Zebrafish stat3 is expressed in restricted tissues during embryogenesis and stat1 rescues cytokine signaling in a STAT1-deficient human cell line.

Transcription factors of the STAT family are required for cellular responses to multiple signaling molecules. After ligand binding-induced activation of cognate receptors, STAT proteins are phosphorylated, hetero- or homodimerize, and translocate to the nucleus. Subsequent STAT binding to specific DNA elements in the promoters of signal-responsive genes alters the transcriptional activity of these loci. STAT function has been implicated in the transduction of signals for growth, reproduction, viral defense, and immune regulation. We have isolated and characterized two STAT homologs from the zebrafish Danio rerio. The stat3 gene is expressed in a tissue-restricted manner during embryogenesis, and larval development with highest levels of transcript are detected in the anterior hypoblast, eyes, cranial sensory ganglia, gut, pharyngeal arches, cranial motor nuclei, and lateral line system. In contrast, the stat1 gene is not expressed during early development. The stat3 gene maps to a chromosomal position syntenic with the mouse and human STAT3 homologs, whereas the stat1 gene does not. Despite a higher rate of evolutionary change in stat1 relative to stat3, the stat1 protein rescues interferon-signaling functions in a STAT1-deficient human cell line, indicating that cytokine-signaling mechanisms are likely to be conserved between fish and tetrapods. Dev Dyn 1999;215:352-370.

Sampling the genomic pool of protein tyrosine kinase genes using the polymerase chain reaction with genomic DNA.

The polymerase chain reaction (PCR), with cDNA as template, has been widely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest, this method offers an opportunity to sample the genome for new members irrespective of their expression pattern.

Mitogen activation of human peripheral T lymphocytes induces the formation of new cyclic AMP response element-binding protein nuclear complexes.

A large body of evidence indicates that experimental agents which raise cellular cAMP levels inhibit T cell growth and division. By contrast, many studies have reported that mitogen activation of T cells increases cAMP levels, implying a positive physiological role for cAMP in the activation process. In the present study we demonstrate that mitogen activation of human peripheral T lymphocytes induces nuclear factors that form complexes with cyclic AMP response element-binding protein (CREB). Four complexes are identified by the electrophoretic mobility shift assay, two of which are induced by mitogen activation. All four complexes contain CREB and are bound to the cAMP response element (CRE) core sequence (TGACGTCA), as indicated by antibody and oligonucleotide competition experiments. Binding of the four complexes to CRE is prevented by dephosphorylation of nuclear extracts and is restored by rephosphorylation with cAMP-dependent protein kinase or endogenous kinases. Similar complexes are detected in nuclear extracts of Jurkat cells. Mitogen induction of the electrophoretic mobility shift assay complexes is not accounted for by protein phosphorylation or by induction of CREB. Rather, the data indicate that mitogen increases the levels of a nuclear factor(s) that dimerizes with CREB. Induction of new CREB complexes implies a physiological role for cAMP in mitogen activation of T lymphocytes.

The effects of cAMP on the expression of glycolytic isozymes in activated peripheral human T lymphocytes.

Forskolin, an activator of adenylate cyclase, inhibits mitogen induction of glycolytic isozymes in human peripheral T cells. Inhibition of lactate dehydrogenase isozyme activity is correlated with lowered mRNA levels, suggesting a transcriptional block in the presence of increased cAMP. Forskolin added with the mitogen, or 12-24 h after the mitogen, strongly inhibits isozyme expression and DNA synthesis, and causes cells to accumulate in the G0 or early G1 phase of the cell cycle. The data suggest that DNA synthesis and isozyme expression are both inhibited by a cAMP-sensitive step(s) in the early activation or progression phase.

Regulation of the expression of lactate dehydrogenase isozymes in human lymphocytes.

Mitogen activation of human peripheral lymphocytes leads to a switch in the isozymes of LDH; resting cells contain low activities of only the B4 and B3A forms, whereas activated cells contain high activities of the A4 and A3B forms. B4 LDH is not altered in activated cells. In this study we show that the appearance of the A subunits occurs concomitantly with a several fold increase in the steady state levels of LDH-A mRNA. Responses in LDH-A mRNA are observed within 12 hrs of activation, and are, thus, associated with the G0/G1 transition or with early G1 (Marjanovic et al. Exp. Cell Res. (1991) 193: 425-431). Maximal expression of LDH-A mRNA requires both phorbol ester and concanavalin A, implying a complex regulatory pathway involving cascade systems activated through both the antigen receptor (TR) and protein kinase C.