Sequence complexity of the S receptor kinase gene family in Brassica

A genomic library from an S ₂₉/S ₂₉ self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650–700 by region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG ₂₉ cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.     

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.     

Sequence complexity of the S receptor kinase gene family in Brassica

A genomic library from an S ₂₉/S ₂₉ self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650–700 by region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG ₂₉ cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.     

Three members of the S multigene family are linked to the S locus of Brassica

Two self-incompatibility genes in Brassica, SLG and SRK (SLG encodes a glycoprotein; SRK encodes a receptor-like kinase), are included in the S multigene family. Products of members of the S multigene family have an SLG-like domain (S domain) in common, which may function as a receptor. In this study, three clustered members of the S multigene family, BcRK1, BcRL1 and BcSL1, were characterized. BcRK1 is a putative functional receptor kinase gene expressed in leaves, flower buds and stigmas, while BcRL1 and BcSL1 are considered to be pseudogenes because deletions causing frameshifts were identified in these sequences. Sequence and expression pattern of BcRK1 were most similar to those of the Arabidopsis receptor-like kinase gene ARK1, indicating that BcRK1 might have a function similar to that of ARK1, in processes such as cell expansion or plant growth. Interestingly, the region containing BcRK1, BcRL1 and BcSL1 is genetically linked to the S locus and the physical distance between SLG, SRK and the three S-related genes was estimated to be less than 610 kb. Thus the genes associated with self-incompatibility exist within a cluster of S-like genes in the genome of Brassica.     

Three members of the S multigene family are linked to the S locus of Brassica

Two self-incompatibility genes in Brassica, SLG and SRK (SLG encodes a glycoprotein; SRK encodes a receptor-like kinase), are included in the S multigene family. Products of members of the S multigene family have an SLG-like domain (S domain) in common, which may function as a receptor. In this study, three clustered members of the S multigene family, BcRK1, BcRL1 and BcSL1, were characterized. BcRK1 is a putative functional receptor kinase gene expressed in leaves, flower buds and stigmas, while BcRL1 and BcSL1 are considered to be pseudogenes because deletions causing frameshifts were identified in these sequences. Sequence and expression pattern of BcRK1 were most similar to those of the Arabidopsis receptor-like kinase gene ARK1, indicating that BcRK1 might have a function similar to that of ARK1, in processes such as cell expansion or plant growth. Interestingly, the region containing BcRK1, BcRL1 and BcSL1 is genetically linked to the S locus and the physical distance between SLG, SRK and the three S-related genes was estimated to be less than 610 kb. Thus the genes associated with self-incompatibility exist within a cluster of S-like genes in the genome of Brassica. Received: 15 April 1997 / Accepted: 13 June 1997     

Existence of a Common Glycoprotein Homologous to S-Glycoproteins in Two Self-incompatible Homozygotes of Brassica campestris

S-Glycoproteins (S-locus-specific glycoproteins) in Brassicaspecies are present only in stigmas and thought to play an importantrole in self-incompatibility system. The stigma extract containsalso several other glycoproteins reacting with the antiserumto S-glycoproteins, among which some glycoproteins from S₈S₈-and S₉S₉-homozygotes have the same pI value. Both of the glycoproteinswhich were tentatively termed NS₈- and NS₈S₉-glycoproteins,respectively, were isolated and analyzed. Those were revealedto be identical. Its amino acid sequence was homologous withthe S-glycoproteins in Brassica species. The NS-glycoproteinswere expressed at the same time and only in stigma as S-glycoproteins. (Received July 19, 1988; Accepted September 7, 1988)     

Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea

Summary In Brassica oleracea, the pollen-stigma interaction of self-incompatibility is controlled by a single genetically defined locus designated S. Molecular studies have identified two genes that are tightly linked to the classically defined S locus: The S-Locus Glycoprotein (SLG) gene and the S-Receptor Kinase (SRK) gene. In previous RFLP linkage analyses with probes specific for SLG and SRK, we were unable to identify any recombination events between SLG, SRK, and self-incompatibility phenotype. In this paper, we use pulsed-field gel electrophoresis (PFGE) in conjunction with DNA blot analysis to characterize the S-locus region from two highly divergent self-incompatibility genotypes, S ₂ and S ₆. We establish the physical linkage of SLG and SRK in each genotype, and demonstrate that the two genes are separated by a maximum distance of 220 kb in the S ₆ genotype and 350 kb in the S ₂ genotype. Furthermore, a comparison of the data from the two genotypes reveals that a high level of polymorphism exists across the entire S-locus region.     

Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs

Polymorphism of SLG (the S-locus glycoprotein gene) in Brassica campestris was analyzed by PCR-RFLP using SLG-specific primers. Nucleotide sequences of PCR products from 15 S genotypes were determined in order to characterise the exact DNA fragment sizes detected in the PCR-RFLP analysis. Forty-seven lines homozygous for 27 S-alleles were used as plant material. One combination of primers, PS5 + PS 15, which had a nucleotide sequence specific to a class-I SLG, gave amplification of a single DNA fragment of approximately 1.3kb from the genomic DNA of 15 S genotypes. All the DNA fragments showed different electrophroetic profiles from each other after digestion with MboI or MspI. Different lines having the same S genotype had an identical electrophoretic profile even between the lines collected in Turkey and in Japan. Another class-I SLG-specific primer, PS 18, gave amplification of a 1.3-kb DNA fragment from three other S genotypes in combination with PS 15, and the PCR product also showed polymorphism after cleavage with the restriction endonucleases. Genetic analysis, Southern-hybridization analysis, and determination of the nucleotide sequences of the PCR products suggested that the DNA fragments amplified with these combinations of primers are class-I SLGs. Expected DNA fragment sizes in the present PCR-RFLP condition were calculated from the determined nucleotide sequence of SLG PCR products. A single DNA fragment was also amplified from six S genotypes by PCR with a combination of primers, PS3 + PS21, having a nucleotide sequence specific to a class-II SLG. The amplified DNA showed polymorphisnm after cleavage with restriction endonucleases. The cleaved fragments were detected by Southern-hybridization analysis using a probe of S ⁵ SLG cDNA, a class-IISLG. Partial sequencing revealed a marked similarity of these amplified DNA fragments to a class-II SLG, demonstrating the presence of class-I and class-II S alleles also in B. campestris. The high SLG polymorphism detected by the present investigation suggests the usefulness of the PCR-RFLP method for the identification of S alleles in breeding lines and for listing S alleles in B. campestris.     

Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway

N-Linked protein glycosylation in most eukaryotic cells initiateswith the transfer of the oligosaccharide Glc₃Man₉GlcNAc₂ fromthe lipid carrier dolichyl pyrophosphate to selected asparagineresidues. In the yeast Saccharomyces cerevisiae, alg mutationswhich affect the assembly of the lipid-linked oligosaccharideat the membrane of the endoplasmic reticulum result in the accumulationof lipid-linked oligosaccharide intermediates and a hypoglycosylationof proteins. Exploiting the synthetic growth defect of alg mutationsin combination with mutations affecting oligosaccharyl transferaseactivity (Stagljar et al., 1994), we have isolated the ALG6locus. alg6 mutants accumulate lipid-linked Man₉GlcNAc₂, suggestingthat this locus encodes an endoplasmic glucosyltransferase.Alg6p has sequence similarity to Alg8p, a protein required forglucosylation of Glc₁Man⁹GlcNAc². Saccharomyces cerevisiae endoplasmic reticulum glycosyltransferase dolichol     

Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents

Cell shrinkageis an early prerequisite in programmed cell death, and cytoplasmicK⁺ is a dominant cation that controls intracellular ionhomeostasis and cell volume. Blockade of K⁺ channelsinhibits apoptotic cell shrinkage and attenuates apoptosis. We examined whether apoptotic repressor with caspase recruitment domain (ARC), an antiapoptotic protein, inhibits cardiomyocyte apoptosis by reducing K⁺ efflux throughvoltage-gated K⁺ (Kv) channels. In heart-derived H9c2cells, whole cell Kv currents (I_K(V)) wereisolated by using Ca²⁺-free extracellular (bath) solutionand including 5 mM ATP and 10 mM EGTA in the intracellular (pipette)solution. Extracellular application of 5 mM 4-aminopyridine (4-AP), ablocker of Kv channels, reversibly reduced I_K(V)by 50-60% in H9c2 cells. The remaining currents during 4-APtreatment may be generated by K⁺ efflux through4-AP-insensitive K⁺ channels. Overexpression of ARC inheart-derived H9c2 cells significantly decreasedI_K(V), whereas treatment with staurosporine, apotent apoptosis inducer, enhanced I_K(V)in wild-type cells. The staurosporine-induced increase inI_K(V) was significantly suppressed and thestaurosporine-mediated apoptosis was markedly inhibited incells overexpressing ARC compared with cells transfected with thecontrol neomycin vector. These results suggest that theantiapoptotic effect of ARC is, in part, due to inhibition of Kvchannels in cardiomyocytes.

     

Analysis of S-locus and expression of S-alleles of self-compatible rapid-cycling Brassica oleracea ‘TO1000DH3’

Brassica oleracea is a strictly self-incompatible (SI) plant, but rapid-cycling B. oleracea ‘TO1000DH3’ is self-compatible (SC). Self-incompatibility in Brassicaceae is controlled by multiple alleles of the S-locus. Three S-locus genes, S-locus glycoprotein (SLG), S-locus receptor kinase (SRK) and S-locus protein 11 or S-locus cysteine-rich (SP11/SCR), have been reported to date, all of which are classified into class I and II. In this study, we investigated the molecular mechanism behind alterations of SI to SC in rapid-cycling B. olerace ‘TO1000DH3’. Class I SRK were identified by genomic DNA PCR and PCR-RFLP analysis using SRK specific markers and found to be homozygous. Cloning and sequencing of class I SRK revealed a normal kinase domain without any S-domain/transmembrane domain. Moreover, S-locus sequencing analysis revealed only an SLG sequence, but no SP11/SCR. Expression analysis showed no SRK expression in the stigma, although other genes involved in the SI recognition reaction (SLG, MLPK, ARC1, THL) were found to have normal expression in the stigma. Taken together, the above results suggest that structural aberrations such as deletion of the SI recognition genes may be responsible for the breakdown of SI in rapid-cycling B. oleracea ‘TO1000DH3’.     

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺ levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀ values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_i increase. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_i increase and ROS generation, and the [Ca²⁺]_i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca²⁺]_i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca²⁺]_i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca²⁺]_i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca²⁺]_i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species     

Molecular Cloning and Characterization of S-Adenosyl-L-Methionine:Scoulerine-9-O-Methyltransferase from Cultured Cells of Coptis japonica

S-Adenosyl-L-methionine : scoulerine-9-O-methyltransferase (SMT)catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionineto the 9-hydroxyl group of scoulerine during the biosynthesisof berberine. We have isolated functionally active cDNA clones(pCJSMTs) from a cDNA library prepared from cultured cells ofCoptis japonica. The longest cDNA insert (pCJSMT1) had an openreading frame that encoded 351 amino acids, but the calculatedmolecular mass (38,364 Da) of the deduced product was slightlylower than the experimentally determined molecular mass of purifiedSMT. Rapid amplification of the 5' end of the cDNA indicatedthat the full-length cDNA of SMT consisted of 1,458 nucleotidesthat encoded 381 amino acids. When the full-length cDNA wasexpressed in E. coli, the molecular mass of the expressed SMTwas greater than that of native SMT in Coptis cells. This resultsuggests that SMT might be produced in a pre-mature form andprocessed post-translationally. SMT was also found to exhibitsequence homology to other O-methyltransferases from plantsand N-terminal region of the SMT polypeptide appeared to benecessary for enzymatic activity. ¹Present address: High Quality Life Research Laboratories, SumitomoMetal Industries, Ltd., 3-5 Hikaridai, Seika, Sourakugun, Kyoto,619-02 Japan ²Present address: Suntory Research Center, 1-1-1 Wakayamadai,Shimamoto, Mishima-gun, Osaka, 618 Japan ³Present address: Department of Cell Biology, The Scripps ResearchInstitute, La Jolla, CA 92037 U.S.A.     

Does the genome of Corylus avellana L. contain sequences homologous to the self-incompatibility gene of Brassica?

Self-incompatibility is a genetic mechanism enforcing cross-pollination in plants. Hazelnut (Corylus avellana L.) expresses the sporophytic type of self-incompatibility, for which the molecular genetic basis is characterized only in Brassica. The hypothesis that the hazelnut genome contains homologs of Brassica self-incompatibility genes was tested. The S-locus glycoprotein gene (SLG) and the kinase-encoding domain of the S-receptor kinase (SRK) gene of B. oleracea L. were used to probe blots of genomic DNA from six genotypes of hazelnut. Weak hybridization with the SLG probe was detected for all hazelnut genotypes tested; however, no hybridization was detected with PCR-generated probes corresponding to two conserved regions of the SLG gene. One of these PCR probes included the region of SLG encoding the 11 invariant cysteine residues that are an important structural feature of all S-family genes. The present evidence suggests that hazelnut DNA hybridizing to SLG differs significantly from the Brassica gene, and that the S-genes cloned from Brassica will not be useful for exploring self-incompatibility in hazelnut.     

The Conductance of the Plasmalemma for CO2

Permeability coefficients (P_S values) for CO₂ of the plasmamembrane (PM) of the unicellular green algae Eremosphaera viridis,Dunaliella parva, and Dunaliella acidophila, and of mesophyllprotoplasts isolated from Valerianella locusta were determinedfrom ¹⁴CO₂ uptake experiments using the rapid separation ofcells by the silicone oil layer centrifugation technique. Theexperimental P_S values were compared with calculated numbersobtained by interpolation of Collander plots, which are basedon lipid solubility and molecular size, for D. parva cells,mesophyll protoplasts isolated from Spinacia oleracea, mesophyllcells and guard cells of Valerianella, and guard cell protoplastsisolated from Vicia faba. The conductivity of algal plasma membranes for CO₂ varies between0.1 and 9 ? 10^–6 m s^–1, whereas for the plasmalemmaof cells and protoplasts isolated from leaves of higher plantsvalues between 0.3 and 11 ? 10^–6 m s^–1 were measured.By assuming that these measurements are representative for plantsand algae in general, it is concluded that the CO₂ conductivityof algal PM is of the same order of magnitude as that of thehigher plant cell PM. P_s values of plasma membranes for CO₂are lower than those for SO₂, but are in the same order of magnitudeas those measured for H₂O. On the basis of these results itis concluded that theoretical values of about 3000 ? 10^–6m s^–1 believed to be representative for higher plant cells(Nobel, 1983) and which are frequently used for computer-basedmodels of photosynthesis, lack experimental confirmation andrepresent considerable overestimations. However, with severalsystems, including higher plant cells, the conductance of thePM for CO₂ was significantly higher in light than in darkness.This suggests that in light, additional mechanisms for CO₂ uptakesuch as facilitated diffusion or active uptake may operate inparallel with diffusional uptake. Key words: Conductivity, CO₂, permeability coefficient, photosynthesis, plasmalemma