Recombination studies between trpOc bacteria and the transducing phages have yielded information that can be used to order the trp end points of some phages and to provide an estimate of the size of the trp promoter region.

Tryptophan-transducing bacteriophages: in vitro studies with restriction endonucleases HindII + III and Escherichia coli ribonucleic acid polymerase.

The HindII + III restriction enzyme fragmentation pattern of various lambda-phi80trp deoxyribonucleic acid molecules is presented. An analysis of deoxyribonucleic acid molecules carrying deletions ending within the trp regulatory elements and a deoxyribonucleic acid molecule carrying a deletion within trpE indicates that a fragment of 8.3 X 10(5) daltons contains at least part of the trp promoter, the entire trp leader region, and part of the trpE gene. The observation that ribonucleic acid polymerase, when present in the HindII + III digestion mixture, results in the fusion of this 8.3 X 10(5)-dalton fragment to the preceding bacterial fragment suggests that HindII + III cuts within trpP.

Identification of the N gene protein of bacteriophage lambda.

The N gene protein, pN, of bacteriophage lambda stimulates early gene transcription by allowing mRNA chain elongation to proceed into genes distal to transcription termination sites normally recognized by the Escherichia coli transcription termination protein rho. pN has previously eluded detection on sodium dodecyl sulfate/polyacrylamide gels because of its small size, its instability, and the difficulty of distinguishing pN itself both from host proteins and from other early lambda proteins whose synthesis depends on pN action. These problems have now been overcome and we find that the major form of pN present in crude cell extracts of infected cells has an apparent molecular weight of 13,500. lambdabio256, a deletion-substitution mutant terminating in N, codes for a shorter pN of molecular weight 12,500. A nonsense fragment of 10,500 molecular weight coded by lambdaN(am7) has also been identified. These conclusions are based on examination of the electrophoretic profiles of the proteins synthesized after infection of UV-irradiated E. coli by various lambdaN(-) temperature-sensitive, nonsense, and deletion-substitution mutants. It has also been possible to distinguish pN itself from other early lambda polypeptides by infecting ron(-) cells with either lambdaN(mar) phage allowing pN synthesis but not pN action or lambdaN(am) phage defective in pN synthesis and pN action. Our results together with previous data are discussed with respect to the possible existence of multiple molecular weight forms of pN and the location of the coding sequences in the N gene region.

Computer-assisted fluorescence identification of colon cancer in rats.

Hematoporphyrin derivative (HPD) fluorescence is highly sensitive in identifying colon cancers in the rat. Its specificity, however, is compromised by HPD accumulation in lymphoid follicles. We developed a computer-assisted method to distinguish lymphoid from malignant tissue using HPD fluorescence. Colon cancer was induced in rats by weekly injections of 1,2-dimethylhydrazine. Twenty-four hours after intravenous HPD injection, the animals were sacrificed and the colonic surface was illuminated with a blue light (340-410 nm). Computer images were examined for characteristic patterns of fluorescence at 632 nm. The ratio of fluorescence intensity between lesions and adjacent background areas was also determined. The fluorescent areas were then coded and examined histologically. Eighty-one lesions (21 cancers, 60 lymphoid follicles) were identified. Only two lesions (one cancer, one lymphoid follicle) were incorrectly identified using computer images (concordance rate = 98%). The lesion:background fluorescence ratio was higher in malignant tissue than lymphoid follicles, but significant overlap was seen. These results indicate that computer-enhanced images of HPD fluorescence can distinguish malignant from lymphoid tissue in the rat colon and may have a potential role in the diagnosis of colon cancer.

RNA polymerase binding sites in lambdaplac5 DNA.

The in vitro binding of the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) to fragments of lambdaplac5 DNA generated by restriction endonucleases HindII and HindIII has been studied by a filter binding technique. The results are consistent with RNA polymerase binding at p(R)', the INT promoter (p(I)), several sites in the b2 region, the mis promoter, the oop promoter (or p(O)), and p(rm). Binding was also observed on some fragments that are not known to contain active promoters, including the fragment from the cIII-t(L) region. Some of these binding reactions might also be explained by interaction of RNA polymerase with termination sites. Additional polymerase binding sites have been detected by examining which HindII and HindIII sites were not cleaved when digestion was performed after RNA polymerase had been bound to the DNA. This technique revealed polymerase binding at p(L), at p(R), at a site between R and cos, and at a site at the junction of the gamma and cIII-t(L) fragments. A comparison of the location of polymerase binding fragments with the partial denaturation map of the lambda genome indicates that RNA polymerase binding sites are located within A-T rich regions. It is suggested that RNA polymerase binding is a function both of specific sequences (where recognition occurs) and of the base composition of the surrounding regions (which affects the stability of the helix at the specific site).