A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana.

We describe here the cloning and characterization of a cDNA encoding a protein kinase that has high sequence homology to members of the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK or MEKK) family; this cDNA is named cATMEKKI (Arabidopsis thaliana MAP kinase or ERK kinase kinase 1). The catalytic domain of the putative ATMEKK1 protein shows approximately 40% identity with the amino acid sequences of the catalytic domains of MAPKKKs (such as Byr2 from Schizosaccharomyces pombe, Ste11 from Saccharomyces cerevisiae, Bck1 from S. cerevisiae, MEKK from mouse, and NPK1 from tobacco). In yeast cells that overexpress ATMEKK1, the protein kinase replaces Ste11 in responding to mating pheromone. In this study, the expression of three protein kinases was examined by Northern blot analyses: ATMEKK1 (structurally related to MAPKKK), ATMPK3 (structurally related to MAPK), and ATPK19 (structurally related to ribosomal S6 kinase). The mRNA levels of these three protein kinases increased markedly and simultaneously in response to touch, cold, and salinity stress. These results suggest that MAP kinase cascades, which are thought to respond to a variety of extracellular signals, are regulated not only at the posttranslational level but also at the transcriptional level in plants and that MAP kinase cascades in plants may function in transducing signals in the presence of environmental stress.

Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase.

We have cloned the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to remove the two C-4 methyl groups leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzy-mosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 309-amino acid polypeptide (calculated molecular mass, 36.48 kDa). The amino acid sequence shows a C-terminal endoplasmic reticulum retrieval signal KKXX and three histidine-rich clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The sterol profile of an ERG25 disruptant was consistent with the erg25 allele obtained by mutagenesis.

Cloning and expression of a gene encoding an integrin-like protein in Candida albicans.

The existence of integrin-like proteins in Candida albicans has been postulated because monoclonal antibodies to the leukocyte integrins alpha M and alpha X bind to blastospores and germ tubes, recognize a candidal surface protein of approximately 185 kDa, and inhibit candidal adhesion to human epithelium. The gene alpha INT1 was isolated from a library of C. albicans genomic DNA by screening with a cDNA probe from the transmembrane domain of human alpha M. The predicted polypeptide (alpha Int1p) of 188 kDa contains several motifs common to alpha M and alpha X: a putative I domain, two EF-hand divalent cation-binding sites, a transmembrane domain, and a cytoplasmic tail with a single tyrosine residue. An internal RGD tripeptide is also present. Binding of anti-peptide antibodies raised to potential extracellular domains of alpha Int1p confirms surface localization in C. albicans blastopores. By Southern blotting, alpha INT1 is unique to C. albicans. Expression of alpha INT1 under control of a galactose-inducible promoter led to the production of germ tubes in haploid Saccharomyces cerevisiae and in the corresponding ste12 mutant. Germ tubes were not observed in haploid yeast transformed with vector alone, in transformants expressing a galactose-inducible gene from Chlamydomonas, or in transformants grown in the presence of glucose or raffinose. Transformants producing alpha Int1p bound an anti-alpha M monoclonal antibody and exhibited enhanced aggregation. Studies of alpha Int1p reveal novel roles for primitive integrin-like proteins in adhesion and in STE12-independent morphogenesis.

Identification of a putative estrogen response element in the gene encoding brain-derived neurotrophic factor.

We have been studying the role and mechanism of estrogen action in the survival and differentiation of neurons in the basal forebrain and its targets in the cerebral cortex, hippocampus, and olfactory bulb. Previous work has shown that estrogen-target neurons in these regions widely coexpress the mRNAs for the neurotrophin ligands and their receptors, suggesting a potential substrate for estrogen-neurotrophin interactions. Subsequent work indicated that estrogen regulates the expression of two neurotrophin receptor mRNAs in prototypic peripheral neural targets of nerve growth factor. We report herein that the gene encoding the neurotophin brain-derived neurotrophic factor (BDNF) contains a sequence similar to the canonical estrogen response element found in estrogen-target genes. Gel shift and DNA footprinting assays indicate that estrogen receptor-ligand complexes bind to this sequence in the BDNF gene. In vivo, BDNF mRNA was rapidly up-regulated in the cerebral cortex and the olfactory bulb of ovariectomized animals exposed to estrogen. These data suggest that estrogen may regulate BDNF transcription, supporting our hypothesis that estrogen may be in a position to influence neurotrophin-mediated cell functioning, by increasing the availability of specific neurotrophins in forebrain neurons.

A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses.

To examine the in vivo role(s) of type I interferons (IFNs) and to determine the role of a component of the type I IFN receptor (IFNAR1) in mediating responses to these IFNs, we generated mice with a null mutation (-/-) in the IFNAR1 gene. Despite compelling evidence for modulation of cell proliferation and differentiation by type I IFNs, there were no gross signs of abnormal fetal development or morphological changes in adult IFNAR1-/- mice. However, abnormalities of hemopoietic cells were detected in IFNAR1 -/- mice. Elevated levels of myeloid lineage cells were detected in peripheral blood and bone marrow by staining with Mac-1 and Gr-1 antibodies. Furthermore, bone marrow macrophages from IFNAR1 -/- mice showed abnormal responses to colony-stimulating factor 1 and lipopolysaccharide. IFNAR1 -/- mice were highly susceptible to viral infection: viral titers were undetected 24 hr after infection of IFNAR1 +/+ mice but were extremely high in organs of IFNAR1 -/- mice, demonstrating that the type I IFN system is a major acute antiviral defence. In cell lines derived from IFNAR1 -/- mice, there was no signaling in response to IFN-alpha or -beta as measured by induction of 2'-5' oligoadenylate synthetase, antiviral, or antiproliferative responses. Importantly, these studies demonstrate that type I IFNs function in the development and responses of myeloid lineage cells, particularly macrophages, and that the IFNAR1 receptor component is essential for antiproliferative and antiviral responses to IFN-alpha and -beta.

Mutations in the gene encoding the alpha subunit of the rod cGMP-gated channel in autosomal recessive retinitis pigmentosa.

Mutations in the genes encoding two proteins of the retinal rod phototransduction cascade, opsin and the beta subunit of rod cGMP phosphodiesterase, cause retinitis pigmentosa (RP) in some families. Here we report defects in a third member of this biochemical pathway in still other patients with this disease. We screened 94 unrelated patients with autosomal dominant RP and 173 unrelated patients with autosomal recessive RP for mutations in the gene encoding the alpha subunit of the rod cGMP-gated cation channel. Five mutant sequences cosegregated with disease among four unrelated families with autosomal recessive RP. Two of these were nonsense mutations early in the reading frame (Glu76End and Lys139End) and one was a deletion encompassing most if not all of the transcriptional unit; these three alleles would not be expected to encode a functional channel. The remaining two mutations were a missense mutation (Ser316Phe) and a frameshift [Arg654(1-bp del)] mutation truncating the last 32 aa in the C terminus. The latter two mutations were expressed in vitro and found to encode proteins that were predominantly retained inside the cell instead of being targeted to the plasma membrane. We conclude that the absence or paucity of functional cGMP-gated cation channels in the plasma membrane is deleterious to rod photoreceptors and is an uncommon cause of RP.

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.

Abscisic acid (ABA) modulates the activities of three major classes of ion channels--inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels--at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA. In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior--including channel insensitivity to ABA-dependent alkalinization of pHi--as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

Cloning, expression, and function of TFC5, the gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIB.

TFC5, the unique and essential gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been cloned. It encodes a 594-amino acid protein (67,688 Da). Escherichia coli-produced B" has been used to reconstitute entirely recombinant TFIIIB that is fully functional for TFIIIC-directed, as well as TATA box-dependent, DNA binding and transcription. The DNase I footprints of entirely recombinant TFIIIB, composed of B", the 67-kDa Brf, and TATA box-binding protein, and TFIIIB reconstituted with natural B" are indistinguishable. A truncated form of B" lacking 39 N-terminal and 107 C-terminal amino acids is also functional for transcription.

Hippocampal long-term depression and depotentiation are defective in mice carrying a targeted disruption of the gene encoding the RI beta subunit of cAMP-dependent protein kinase.

The cAMP-dependent protein kinase (PKA) has been shown to play an important role in long-term potentiation (LTP) in the hippocampus, but little is known about the function of PKA in long-term depression (LTD). We have combined pharmacologic and genetic approaches to demonstrate that PKA activity is required for both homosynaptic LTD and depotentiation and that a specific neuronal isoform of type I regulatory subunit (RI beta) is essential. Mice carrying a null mutation in the gene encoding RI beta were established by use of gene targeting in embryonic stem cells. Hippocampal slices from mutant mice show a severe deficit in LTD and depotentiation at the Schaffer collateral-CA1 synapse. This defect is also evident at the lateral perforant path-dentate granule cell synapse in RI beta mutant mice. Despite a compensatory increase in the related RI alpha protein and a lack of detectable changes in total PKA activity, the hippocampal function in these mice is not rescued, suggesting a unique role for RI beta. Since the late phase of CA1 LTP also requires PKA but is normal in RI beta mutant mice, our data further suggest that different forms of synaptic plasticity are likely to employ different combinations of regulatory and catalytic subunits.

Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor.

The very low density lipoprotein (VLDL) receptor is a recently cloned member of the low density lipoprotein (LDL) receptor family that mediates the binding and uptake of VLDL when overexpressed in animal cells. Its sequence is 94% identical in humans and rabbits and 84% identical in humans and chickens, implying a conserved function. Its high level expression in muscle and adipose tissue suggests a role in VLDL triacylglycerol delivery. Mutations in the chicken homologue cause female sterility, owing to impaired VLDL and vitellogenin uptake during egg yolk formation. We used homologous recombination in mouse embryonic stem cells to produce homozygous knockout mice that lack immunodetectable VLDL receptors. Homozygous mice of both sexes were viable and normally fertile. Plasma levels of cholesterol, triacylglycerol, and lipoproteins were normal when the mice were fed normal, high-carbohydrate, or high-fat diets. The sole abnormality detected was a modest decrease in body weight, body mass index, and adipose tissue mass as determined by the weights of epididymal fat pads. We conclude that the VLDL receptor is not required for VLDL clearance from plasma or for ovulation in mice.

A gene encoding a phosphatidylinositol-specific phospholipase C is induced by dehydration and salt stress in Arabidopsis thaliana.

A cDNA corresponding to a putative phosphatidylinositol-specific phospholipase C (PI-PLC) in the higher plant Arabidopsis thaliana was cloned by use of the polymerase chain reaction. The cDNA, designated cAtPLC1, encodes a putative polypeptide of 561 aa with a calculated molecular mass of 64 kDa. The putative product includes so-called X and Y domains found in all PI-PLCs identified to date. In mammalian cells, there are three types of PI-PLC, PLC-beta, -gamma, and -delta. The overall structure of the putative AtPLC1 protein is most similar to that of PLC-delta, although the AtPLC1 protein is much smaller than PLCs from other organisms. The recombinant AtPLC1 protein synthesized in Escherichia coli was able to hydrolyze phosphatidylinositol 4,5-bisphosphate and this activity was completely dependent on Ca2+, as observed also for mammalian PI-PLCs. These results suggest that the AtPLC1 gene encodes a genuine PI-PLC of a higher plant. Northern blot analysis showed that the AtPLC1 gene is expressed at very low levels in the plant under normal conditions but is induced to a significant extent under various environmental stresses, such as dehydration, salinity, and low temperature. These observations suggest that AtPLC1 might be involved in the signal-transduction pathways of environmental stresses and that an increase in the level of AtPLC1 might amplify the signal, in a manner that contributes to the adaptation of the plant to these stresses.

Mutation spectrum of the gene encoding the beta subunit of rod phosphodiesterase among patients with autosomal recessive retinitis pigmentosa.

Mutations in the gene encoding the beta subunit of rod cGMP phosphodiesterase are known causes of photoreceptor degeneration in two animal models of retinitis pigmentosa, the rd (retinal degeneration) mouse and the Irish setter dog with rod/cone dysplasia. Here we report a screen of 92 unrelated patients with autosomal recessive retinitis pigmentosa for defects in the human homologue of this gene. We identified seven different mutations that cosegregate with the disease. They were found among four patients with each patient heterozygously carrying two mutations. All of these mutations are predicted to affect the putative catalytic domain, probably leading to a decrease in phosphodiesterase activity and an increase in cGMP levels within rod photoreceptors. Mutations in the gene encoding the beta subunit of rod phosphodiesterase are the most common identified cause of autosomal recessive retinitis pigmentosa, accounting for approximately 4% of cases in North America.

Induction of the gene encoding mucosal vascular addressin cell adhesion molecule 1 by tumor necrosis factor alpha is mediated by NF-kappa B proteins.

Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is involved in trafficking of lymphocytes to mucosal endothelium. Expression of MAdCAM-1 is induced in the murine endothelial cell line bEnd.3 by tumor necrosis factor alpha (TNF-alpha), interleukin 1, and bacterial lipopolysaccharide. Here we show that TNF-alpha enhances expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter, confirming transcriptional regulation of MAdCAM-1. Mutational analysis of the promoter indicates that a DNA fragment extending from nt -132 to nt +6 of the gene is sufficient for TNF-alpha inducibility. Two regulatory sites critical for TNF-alpha induction were identified in this region. DNA-binding experiments demonstrate that NF-kappa B proteins from nuclear extracts of TNF-alpha-stimulated bEnd.3 cells bind to these sites, and transfection assays with promoter mutants of the MAdCAM-1 gene indicate that occupancy of both sites is essential for promoter function. The predominant NF-kappa B binding activity detected with these nuclear extracts is a p65 homodimer. These findings establish that, as with other endothelial cell adhesion molecules, transcriptional induction of MAdCAM-1 by TNF-alpha requires activated NF-kappa B proteins.

Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; p = 1.18 g CM-3) and a low-density fraction (LDMV; rho=1.12 g cm(-3)). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.

Hypermethylation of the 5 ' CpG island of the gene encoding the serine protease Testisin promotes its loss in testicular tumorigenesis

The Testisin gene (PRSS21) encodes a glycosylphosphatidylinositol (GPI)-linked serine protease that exhibits testis tissue-specific expression. Loss of Testisin has been implicated in testicular tumorigenesis, but its role in testis biology and tumorigenesis is not known. Here we have investigated the role of CpG methylation in Testisin gene inactivation and tested the hypothesis that Testisin may act as a tumour suppressor for testicular tumorigenesis. Using sequence analysis of bisulphite-treated genomic DNA, we find a strong relationship between hypermethylation of a 385 bp 50 CpG rich island of the Testisin gene, and silencing of the Testisin gene in a range of human tumour cell lines and in 100% (eight/eight) of testicular germ cell tumours. We show that treatment of Testisin-negative cell lines with demethylating agents and/or a histone deacetylase inhibitor results in reactivation of Testisin gene expression, implicating hypermethylation in Testisin gene silencing. Stable expression of Testisin in the Testisin-negative Tera-2 testicular cancer line suppressed tumorigenicity as revealed by inhibition of both anchorage-dependent cell growth and tumour formation in an SCID mouse model of testicular tumorigenesis. Together, these data show that loss of Testisin is caused, at least in part, by DNA hypermethylation and histone deacetylation, and suggest a tumour suppressor role for Testisin in testicular tumorigenesis.