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G Smant

Publications and source records attributed to G Smant.

11 recordsLinked to original sources
PubMed2001

GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP.

The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power of linking cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-AFLP, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. We have developed a computer program (GenEST) that predicts the sizes of virtual transcript-derived fragments (TDFs) of in silico-digested cDNA sequences retrieved from databases. The vast majority of the resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. Sequencing of the corresponding bands excised from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostochiensis. Various novel pathogenicity factors were identified by combining ESTs from the infective stage juveniles with expression profiles of approximately 4000 genes in five developmental stages produced by cDNA-AFLP.

Animals↗
PubMed2001

Functional screening yields a new beta-1,4-endoglucanase gene from Heterodera glycines that may be the product of recent gene duplication.

Clones with secreted cellulolytic activity were identified when a cDNA library constructed from poly A(+) RNA of preparasitic second-stage juveniles of Heterodera glycines, the soybean cyst nematode, was expressed in the Escherichia coli SOLR strain and overlaid with a carboxymethylcellulose (CMC) substrate. Twenty CMC-degrading clones were analyzed, and all were either identical or strongly similar to a beta-1,4-endoglucanase gene (HG-eng-2), previously isolated from H. glycines. A subgroup of identical "HG-eng-2-like" clones had considerable differences in the 5' untranslated region compared with HG-eng-2 and were designated HG-eng-3. One H. glycines genomic clone contained HG-eng-2 and HG-eng-3 full-length genes, separated by a distance of approximately 8 kb, and a second genomic clone contained two copies of HG-eng-2, separated by approximately 6.5 kb, suggesting the presence of endoglucanase gene clusters in H. glycines. The HG-eng-2 and HG-eng-3 genes were in opposite transcriptional orientation, with considerable nucleotide differences in their 5' flanking regions. The highly conserved nucleotide sequence in the introns and exons and their close proximity within the genome suggest that HG-eng-2 and HG-eng-3 are the products of recent gene duplication and inversion.

Amino Acid Sequence↗
PubMed2000

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis.

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

Animals↗
PubMed1999

In planta localization of a beta-1,4-endoglucanase secreted by Heterodera glycines.

Polyclonal sera specific to beta-1,4-endoglucanases (cellulases) synthesized in the subventral esophageal gland cells of the soybean cyst nematode, Heterodera glycines, were used to provide the first identification of a nematode esophageal gland protein that is secreted into host plant tissue. Sera generated to proteins encoded by Hg-eng-1 and Hg-eng-2 (endoglucanases) did not cross-react with soybean root proteins on Western blots (immunoblots) or in immunofluorescence microscopy of noninoculated (control) soybean root sections. In cross sections of soybean roots at 24 h after inoculation of roots with second-stage juveniles of H. glycines, HG-ENG-1 was localized within the nematode's subventral gland cells and was not detected in root tissue. HG-ENG-2 was localized within the subventral gland cells and was secreted from the juvenile's stylet into root cortical tissue at 24 h after inoculation of roots with second-stage juveniles of H. glycines. HG-ENG-2 was localized along the juvenile's migratory path through the root cortex.

Animals↗
PubMed1999

Naturally induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells.

Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones alpha-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (< 3 kDa) was shown to be responsible for the observed effect. This mitogenic oligopeptide(s) is functionally dissimilar to auxin and cytokinin and, in addition, it does not change the sensitivity of the protoplasts toward these phytohormones. In combination with the mitogen phytohemagglutinin (PHA), cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from nontarget tissue--these nematodes normally invade the roots of potato plants--suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low-molecular-weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.

Adenine↗
PubMed1999

Developmental expression of secretory beta-1,4-endoglucanases in the subventral esophageal glands of Heterodera glycines.

Two beta-1,4-endoglucanases (EGases), Hg-eng-1 and Hg-eng-2, were recently cloned from the soybean cyst nematode, Heterodera glycines, and their expression was shown in the subventral esophageal glands of hatched second-stage juveniles (J2). We examined the expression of these EGases in the subventral glands of all post-embryonic life stages of H. glycines by in situ hybridization and immunolocalization. The first detectable accumulation of EGase mRNAs occurred in the subventral glands of unhatched J2. EGase transcripts remained detectable in J2 after hatching and during subsequent root invasion. However, in late parasitic J2 and third-stage juveniles (J3), the percentage of individuals that showed EGase transcripts decreased. In female fourth-stage juveniles and adult females, EGase transcripts were no longer detected in the subventral glands. EGase hybridization signal reappeared in unhatched males coiled within the J3 cuticle, and transcripts were also present in the subventral glands of migratory adult males. Immunofluorescence labeling showed that EGase translation products are most abundantly present in the subventral glands of preparasitic J2, migratory parasitic J2, and adult males. The presence of EGases predominantly in the migratory stages suggests that the enzymes are used by the nematodes to soften the walls of root cells during penetration and intracellular migration.

Animals↗
PubMed1998

Cloning of a trans-spliced glyceraldehyde-3-phosphate-dehydrogenase gene from the potato cyst nematode Globodera rostochiensis and expression of its putative promoter region in Caenorhabditis elegans.

Reverse genetics to determine the relative importance of individual pathogenicity factors of the potato cyst nematode Globodera rostochiensis depends, apart from an efficient transformation protocol for this obligatory plant parasite, on the availability of an efficient promoter. PCR-based cloning was used to isolate a cDNA encoding glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, a crucial enzyme in glycolysis and gluconeogenesis; this gene was designated gpd) and its 5'-flanking region. The cDNA includes 1047 nucleotides encoding an open reading frame that shows high homology with GAPDHs from Caenorhabditis elegans and other species. Analysis of the 745 bp 5'-flanking region of the gpd gene showed no homology with a similar region in C. elegans. In this region several eukaryotic promoter elements are present. 5' Rapid amplification of cDNA ends revealed this gene was trans-spliced with a SL1 spliced leader. The 5'-flanking region of the gpd gene was fused to green fluorescent protein reporter gene and microinjected into the gonads of C. elegans. Green fluorescent protein expression, under the transcriptional control of the 5'-flanking region of gpd, was mainly observed in body wall muscles of transgenic animals. This putative promoter region of GAPDH could be a valuable tool to drive gene expression in transgenic G. rostochiensis and other related plant-parasitic nematode species.

Amino Acid Sequence↗
PubMed1998

Genomic organization of four beta-1,4-endoglucanase genes in plant-parasitic cyst nematodes and its evolutionary implications.

The genomic organization of genes encoding beta-1,4-endoglucanases (cellulases) from the plant-parasitic cyst nematodes Heterodera glycines and Globodera rostochiensis (HG-eng1, Hg-eng2, GR-eng1, and GR-eng2) was investigated. HG-eng1 and GR-eng1 both contained eight introns and structural domains of 2151 and 2492bp, respectively. HG-eng2 and GR-eng2 both contained seven introns and structural domains of 2324 and 2388bp, respectively. No significant similarity in intron sequence or size was observed between HG-eng1 and HG-eng2, whereas the opposite was true between GR-eng1 and GR-eng2. Intron positions among all four cyst nematode cellulase genes were conserved identically in relation to the predicted amino acid sequence. HG-eng1, GR-eng1, and GR-eng2 had several introns demarcated by 5'-GCellipsisAG-3' in the splice sites, and all four nematode cellulase genes had the polyadenylation and cleavage signal sequence 5'-GAUAAA-3'-both rare occurences in eukaryotic genes. The 5'- flanking regions of each nematode cellulase gene, however, had signature sequences typical of eukaryotic promoter regions, including a TATA box, bHLH-type binding sites, and putative silencer, repressor, and enhancer elements. Database searches and subsequent phylogenetic comparison of the catalytic domain of the nematode cellulases placed the nematode genes in one group, with Family 5, subfamily 2, glycosyl hydrolases from Scotobacteria and Bacilliaceae as the most homologous groups. The overall amino acid sequence identity among the four nematode cellulases was from 71 to 83%, and the amino acid sequence identity to bacterial Family 5 cellulases ranged from 33 to 44%. The eukaryotic organization of the four cyst nematode cellulases suggests that they share a common ancestor, and their strong homology to prokaryotic glycosyl hydrolases may be indicative of an ancient horizontal gene transfer.

3' Untranslated Regions↗
PubMed1998

Endogenous cellulases in animals: isolation of beta-1, 4-endoglucanase genes from two species of plant-parasitic cyst nematodes.

beta-1,4-Endoglucanases (EGases, EC 3.2.1.4) degrade polysaccharides possessing beta-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37-44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi, Clostridium acetobutylicum, and Bacillus subtilis. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3' end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.

Amino Acid Sequence↗
PubMed1996

Secretory granule proteins from the subventral esophageal glands of the potato cyst nematode identified by monoclonal antibodies to a protein fraction from second-stage juveniles.

Sodium dodecyl sulfate-extracted proteins from second-stage juveniles (J2) of the potato cyst nematode Globodera rostochiensis were fractionated by preparative continuous flow electrophoresis, and monoclonal antibodies (MAbs) were raised against the 38- to 40.5-kDa protein fraction. Screening of the hybridoma culture fluids by immunofluorescence microscopy of J2 resulted in the identification of 12 MAbs that bound specifically to the subventral esophageal glands. On Western blots of J2 these MAbs identified four protein bands with apparent molecular masses of 30, 31, 39, and 49 kDa. Immunoelectron microscopy with one of these MAbs showed an intense labeling of the electron dense core of the secretory granules in the subventral gland cells of J2. It is concluded that one or more of these proteins are localized within these secretory granules. Immunofluorescence microscopy of J2 from other plant parasitic nematode species showed that most of these MAbs also bind to the subventral glands of G. pallida and G. tabacum but not of Heterodera schachtii, H. glycines, Meloidogyne incognita, or M. hapla.

Animals↗