FoEG1, a secreted glycoside hydrolase family 12 protein from Fusarium oxysporum,triggers cell death and modulates plant immunity

Fusarium oxysporum is an important soilborne fungal pathogen with many different formae speciales that can colonize the plant vascular system and cause serious crop wilt disease worldwide. We found a glycoside hydrolase family 12 protein FoEG1, secreted by F. oxysporum, that acted as a pathogen-associated molecular pattern (PAMP) targeting the apoplast of plants to induce cell death. Purified FoEG1 protein triggered cell death in different plants and induced the plant defence response to enhance the disease resistance of plants. The ability of FoEG1 to induce cell death was mediated by leucine-rich repeat (LRR) receptor-like kinases BAK1 and SOBIR1, and this ability was independent of its hydrolase activity. The mutants of cysteine residues did not affect the ability of FoEG1 to induce cell death, and an 86 amino acid fragment from amino acid positions 144 to 229 of FoEG1 was sufficient to induce cell death in Nicotiana benthamiana. In addition, the expression of FoEG1 was strongly induced in the early stage of F. oxysporum infection of host plants, and FoEG1 deletion or loss of enzyme activity reduced the virulence of F. oxysporum. Therefore, our results suggest that FoEG1 can contribute to the virulence of F. oxysporum depending on its enzyme activity and can also act as a PAMP to induce plant defence responses.     

hUNC93B1: a novel human gene representing a new gene family and encoding an unc-93-like protein

We have identified a novel human gene UNC93B1 encoding a protein related to unc-93 of Caenorhabditis elegans. The combined sequence derived from several cDNA clones is 2282 bp and comparison with genomic sequence shows that the gene contains 11 exons. The longest open reading frame encodes a deduced sequence of 597 amino acids. Homology analysis shows that the hUNC93B1 gene is highly conserved and related to sequences in Arabidopsis thaliana, C. elegans, Drosophila melanogaster, chicken and mouse. Structural analysis of the deduced amino acid sequence of hUNC93B1 points to possible existence of multiple membrane-spanning domains. hUNC93B1 protein also displays some similarities to the bacterial ABC-2 type transporter signature and to ion transporters of Deinococcus radiodurans and Helicobacter pylori. As revealed by Northern analysis, the level of expression varies significantly between tissues, with the highest level detected in the heart. The gene was mapped to chromosomal band 11q13 by fluorescence in situ hybridization. We suggest that this gene is a member of a novel hUNC93B-related gene family.     

Expression of MUL, a gene encoding a novel RBCC family ring-finger protein, in human and mouse embryogenesis

In mammals, the SWI/SNF complex is involved in chromatin remodelling in a wide range of cellular events for which regulatory factors require access to DNA. In the present study, we analyzed in early postimplantation mouse embryos the expression pattern of BRM (SNF2alpha) and BRG1 (SNF2beta), which are both ATPase subunits of this complex. Contrarily to the previous studies conducted in adult mice, showing the ubiquitous and overlapping expressions of BRM and BRG1, we show that BRM expression is restricted to mesodermal tissues involved in early vasculogenesis and heart morphogenesis.     

Characterization of a modular, cell-surface protein and identification of a new gene family in the diatom Thalassiosira pseudonana

We report the characterization of a cell-surface protein isolated from copper-stressed cells of the centric diatom Thalassiosira pseudonana Hasle and Heimdal (CCMP 1335). This protein has an apparent molecular weight of 100kDa and is highly acidic. The 100kDa protein (p100) sequence is comprised almost entirely of a novel domain termed TpRCR for T. pseudonana repetitive cysteine-rich domain, that is repeated 8 times and that contains conserved aromatic, acidic, and potential metal-binding amino acids. The analysis of the T. pseudonana genome suggests that p100 belongs to a large family of modular proteins that consist of a variable number of TpRCR domain repeats. Based on cell surface biotinylation and antibody data, p100 appears to migrate more rapidly with SDS-PAGE when extracted from cells exposed to high levels of copper; however, the discovery of a large family of TpRCR domain-containing proteins leaves open the possibility that the antibody may be cross-reacting with members of this protein family that are responding differently to copper. The response of the gene encoding p100 at the mRNA level during synchronized progression through the normal cell cycle is similar to previously characterized genes in T. pseudonana encoding cell wall proteins called silaffins.     

The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family

The surface glycoproteins T4 and T8 define different functional subsets of T lymphocytes and may act as recognition molecules mediating appropriate interactions between the T cell and its target. Previously we employed gene transfer and subtractive hybridization to isolate a T8 cDNA; now we have isolated and sequenced a cDNA clone encoding the T4 molecule. The deduced protein sequence reveals that T4 is an integral membrane protein that shares significant amino acid and structural homologies with members of the immunoglobulin supergene family. The overall structure of T4 consists of an N-terminal variable (V)-like domain, a joining (J)-like region, a third extracellular domain, a membrane-spanning region homologous to class II MHC beta-chains, and a highly charged cytoplasmic domain. Comparison of the protein sequences deduced from the T4 and T8 cDNAs reveals structural similarities consistent with their postulated role as recognition molecules, as well as differences suggesting that the two proteins recognize different structures on the target cell.     

Specific expression of a gene encoding a novel calcium-binding protein, CAF-1, during transition of Dictyostelium cells from growth to differentiation

The gene expressions involved in the transition from cell proliferation to differentiation were analyzed, using synchronized Dictyostelium discoideum Ax-2 cells and the differential plaque hybridization method. As one of the genes (cDNA) specifically expressed when Ax-2 cells were starved just before the putative shift (PS)-point (putative shift point; a switchover point from growth to differentiation in the cell cycle), calfumirin-1 ( CAF-1 ) was cloned, which encoded a novel calcium-binding protein with E-F hand. Although CAF-1 mRNA was slightly expressed in vegetatively growing cells, the expression was markedly increased in response to starvation of cells just before the PS-point. Northern analysis using non-synchronized Ax-2 cells showed that the CAF-1 mRNA is predominantly expressed within a few hours of starvation. Such a starvation-induced early expression of the CAF-1 mRNA raised a possibility that CAF-1 might be one of Ca²⁺-binding proteins involved in the phase-shift of cells from growth to differentiation.     

Expression of a gene encoding a ribosomal p40 protein and identification of an active promoter site

The promoter of a gene encoding a ribosome-associated protein of 40 kDa from Arabidopsis thaliana (A-p40) was sequenced and the expression of the gene studied. A-p40 was expressed in the same organs and with the same variations as the eukaryotic elongation factor 1 (eEF1A), another gene coding for a protein involved in translation Arabidopsis plants transformed with a -glucuronidase (GUS) gene driven by the A-p40 promoter confirm that A-p40 is expressed in actively dividing and growing cells. eEF1A promoter-GUS fusions have the same pattern of expression. Comparison of cis-acting elements from A-p40 and eEF1A revealed some common elements. A-p40 promoter deletions and transient gene expression in transfected Arabidopsis protopasts allowed the identification of trap40, a cis-acting element regulating gene expression. Gel retardation experiments indicate that eEF1A and A-p40 are regulated by different cis-acting elements. The role of such elements is discussed.     

Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15. Structure of the gene encoding transglutaminase X and a novel gene family member, transglutaminase Z

We isolated and characterized the gene encoding human transglutaminase (TG)(X) (TGM5) and mapped it to the 15q15.2 region of chromosome 15 by fluorescence in situ hybridization. The gene consists of 13 exons separated by 12 introns and spans about 35 kilobases. Further sequence analysis and mapping showed that this locus contained three transglutaminase genes arranged in tandem: EPB42 (band 4.2 protein), TGM5, and a novel gene (TGM7). A full-length cDNA for the novel transglutaminase (TG(Z)) was obtained by anchored polymerase chain reaction. The deduced amino acid sequence encoded a protein with 710 amino acids and a molecular mass of 80 kDa. Northern blotting showed that the three genes are differentially expressed in human tissues. Band 4.2 protein expression was associated with hematopoiesis, whereas TG(X) and TG(Z) showed widespread expression in different tissues. Interestingly, the chromosomal segment containing the human TGM5, TGM7, and EPB42 genes and the segment containing the genes encoding TG(C),TG(E), and another novel gene (TGM6) on chromosome 20q11 are in mouse all found on distal chromosome 2 as determined by radiation hybrid mapping. This finding suggests that in evolution these six genes arose from local duplication of a single gene and subsequent redistribution to two distinct chromosomes in the human genome.     

The OSBP-related proteins: a novel protein family involved in vesicle transport,cellular lipid metabolism,and cell signalling

Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man. The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain. The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids. Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region. The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins. It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries. Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events.     

Isolation,characterization, and chromosomal mapping of a novel cDNA clone encoding human selenium binding protein

We have isolated the full-length human 56 kDa selenium binding protein (hSP56) cDNA clone, which is the human homolog of mouse 56 kDa selenium binding protein. The cDNA is 1,668 bp long and has an open reading frame encoding 472 amino acids. The calculated molecular weight is 52.25 kDa and the estimated isoelectric point is 6.13. Using Northern blot hybridization, we found that this 56 kDa selenium binding protein is expressed in mouse heart with an intermediate level between those found in liver/lung/kidney and intestine. We have also successfully expressed hSP56 in Escherichia coli using the expression vector-pAED4. The hSP56 gene is located at human chromosome 1q21–22. J. Cell. Biochem. 64:217–224. © 1997 Wiley-Liss, Inc.     

Disruption of gene SPL35, encoding a novel CUE domain‐containing protein,leads to cell death and enhanced disease response in rice

Lesion mimic mutants that exhibit spontaneous hypersensitive response (HR)‐like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL‐PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H₂O₂, up‐regulated expression of defence‐related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain‐containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta‐COP1 and Delta‐COP2 through the CUE domain, and down‐regulation of these interacting proteins also cause development of HR‐like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.     

Identification and expression of two novel cytochrome P450 genes, belonging to CYP4 and a new CYP331 family, in the polychaete Capitella capitata sp.I

The polychaete Capitella capitata sp.I has a high capacity to metabolize polycyclic aromatic hydrocarbons (PAHs) which are among the most hazardous environmental pollutants with significant biological effects. In the present study, two novel cytochrome P450 (CYP) genes were identified in this species. One was named CYP331A1, the first member of a new family of CYP331, and the other CYP4AT1 is the first member of a new subfamily CYP4AT. Both of these genes are constitutively expressed in the worms and detectable by RT-PCR. The expression of CYP331A1 mRNA was observed to be more sensitive to PAH exposure than CYP4AT1, which indicated that CYP331A1 should play a more important role than CYP4AT1 in PAH metabolism in this species. Considering the importance of C. capitata sp.I in taking up PAH and other organic pollutants from contaminated marine sediments with the potential for subsequent food-chain transfer, our results are important for understanding the molecular basis of biotransformation and detoxification in this invertebrate, and also have evolutionary significance for understanding the diversity and history of the CYP superfamily.     

Molecular cloning, physical mapping, and expression analysis of a novel gene, BCL2L12, encoding a proline-rich protein with a highly conserved BH2 domain of the Bcl-2 family

Members of the Bcl-2 family of apoptosis-regulating proteins contain at least one of the four evolutionarily conserved domains, termed BH1, BH2, BH3, or BH4. Here, we report the identification, cloning, physical mapping, and expression pattern of BCL2L12, a novel gene that encodes a BCL2-like proline-rich protein. Proline-rich sites have been shown to interact with Src homology region 3 (SH3) domains of several tyrosine kinases, mediating their oncogenic potential. This new gene maps to chromosome 19q13.3 and is located between the IRF3 and the PRMT1/HRMT1L2 genes, close to the RRAS gene. BCL2L12 is composed of seven coding exons and six intervening introns, spanning a genomic area of 8.8 kb. All of the exon-intron splice sites conform to the consensus sequence for eukaryotic splice sites. The BCL2L12 protein is composed of 334 amino acids, with a calculated molecular mass of 36.8 kDa and an isoelectric point of 9.45. The BCL2L12 protein contains one BH2 homology domain, one proline-rich region similar to the TC21 protein and, five consensus PXXP tetrapeptide sequences. BCL2L12 is expressed mainly in breast, thymus, prostate, fetal liver, colon, placenta, pancreas, small intestine, spinal cord, kidney, and bone marrow and to a lesser extent in many other tissues. We also identified one splice variant of BCL2L12 that is primarily expressed in skeletal muscle.