Structure, expression, and functional analysis of the gene coding for calmodulin in the chytridiomycete Blastocladiella emersonii

The single calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete Blastocladiella emersonii were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0.7-kb mRNA species encoding a predicted protein 91% identical to human CaM. B. emersonii CaM has been expressed in Escherichia coli as a fusion protein with gluthatione S-transferase (GST) and purified by affinity chromatography and cleavage from the GST portion using a site-specific protease. In the presence of Ca(2+), B. emersonii CaM exhibited a shift in apparent molecular mass similar to that observed with bovine CaM and was able to activate the autophosphorylation of CaM-dependent protein kinase II (CaMKII) from rat brain. CaM expression is developmentally regulated in B. emersonii, with CaM mRNA and protein concentrations increasing during sporulation to maximum levels observed just prior to the release of the zoospores into the medium. Both CaM protein and mRNA levels decrease drastically at the zoospore stage, increasing again during germination. The CaM antagonists compound 48/80, calmidazolium, and W7 were shown to completely inhibit B. emersonii sporulation when added to the cultures at least 120, 150, and 180 min after induction, respectively. All these drugs also inhibited growth and zoospore production in this fungus. The Ca(2+) channel blocker TMB-8 and the CaMKII inhibitor KN93 completely inhibited sporulation if added up to 60 min after induction of this stage, but only KN93 affected fungal growth. The data presented suggest that the Ca(2+)-CaM complex and CaMKII play an important role during growth and sporulation in B. emersonii.     

Segregation of Na(+)-channel gene expression during neuronal-glial branching of a rat PNS-derived stem cell line, RT4-AC.

RT4 is a family of cell lines isolated from an ethylnitrosourea-induced rat peripheral neurotumor. RT4-AC cells express both excitable membrane and glial cell properties. In a process called cell-type conversion, RT4-AC cells segregate these properties to generate three distinct derivative cell types which have been classified as either neuronal (RT4-E and RT4-B) or glial (RT4-D). In this report we demonstrate that: (1) upon cell-type conversion, Na(+)-channel mRNA expression segregates primarily with the RT4 neuronal derivatives, (2) the SkM2 Na(+)-channel gene, which was originally isolated from rat muscle cDNA libraries, is the predominant gene expressed by the RT4 neuronal derivatives, (3) the three rat brain Na(+)-channel genes I, II, and III and the muscle-derived SkM1 gene are not the principal Na(+)-channel genes involved in the segregation, although very low levels of message of these genes are detected, and (4) the RT4 glial derivative expresses slightly higher levels of message from rat brain genes I and II than the neuronal derivatives. Since the RT4 cell lines were derived from a peripheral neurotumor these results present the possibility that the SkM2 gene may be important in vivo in the rat peripheral nervous system.     

Germline V genes encode viable motheaten mouse autoantibodies against thymocytes and red blood cells

Autoantibodies against thymocytes and RBC may contribute to the pathophysiology of homozygous viable motheaten (mev) autoimmune disease. Whether the production of these autoantibodies in mev mouse results from polyclonal nonspecific B cell activation or specific Ag-driven stimulation is not known. To understand the mechanisms involved in the induction of antithymocyte autoantibody response in mev mouse, we have studied the fine antigenic specificity, structure, and origin of three antithymocyte autoantibodies derived from mev splenic B cell hybridomas. Western blot analysis showed that these mAb bind to polypeptides of 33 and 105 kDa present in RBC and thymocytes, respectively. Additional specificities for the epitopes present in other polypeptides distinguished these three autoantibodies. Northern hybridization and flow microfluorimetry analysis indicated that these hybridomas are derived from the Ly1+ B cell subset. These autoreactive Ly-1 B cell hybridomas, chosen on the basis of their specificity, expressed L chain V genes from a single VK family (VK9) and VH genes from J606 and S107 families. Hybridomas UN34.11 and UN42.5 expressed the VK9 gene identical to that used by peritoneal Ly1+ B cells from various mouse strains and malignant B lymphoma cells secreting anti-mouse RBC treated with proteolytic enzyme bromelin and anti-SRBC antibodies. The third hybridoma, S2-14.2, used a VK9 gene identical to that expressed by MOPC41. None of the VK genes encoding these autoantibodies showed any somatic mutations. In the case of VH genes, the two hybridomas UN42.5 and S2-14.2 derived from two separate fusions, used identical VH genes from the J606 family. The third hybridoma UN34.11 used unmutated V11 germline VH gene, a member of the S107 family. Southern hybridizations, using oligonucleotide probes specific for CDR1 and CDR2, showed that the VH genes encoding the J606 autoantibodies were derived from a germline gene found in the 6.7-kb fragment of EcoRI-digested germline DNA. This germline VH gene is distinct from VH22.1 germline gene that codes for antigalactan antibodies. Sequence analysis of this gene showed perfect homology with the rearranged VH genes confirming the lack of somatic mutations. Thus, our data demonstrate that antithymocyte antibody response occurring in mev mouse is polyclonal and it involves Ly-1 B cells expressing unmutated germline VH and VK genes. These results indicate that antigen driven stimulation may not play an important role in the induction of anti-thymocyte antibody response in mev mouse.     

Housekeeping Na,K-ATPase alpha 1 subunit gene promoter is composed of multiple cis elements to which common and cell type-specific factors bind.

Na,K-ATPase alpha 1 subunit gene (ATP1A1) is one of the housekeeping genes involved in homeostasis of Na+ and K+ in all animal cells. We identified and characterized the cis-acting elements that regulate the expression of ATP1A1. The region between -155 and -49 was determined as a positive regulatory region in five cultured cell lines of different tissue origins (MDCK, B103, L6, 3Y1, and HepG2). The region was divided into three subregions: from -120 to -106 (including the Sp1 binding site), from -102 to -61, and from -58 to -49 (including an Sp1 consensus sequence). Cell type-specific factors binding to the middle subregion (from -102 to -61) were detected by gel retardation analysis, using nuclear extracts prepared from MDCK and B103 cells. Two gel retardation complexes were formed in the B103 nuclear extract, and three were formed in the MDCK nuclear extract. DNA binding regions of these factors were located at -88 to -69 and differed from each other in DNase I footprinting experiments. These factors also showed different binding characteristics in gel retardation competition and methylation interference experiments. The identified cis element was named the ATP1A1 regulatory element. The core sequence of this element is found in several other genes involved in cellular energy metabolism, suggesting that the sequence is a common regulatory element responsive to the state of energy metabolism.     

1,25 dihydroxyvitamin D3 and dexamethasone induce the cyclooxygenase 1 gene in osteoclast-supporting stromal cells.

Commitment of members of the monocyte/macrophage family to the bone resorptive phenotype, in vitro, requires contact, of these osteoclast precursors, with osteoblasts or related stromal cells. The osteoclast-inductive properties of these stromal cells are typically expressed, however, only in the presence of steroid hormones such as 1,25 dihydroxyvitamin D (1,25D3) and dexamethasone (DEX). To gain insight into the means by which steroid treated accessory cells induce osteoclast differentiation we asked, using differential RNA display (DRD), if gene expression by this stromal cell population differs from that of their untreated, non-osteoclastogenic counterpart. We identified four known genes specifically expressed by 1,25D3/DEX-treated ST2 stromal cells: 1) a family of rat organic anion transporters, 2) Na/K ATPase ss-subunit, 3) tazarotene-induced gene 2 (TIG2), and 4) prostaglandin G/H synthase I, or cyclooxygenase 1 (Cox-1). The regulation of these genes in 1,25D3/DEX-treated ST2 cells was demonstrated by Northern blot analysis of treated (osteoclast-supporting) and untreated (non-osteoclast-supporting) ST2 cells; the genes have a limited and specific tissue mRNA expression pattern. Northern blot analysis of treated and untreated ST2 cell total RNA using either a DRD-derived Cox-1 cDNA or a Cox-1 specific oligonucleotide confirmed the steroid regulation of Cox-1 mRNA. Surprisingly, there is no detectable expression by untreated or steroid exposed ST2 cells, of Cox-2, the classical regulated cyclooxygenase isoform. In contrast to 1, 25D3/DEX, serum treatment rapidly induces Cox-2 mRNA, substantiating the capacity of ST2 cells to express the gene. These data establish that steroid induction of the osteoclastogenic properties of stromal cells is attended by Cox gene expression, a phenomenon consistent with the capacity of eicosinoids to impact the resorptive process. The response of osteoclast-supporting ST2 cells to 1,25D3/DEX treatment may be one prostaglandin-mediated event which specifically involves Cox-1 regulation.     

Differential DNA binding properties of three human homeodomain proteins.

The products of three human homeobox containing (HOX) genes, 2C, 3C and 4B, were produced in insect cells using the Baculovirus expression system and purified to near homogeneity. In this system we observed that the DNA binding forms of the three proteins are not glycosylated. HOX 3C and 4B are phosphorylated in insect cells, while HOX 2C is not. The three HOX proteins bind to a DNA sequence known to be a target site for Antennapedia protein with a very similar affinity (Kd = 1-2 x 10(-9) M). We then measured their binding properties to four human sequences present in the HOX 3D, 4C, 1C and 4B promoters. Two of these sequences have been reported to be binding sites for HOX proteins. HOX 2C, 3C and 4B behaved quite differently, showing low affinity for promoters of genes located upstream from their own gene in the HOX clusters and a higher affinity for regulatory sequences of their own gene and downstream HOX genes.     

Knockout B lymphoma cell lines as biochemical tools to explore multiple signalling pathways

Studies on B lymphocyte signalling pathways using B lymphocytes from genetically modified mice have the disadvantages of primary cell polyclonality and finite life span. B lymphoma cell lines have been generated from mice with targeted mutations in the oct-2, OBF-1, vav-1 and btk genes, as a model system that lacks these limitations and possesses additional potential for experimental manipulation. To assess their utility, activation of the B cell receptor using anti- micro, the Toll-like receptor-4 using lipopolysaccharide and the interleukin-4 receptor were assessed in these cell lines. Differential tyrosine phosphorylation of intracellular proteins was measured in the wild-type controls compared to the corresponding mutant cell lines after B cell receptor stimulation. Intracellular calcium (Ca2+i) was mobilized in the control cell lines but not in the OBF-1 and Vav1-deficient cells, while Xid B cell lines (btk mutant) showed a reduced Ca2+ mobilization. Extracellular signal-regulated kinase 1/2 phosphorylation in response to anti- micro or lipopolysaccharide stimulation was significantly reduced in Vav1-deficient cells. Interleukin-4 stimulation of wild-type cells resulted in a 2-3-fold increase in Stat-6 phosphorylation. These results indicate that the cell lines mimic the biochemical responses of the corresponding primary B cells. They therefore represent a useful model system to investigate the regulation and roles of these and other gene products in B cell signal transduction and activation.