PubMed Health⌕ Search

Biomedical subjects

J M Cock

Publications and source records attributed to J M Cock.

At least 19 recordsLinked to original sources
PubMed2001

The S-locus receptor kinase is inhibited by thioredoxins and activated by pollen coat proteins.

The self-incompatibility response in Brassica allows recognition and rejection of self-pollen by the stigmatic papillae. The transmembrane S-locus receptor kinase (SRK), a member of the receptor-like kinase superfamily in plants, mediates recognition of self-pollen on the female side, whereas the S-locus cysteine-rich protein (SCR) is the male component of the self-incompatibility response. SCR is presumably located in the pollen coat, and is thought to be the SRK ligand. Although many receptor-like kinases have been isolated in plants, the mechanisms of signal transduction mediated by these molecules remain largely unknown. Here we show that SRK is phosphorylated in vivo within one hour of self-pollination. We also show that, in vitro, autophosphorylation of SRK is prevented by the stigma thioredoxin THL1 in the absence of a ligand. This inhibition is released in a haplotype-specific manner by the addition of pollen coat proteins. Our data indicate that SRK is inhibited by thioredoxins and activated by pollen coat proteins.

Brassica↗
PubMed2001

Further analysis of the interactions between the Brassica S receptor kinase and three interacting proteins (ARC1, THL1 and THL2) in the yeast two-hybrid system.

The yeast two-hybrid system was used to further characterize the interactions between the Brassica S receptor kinase (SRK) and three putative substrates, ARC1 and the two thioredoxin h proteins, THL1 and THL2. Interactions were generally detectable with kinase domains of both Class I and Class II SRKs. Chimeric constructs were made between the SRK910 kinase domain and the non-interacting Arabidopsis RLK5 kinase domain. Only one chimeric construct, SRR2, interacted with THL1 and THL2, while none of the chimeras were able to interact with ARC1. SRR2 is largely made up of RLK5 kinase domain with the N-terminal end being derived from the SRK910 kinase domain and was the only chimeric construct that retained kinase activity. Deletion or substitution of a conserved cysteine at the N-terminal end of the SRK910 kinase domain resulted in loss of interaction with THL1 and THL2, while the addition of this cysteine to a related receptor kinase, SFR1, conferred the ability to interact with the thioredoxin h proteins. In addition, substitution of the cysteines in the THL1 active site abolished the interaction. Lastly, the two Arabidopsis thioredoxin h clones most closely related to THL1 and THL2 were found to interact with the SRK kinase domains. Thus, the nature of the interaction of the thioredoxin h clones with SRK involves the reducing activity of these proteins and is restricted to the class of thioredoxin h proteins which have the variant CPPC active site.

Amino Acid Sequence↗
PubMed2001

Two large Arabidopsis thaliana gene families are homologous to the Brassica gene superfamily that encodes pollen coat proteins and the male component of the self-incompatibility response.

The male component of the self-incompatibility response in Brassica has recently been shown to be encoded by the S locus cysteine-rich gene (SCR). SCR is related, at the sequence level, to the pollen coat protein (PCP) gene family whose members encode small, cysteine-rich proteins located in the proteo-lipidic surface layer (tryphine) of Brassica pollen grains. Here we show that the Arabidopsis genome includes two large gene families with homology to SCR and to the PCP gene family, respectively. These genes are poorly predicted by gene-identification algorithms and, with few exceptions, have been missed in previous annotations. Based on sequence comparison and an analysis of the expression patterns of several members of each family, we discuss the possible functions of these genes. In particular, we consider the possibility that SCR-related genes in Arabidopsis may encode ligands for the S gene family of receptor-like kinases in this species.

Alleles↗
PubMed2001

Intrahaplotype polymorphism at the Brassica S locus.

The S locus receptor kinase and the S locus glycoproteins are encoded by genes located at the S locus, which controls the self-incompatibility response in Brassica. In class II self-incompatibility haplotypes, S locus glycoproteins can be encoded by two different genes, SLGA and SLGB. In this study, we analyzed the sequences of these genes in several independently isolated plants, all of which carry the same S haplotype (S(2)). Two groups of S(2) haplotypes could be distinguished depending on whether SRK was associated with SLGA or SLGB. Surprisingly, SRK alleles from the two groups could be distinguished at the sequence level, suggesting that recombination rarely occurs between haplotypes of the two groups. An analysis of the distribution of polymorphisms along the S domain of SRK showed that hypervariable domains I and II tend to be conserved within haplotypes but to be highly variable between haplotypes. This is consistent with these domains playing a role in the determination of haplotype specificity.

Base Sequence↗
PubMed2000

The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta.

To gain further insight into the mode of action of S-locus receptor kinase (SRK), a receptor-like kinase involved in the self-incompatibility response in Brassica, different recombinant SRK proteins have been expressed in a membranous environment using the insect cell/baculovirus system. Recombinant SRK proteins exhibited properties close to those of the endogenous stigmatic SRK protein and were found to autophosphorylate on serine and threonine residues in insect cell microsomes. Autophosphorylation was constitutive because it did not require the presence of pollen or stigma extracts in the phosphorylation buffer. Phosphorylation was shown to occur in trans, suggesting the existence of constitutive homooligomers of membrane-anchored recombinant SRK. To investigate the physiological relevance of these results, we have examined the oligomeric status of SRK in planta in cross-linking experiments and by velocity sedimentation on sucrose gradients. Our data strongly suggest that SRK is associated both with other SRK molecules and other stigma proteins in nonpollinated flowers. These findings may have important implications for our understanding of self-pollen signaling.

Biopolymers↗
PubMed1999

Membrane proteins involved in pollen-pistil interactions.

Self-incompatibility (SI) is a widespread mechanism in angiosperms which prevents self-fertilization. This mechanism relies on cell-cell interactions between pollen and pistil. Among the different SI systems that have been reported, two have been particularly investigated: the gametophytic system of Solanaceae and the sporophytic system of Brassicaceae. In these two families, although the molecular bases of SI response are different, secreted and/or membrane-anchored proteins are required for self-pollen rejection. Interestingly, these proteins exhibit two functions: recognition and a catalytic activity. In this review article, we present recent advances which permit a better understanding of how these proteins control the male/female recognition event associated with the SI response.

Animals↗
PubMed1999

Identification of a gene linked to the Brassica S (self-incompatibility) locus by differential display.

Self-incompatibility in Brassica is controlled by a complex locus, the S locus, that includes several expressed genes. Two S locus genes, SLG and SRK, are expressed in the stigma and have been implicated in self-pollen recognition. The male component of this recognition system is also predicted to be encoded by a gene at the S locus but this gene has not been identified to date. In this study, we have used differential display to screen for polymorphic, S-locus-linked genes that are expressed in anthers. This approach has allowed the identification of a gene, named S5J, which was shown to segregate completely with the S locus. We discuss the possible role of this gene in the self-incompatibility response and evaluate the utility of differential display for the identification of genes at specific genetic loci.

Base Sequence↗
PubMed1999

The S15 self-incompatibility haplotype in Brassica oleracea includes three S gene family members expressed in stigmas.

Self-incompatibility in Brassica is controlled by a single, highly polymorphic locus that extends over several hundred kilobases and includes several expressed genes. Two stigma proteins, the S locus receptor kinase (SRK) and the S locus glycoprotein (SLG), are encoded by genes located at the S locus and are thought to be involved in the recognition of self-pollen by the stigma. We report here that two different SLG genes, SLGA and SLGB, are located at the S locus in the class II, pollen-recessive S15 haplotype. Both genes are interrupted by a single intron; however, SLGA encodes both soluble and membrane-anchored forms of SLG, whereas SLGB encodes only soluble SLG proteins. Thus, including SRK, the S locus in the S15 haplotype contains at least three members of the S gene family. The protein products of these three genes have been characterized, and each SLG glycoform was assigned to an SLG gene. Evidence is presented that the S2 and S5 haplotypes carry only one or the other of the SLG genes, indicating either that they are redundant or that they are not required for the self-incompatibility response.

Alleles↗
PubMed1997

Natural antisense transcripts of the S locus receptor kinase gene and related sequences in Brassica oleracea.

Gene expression can be inhibited by antisense RNA transcripts. Although this phenomenon is widely used to analyse gene function in plants, the molecular mechanisms involved are poorly understood. One approach to improving our understanding of antisense gene regulation is to analyse the function of endogenous antisense transcripts. To date, only a small number of plant genes have been shown to be transcribed in both directions and limited information is available concerning the role of natural antisense transcripts in plants. In this study, we have identified several natural antisense transcripts which hybridise to probes derived from the S locus receptor kinase gene (SRK). The RNase protection assay and reverse trancriptase-PCR were used to demonstrate that a proportion of the antisense transcripts are encoded directly by SRK. Using different RNase protection probes, regions of the promoter, exon I (which encodes the S domain) and intron I of SRK were shown to be transcribed in an antisense direction. An antisense SRK transcript was shown to inhibit translation of a sense transcript in vitro. The possible role of antisense SRK transcripts in vivo is discussed.

Antisense Elements (Genetics)↗
PubMed1997

Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase gene in Brassica oleracea.

A receptor-like kinase, SRK, has been implicated in the autoincompatible response that leads to the rejection of self-pollen in Brassica plants. SRK is encoded by one member of a multigene family, which includes several receptor-like kinase genes with patterns of expression very different from that of SRK but of unknown function. Here, we report the characterization of a novel member of the Brassica S gene family, SFR2. RNA gel blot analysis demonstrated that SFR2 mRNA accumulated rapidly in response both to wounding and to infiltration with either of two bacteria: Xanthomonas campestris, a pathogen, and Escherichia coli, a saprophyte. SFR2 mRNA also accumulated rapidly after treatment with salicylic acid, a molecule that has been implicated in plant defense response signaling pathways. A SFR2 promoter and reporter gene fusion was introduced into tobacco and was shown to be induced by bacteria of another genus, Ralstonia (Pseudomonas) solanacearum. The accumulation of SFR2 mRNA in response to wounding and pathogen invasion is typical of a gene involved in the defense responses of the plant. The rapidity of SFR2 mRNA accumulation is consistent with SFR2 playing a role in the signal transduction pathway that leads to induction of plant defense proteins, such as pathogenesis-related proteins or enzymes of phenylpropanoid metabolism.

Base Sequence↗
PubMed1997

A functional S locus anther gene is not required for the self-incompatibility response in Brassica oleracea.

The self-incompatibility (SI) response in Brassica involves recognition of self-pollen by the papillar cells of the stigma and is mediated by the products of genes localized at the S (self-incompatibility) locus. Two S locus genes, SRK and SLG, are thought to encode components of a receptor complex present in the female partner. The putative gene product of SLA, a third S locus-linked gene that is expressed specifically in anthers, is a candidate for the male component of the SI recognition system. The identification of a mutant SLA allele, interrupted by a large insert resembling a retrotransposon, in self-compatible Brassica napus initially suggested that SLA played an essential role in the SI response. In this study, we have characterized an SLA allele from a self-compatible B. oleracea var acephala line and show that it too is interrupted by a large insert. However, analysis of seven B. oleracea var botrytis lines exhibiting both self-compatible and self-incompatible phenotypes showed that these lines carry an S allele very similar or identical to that of the B. oleracea var acephala line and that the SLA gene is interrupted by an insert in all seven lines. The insertion of the putative retrotransposon was shown to interfere with gene expression, with no SLA transcripts being detected by RNA gel blot analysis in a self-incompatible B. oleracea var botrytis line carrying an interrupted SLA gene. These data indicate that a functional SLA gene is not required for the SI response in Brassica.

Amino Acid Sequence↗
PubMed1995

SLR3: a modified receptor kinase gene that has been adapted to encode a putative secreted glycoprotein similar to the S locus glycoprotein.

A new member of the S gene family, SLR3 (S-Locus Related 3), was identified in Brassica oleracea. This gene had a novel pattern of expression compared with previously described members of the family, being expressed in petals, sepals and vegetative apices, in addition to stigmas and anthers. Moreover, use of SLR3-derived probes in RNA blot and RACE-PCR (rapid amplification of cDNA ends-polymerase chain reaction) experiments has identified transcripts of genes closely related to SLR3 in leaves, cotyledons and, at high levels in developing anthers. SLR3 is not linked to the S locus but is linked to two or three closely related genes. Sequence analysis of the SLR3 gene indicates that it is derived from an ancestral receptor kinase gene that has been modified by a series of deletion events. As a result of these modifications, SLR3 is predicted to encode a secreted glycoprotein lacking both transmembrane and kinase domains. The putative SLR3 protein differs from the products of most other S gene family members in that several of the highly conserved cysteines have been lost. Within the S gene family, modification of receptor kinase genes by deletion may represent a general mechanism for the generation of genes encoding secreted glycoproteins.

Amino Acid Sequence↗
PubMed1995

PCR-generated cDNA library of transition-stage maize embryos: cloning and expression of calmodulin genes during early embryogenesis.

One hundred maize zygotic embryos microdissected at the transition stage were used to construct a cDNA library after non-selective PCR (NS-PCR) amplification of whole cDNA populations. The library contains 2.3 x 10(5) recombinants and two different calmodulin cDNAs were cloned using a heterologous probe from petunia. Calmodulin expression was confirmed throughout maize embryogenesis at the mRNA, amplified cDNA and protein levels. Sequence analysis suggests a maize origin for both clones and negligible nucleotide changes linked to PCR. This library is the first described for early plant embryos and represents a breakthrough to isolate genes involved in embryo differentiation.

Amino Acid Sequence↗
PubMed1995

Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene.

The S locus, which controls the self-incompatibility response in Brassica, has been shown to contain at least two genes. SLG encodes a secreted S locus glycoprotein whilst SRK encodes a putative S locus receptor kinase. SRK has been shown potentially to encode a functional kinase and genetic evidence indicates that this gene is essential for the self-incompatibility response. Here the characterization of the SRK and SLG genes of a Brassica line homozygous for the S3 haplotype is described. A 120 kDa glycoprotein was identified in stigmas and several lines of evidence indicated that this protein is encoded by the SRK3 gene. First, the 120 kDa glycoprotein was recognized by antibodies raised against peptides based on the SRK3 gene sequence. Secondly, this protein is polymorphic and, in an F2 population segregating for the S3 haplotype, was expressed only in plants possessing the S3 haplotype. Thirdly, the 120 kDa protein was expressed specifically in stigmas. Finally, the 120 kDa protein was only extracted from stigmas in the presence of detergent indicating that it is anchored in the membrane. SRK has been predicted to encode a transmembrane glycoprotein based on the deduced amino acid sequence. Located on the membrane, SRK is in a position to interface between an extracellular recognition event between pollen and pistil and an intracellular signal transduction pathway which initiates the self-incompatibility response.

Amino Acid Sequence↗
PubMed1995

The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SKR extracellular domain in Brassica oleracea.

Self-incompatibility in Brassica is controlled by the S locus which contains at least two genes. SLG encodes a secreted S locus glycoprotein whilst SRK encodes a putative S locus receptor kinase which consists of three domains: an extracellular domain sharing extensive sequence identity with SLG, transmembrane region, and a cytoplasmic domain exhibiting a serine/threonine protein kinase activity. Here, the existence of truncated forms of the SRK protein corresponding to the extracellular domain of the putative receptor is reported. These proteins were detected by an antibody which recognizes the N-terminus of SRK3 and, in an F2 progeny segregating for the S3 haplotype, were only expressed in plants possessing the S3 haplotype. The truncated SRK proteins were expressed specifically in stigmas but, unlike the membrane-spanning SRK3 protein, were soluble and occurred as four different glycoforms sharing the same amino acid backbone as shown by deglycosylation experiments. Several SRK3 transcripts that may code for these truncated SRK3 proteins have been identified by RACE PCR, stigma cDNA library screening and RNA blot analysis. These transcripts are apparently generated by a combination of alternative splicing and the use of alternative polyadenylation signals. The existence of truncated forms of the S locus receptor kinase highlights some similarities between plant and animal receptor kinases. In animals, soluble extracellular domains of receptors have been described and, in some cases, have been shown to play a role in the modulation of signal transduction. By analogy, the soluble, truncated SRK proteins may play a similar role in the self-incompatibility response.

Amino Acid Sequence↗
PubMed1992

Characterization of the gene encoding the plastid-located glutamine synthetase of Phaseolus vulgaris: regulation of beta-glucuronidase gene fusions in transgenic tobacco.

The gln-delta gene, which encodes the plastid-located glutamine synthetase of Phaseolus vulgaris, was cloned and its promoter region was sequenced. Primer extension analysis was used to map the two major transcription initiation sites which are about 90 nucleotides apart. A fusion of 2.3 kb of the upstream region of the gln-delta gene to the reporter gene uidA encoding beta-glucuronidase was shown to be expressed in the chlorophyllous cell types of leaves and stems and in the root meristem region of transgenic tobacco. Analysis of a series of three 5' promoter deletion fusions revealed the presence of a region essential for promoter activity between -786 and -327 and regions involved in tissue-specific regulation and light regulation between -786 and +43.

Base Sequence↗
PubMed1992

A nitrate reductase gene of the cyanobacterium Synechococcus PCC6301 inferred by heterologous hybridization, cloning and targeted mutagenesis.

DNA probes from the narG gene of Escherichia coli, which encodes the large polypeptide of respiratory nitrate reductase, show cross-hybridization at low stringency to a single region of the genome of the cyanobacterium Synechococcus PCC6301. This segment of cyanobacterial DNA was cloned as the insert of plasmid pDN1 and characterized. RNA complementary to pDN1 was shown to be substantially more abundant in nitrate grown cells of Synechococcus PCC6301 than in ammonium grown cells, thus parallelling the nitrate induction and ammonium repression of nitrate reductase activity in cultures of this cyanobacterium. A mutant of Synechococcus PCC6301 deficient in nitrate reductase activity was obtained after a potentially mutagenic transformation treatment using pDN1 as a donor. This mutant was restored to the wild type phenotype following stable integrative transformation with pDN1 DNA. Taken together these data suggest that pDN1 might encode a polypeptide of nitrate reductase. pDN1 is distinct from three clones of genes involved in nitrate assimilation that were isolated previously from the related cyanobacterium Synechococcus PCC7942 (Kuhlemeier et al., 1984a, J. Bact. 159, 36-41, and 1984b, Gene 31, 109-116).

Amino Acid Sequence↗