A self-fertile mutant of Phalaris produces an S protein with reduced thioredoxin activity

Gametophytic self-incompatibility in the Phalaris coerulescens is controlled by two unlinked genes, S and Z . Isolation of the S gene from the pollen of this grass species indicated that the C terminus has significant hemology with thioredoxin H proteins. The protein from the C terminus, expressed in Escherichia coli , exhibits thioredoxin-like activity. This paper demonstrates that the C terminus of the S protein from an S complete mutant shows significant reduction in thioredoxin activity when compared with the wild-type form. Both pollen and stigma have lost self-incompatibility in this mutant. Close examination of the lesions, which were found only in the C terminus of the mutant gene suggests that the substitution of a serine by an arginine is responsible for the reduced enzymatic activity. The association between reduced activity and the loss of the self-incompatibility provides evidence for a role of thioredoxin activity in the self-incompatibility reaction of this species.     

Expression of 10 S-class SLF-like genes in Nicotiana alata pollen and its implications for understanding the pollen factor of the S locus

The S locus of Nicotiana alata encodes a polymorphic series of ribonucleases (S-RNases) that determine the self-incompatibility (SI) phenotype of the style. The pollen product of the S locus (pollen S) in N. alata is unknown, but in species from the related genus Petunia and in self-incompatible members of the Plantaginaceae and Rosaceae, this function has been assigned to an F-box protein known as SLF or SFB. Here we describe the identification of 10 genes (designated DD1-10) encoding SLF-related proteins that are expressed in N. alata pollen. Because our approach to cloning the DD genes was based on sequences of SLFs from other species, we presume that one of the DD genes encodes the N. alata SLF ortholog. Seven of the DD genes were exclusively expressed in pollen and a low level of sequence variation was found in alleles of each DD gene. Mapping studies confirmed that all 10 DD genes were linked to the S locus and that at least three were located in the same chromosomal segment as pollen S. Finally, the different topologies of the phylogenetic trees produced using available SLF-related sequences and those produced using S-RNase sequences suggests that pollen S and the S-RNase have different evolutionary histories.     

Structural organization of the rat gene for the arginine vasopressin-neurophysin precursor.

The rat arginine vasopressin-neurophysin precursor gene has been isolated from a genomic library cloned in lambda phage Charon 4A. Restriction mapping and nucleotide sequence analysis demonstrated that the gene is 1.85 kilobase pairs long and contains two intervening sequences located in the protein coding region. Exon A encodes a putative signal peptide, the hormone arginine vasopressin and the variable N terminus of the carrier protein neurophysin, exon B encodes the highly conserved middle part of neurophysin and exon C its variable C terminus together with glycoprotein. Thus, the three functional domains of the percursor - arginine, vasopressin, neurophysin, glycoprotein - are encoded on three distinct exons.     

Sequence diversity within the reovirus S2 gene: reovirus genes reassort in nature, and their termini are predicted to form a panhandle motif.

To better understand genetic diversity within mammalian reoviruses, we determined S2 nucleotide and deduced sigma 2 amino acid sequences of nine reovirus strains and compared these sequences with those of prototype strains of the three reovirus serotypes. The S2 gene and sigma 2 protein are highly conserved among the four type 1, one type 2, and seven type 3 strains studied. Phylogenetic analyses based on S2 nucleotide sequences of the 12 reovirus strains indicate that diversity within the S2 gene is independent of viral serotype. Additionally, we found marked topological differences between phylogenetic trees generated from S1 and S2 gene nucleotide sequences of the seven type 3 strains. These results demonstrate that reovirus S1 and S2 genes have distinct evolutionary histories, thus providing phylogenetic evidence for lateral transfer of reovirus genes in nature. When variability among the 12 sigma 2-encoding S2 nucleotide sequences was analyzed at synonymous positions, we found that approximately 60 nucleotides at the 5' terminus and 30 nucleotides at the 3' terminus were markedly conserved in comparison with other sigma 2-encoding regions of S2. Predictions of RNA secondary structures indicate that the more conserved S2 sequences participate in the formation of an extended region of duplex RNA interrupted by a pair of stem-loops. Among the 12 deduced sigma 2 amino acid sequences examined, substitutions were observed at only 11% of amino acid positions. This finding suggests that constraints on the structure or function of sigma 2, perhaps in part because of its location in the virion core, have limited sequence diversity within this protein.     

Molecular characterization of the S locus in two self-incompatible Brassica napus lines.

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region, there are two closely linked genes coding for the S locus glycoprotein (SLG) and S locus receptor kinase (SRK). They appear to comprise the pistil component of the self-incompatibility reaction. SLG and SRK are thought to recognize an unknown pollen component on the incompatible pollen, and the gene encoding this pollen component must also be linked to the SLG and SRK genes. To further our understanding of self-incompatibility, the chromosomal region carrying the SLG and SRK genes has been studied. The physical region between the SLG-910 and the SRK-910 genes in the Brassica napus W1 line was cloned, and a search for genes expressed in the anther revealed two additional S locus genes located downstream of the SLG-910 gene. Because these two genes are novel and are conserved at other S alleles, we designated them as SLL1 and SLL2 (for S locus-linked genes 1 and 2, respectively). The SLL1 gene is S locus specific, whereas the SLL2 gene is not only present at the S locus but is also present in other parts of the genomes in both self-incompatible and self-compatible Brassica ssp lines. Expression of the SLL1 gene is only detectable in anthers of self-incompatible plants and is developmentally regulated during anther development, whereas the SLL2 gene is expressed in anthers and stigmas in both self-incompatible and self-compatible plants, with the highest levels of expression occurring in the stigmas. Although SLL1 and SLL2 are linked to the S locus region, it is not clear whether these genes function in self-incompatibility or serve some other cellular roles in pollen-pistil functions.     

Identification of residues in a hydrophilic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen.

The self-incompatibility response involves S allele-specific recognition between stigmatic S proteins and incompatible pollen. This response results in pollen inhibition. Defining the amino acid residues within the stigmatic S proteins that participate in S allele-specific inhibition of incompatible pollen is essential for the elucidation of the molecular basis of the self-incompatibility response. We have constructed mutant derivatives of the S1 protein from Papaver rhoeas by using site-directed mutagenesis and have tested their biological activity. This has enabled us to identify amino acid residues in the stigmatic S proteins of P. rhoeas that are required for S-specific inhibition of incompatible pollen. We report here the identification of several amino acid residues in the predicted hydrophilic loop 6 of the P. rhoeas stigmatic S1 protein that are involved in the inhibition of S1 pollen. Mutation of the only hypervariable amino acid, which is situated in this loop, resulted in the complete loss of ability of the S protein to inhibit S1 pollen. This clearly demonstrates that this residue plays a crucial role in pollen recognition and may also participate in defining allelic specificity. We have also established the importance of highly conserved amino acids adjacent to this hypervariable site. Our studies demonstrate that both variable and conserved amino acids in the region of the S protein corresponding to surface loop 6 are key elements that play a role in the recognition and inhibition of incompatible pollen in the pollen-pistil self-incompatibility reaction.     

S16, a novel S-RNase allele in the diploid species Solanum chacoense.

Wild potato species have a gametophytic self-incompatibility system controlled by a single multiallelic S locus. In the style, the S-RNase gene codes for an allele-specific ribonuclease that is involved in the rejection of pollen that carries the same S haplotype. This gene has 5 conserved regions (C1-C5) and highly variable regions outside of these areas that play a role in S-RNase allele specificity. In this work, PCR-mediated amplification of genomic DNA from 2 Solanum chacoense accessions was performed using primers designed on the basis of the C1 and C4 conserved regions. By sequencing the PCR products, a new S-RNase allele (S16) was identified in 1 plant of the QBCM argentinian accession. Comparison of the partial sequence (from C2 to C3) of S16 RNase with those of 11 S-RNase genes of other Solanaceae species showed the highest and the lowest similarity scores within the same plant species (respectively, 71% with the S11 and S13 RNase and 35% with the S2 RNase). Differences at the nucleotide level between S16 and S11 RNase alleles are discussed.     

Molecular cloning of two Solanum chacoense S-alleles and a hypothesis concerning their evolution

The polymerase chain reaction (PCR) has been used to clone two S-alleles (S13 and S14) from Solanum chacoense. The two alleles do not cross-hybridize on genomic Southern blots or on northern blots using stylar RNA. Although the S14 message was not detected in a stylar cDNA library prepared from mature flowers, a full-length copy of the S13 coding sequence was isolated by screening with the PCR fragment. We have analysed the sequences of the S13 cDNA and the S14 PCR fragment (60% of the mature protein coding sequence) in the context of S-RNase evolution, and propose that random point mutations may be sufficient to generate new S-alleles. Based on a phylogenetic tree composed of RNase sequences containing the conserved RNase motifs HGLWP and KHGXC, we suggest that gametophytic self-incompatibility genes are RNase genes that have acquired a new function in the gametophytic self-incompatibility system early in the evolution of flowering plants.     

Genomic organization of the S locus: Identification and characterization of genes in SLG/SRK region of S(9) haplotype of Brassica campestris (syn. rapa).

In Brassica, two self-incompatibility genes, encoding SLG (S locus glycoprotein) and SRK (S-receptor kinase), are located at the S locus and expressed in the stigma. Recent molecular analysis has revealed that the S locus is highly polymorphic and contains several genes, i.e., SLG, SRK, the as-yet-unidentified pollen S gene(s), and other linked genes. In the present study, we searched for expressed sequences in a 76-kb SLG/SRK region of the S(9) haplotype of Brassica campestris (syn. rapa) and identified 10 genes in addition to the four previously identified (SLG(9), SRK(9), SAE1, and SLL2) in this haplotype. This gene density (1 gene/5.4 kb) suggests that the S locus is embedded in a gene-rich region of the genome. The average G + C content in this region is 32.6%. An En/Spm-type transposon-like element was found downstream of SLG(9). Among the genes we identified that had not previously been found to be linked to the S locus were genes encoding a small cysteine-rich protein, a J-domain protein, and an antisilencing protein (ASF1) homologue. The small cysteine-rich protein was similar to a pollen coat protein, named PCP-A1, which had previously been shown to bind SLG.     

Sequence diversity in S1 genes and S1 translation products of 11 serotype 3 reovirus strains.

The S1 gene nucleotide sequences of 10 type 3 (T3) reovirus strains were determined and compared with the T3 prototype Dearing strain in order to study sequence diversity in strains of a single reovirus serotype and to learn more about structure-function relationships of the two S1 translation products, sigma 1 and sigma 1s. Analysis of phylogenetic trees constructed from variation in the sigma 1-encoding S1 nucleotide sequences indicated that there is no pattern of S1 gene relatedness in these strains based on host species, geographic site, or date of isolation. This suggests that reovirus strains are transmitted rapidly between host species and that T3 strains with markedly different S1 sequences circulate simultaneously. Comparison of the deduced sigma 1 amino acid sequences of the 11 T3 strains was notable for the identification of conserved and variable regions of sequence that correlate with the proposed domain organization of sigma 1 (M.L. Nibert, T.S. Dermody, and B. N. Fields, J. Virol. 64:2976-2989, 1990). Repeat patterns of apolar residues thought to be important for sigma 1 structure were conserved in all strains examined. The deduced sigma 1s amino acid sequences of the strains were more heterogeneous than the sigma 1 sequences; however, a cluster of basic residues near the amino terminus of sigma 1s was conserved. This analysis has allowed us to investigate molecular epidemiology of T3 reovirus strains and to identify conserved and variable sequence motifs in the S1 translation products, sigma 1 or sigma 1s.     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S2, encoded by the S2 allele, with the expected features of the Sp gene was identified. AhSLF-S2 is located 9 kb downstream of S2 RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S2 are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S2 is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

Self-incompatibility in Petunia inflata: isolation and characterization of cDNAs encoding three S-allele-associated proteins

Summary We have identified three alleles of the S-locus controlling self-incompatibility and their associated pistil proteins in Petunia inflata, a species that displays monofactorial gametophytic self-incompatibility. These S-allele-associated proteins (S-proteins) are pistil specific, and their levels are developmentally regulated. The amino-terminal sequences determined for the three S-proteins are highly conserved and show considerable homology to those of S-proteins from Petunia hybrida, Nicotiana alata and Lycopersicon peruvianum, three other species of the Solanaceae that also exhibit gametophytic self-incompatibility. cDNA clones encoding the three S-proteins were isolated and sequenced. Comparison of their deduced amino acid sequences reveals an average homology of 75.6%, with conserved and variable residue interspersed throughout the protein. Of the 137 conserved residues, 53 are also conserved in the N. alata S-proteins studies so far; of the 64 variable residues, 29 were identified as hypervariable based on calculation of the Similarity Index. There is only one hypervariable region of significant length, and it consists of eight consecutive hypervariable residues. This region correspond approximately to the hypervariable region HV2 identified in N. alata S-proteins. Of the two classes of N. alata S-proteins previously identified, one class exhibits greater homology to the three P. inflata S-proteins reported here than to the other class of N. alata S-proteins.     

SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco.

The S locus receptor kinase (SRK) gene is one of two S locus genes required for the self-incompatibility response in Brassica. We have identified the product of the SRK6 gene in B. oleracea stigmas and have shown that it has characteristics of an integral membrane protein. When expressed in transgenic tobacco, SRK6 is glycosylated and targeted to the plasma membrane. These results provide definitive biochemical evidence for the existence in plants of a plasma membrane-localized transmembrane protein kinase with a known cell-cell recognition function. The timing of SRK expression in stigmas follows a time course similar to that previously described for another S locus-linked gene, the S locus glycoprotein (SLG) gene, and correlates with the ability of stigmas to mount a self-incompatibility response. Based on SRK6 promoter studies, the site of gene expression overlaps with that of SLG and exhibits predominant expression in the stigmatic papillar cells. Although reporter gene studies indicated that the SRK promoter was active in pollen, SRK protein was not detected in pollen, suggesting that SRK functions as a cell surface receptor exclusively in the papillar cells of the stigma.     

Thioredoxin activity in the C terminus of Phalaris S protein

Self-incompatibility in the grass Phalaris coerulescens is controlled by two genes S and Z . Isolation and sequencing of two S alleles showed that they encode proteins that are highly conserved at the C terminus with significant homology to thioredoxin H proteins. In particular, the residues in and around the active site of thioredoxin, Trp-Cys-Gly-Pro-Cys, are perfectly conserved. The C terminus of the S protein has been expressed in Eschericia coli and purified to homogeneity on Ni-NTA resin. Functional assays showed that the protein has thioredoxin activity; it can act as a substrate for E. coli thioredoxin reductase and also catalyse the reduction of insulin by dithiothreitol. The possible role of thioredoxin-like activity of the S protein in mediating the incompatibility reaction in Phalaris is discussed.     

Identification of self-incompatibility (S-) locus linked pollen cDNA markers in Petunia inflata.

Solanaceous type self-incompatibility (SI) is controlled by a single polymorphic locus, termed the S-locus. The only gene at the S-locus that has been characterized thus far is the S-RNase gene, which controls pistil function, but not pollen function, in SI interactions between pistil and pollen. One approach to identifying additional genes (including the pollen S-gene, which controls pollen function in SI) at the S-locus and to study the structural organization of the S-locus is chromosome walking from the S-RNase gene. However, the presence of highly repetitive sequences in its flanking regions has made this approach difficult so far. Here, we used RNA differential display to identify pollen cDNAs of Petunia inflata, a self-incompatible solanaceous species, which exhibited restriction fragment length polymorphism (RFLP) for at least one of the three S-haplotypes (S1, S2, and S3) examined. We found that the genes corresponding to 10 groups of pollen cDNAs are genetically tightly linked to the S-RNase gene. These cDNA markers will expedite the mapping and cloning of the chromosomal region of the Solanaceae S-locus by providing multiple starting points.     

Isolation and characterization of the rat proenkephalin gene

The rat proenkephalin gene has been isolated by molecular cloning and characterized by DNA-sequence analysis. The gene exhibits a structural organization similar to that of the human gene. The nucleotide sequence encoding the biologically active opioid peptides which are generated from the proenkephalin precursor as well as the 3' untranslated region of the mRNA are found on a large exon at the 3' end of the gene (Exon III). The nucleotide sequence encoding the N terminus of the mature protein and its signal peptide are located on Exon II while Exon I encodes the 5' untranslated region of the mRNA. The nucleotide sequence of these exons and their flanking regions has been determined and compared to the human proenkephalin gene. Analysis of the nucleotide sequence homology between the human and rat proenkephalin gene reveals the presence of highly conserved regions within both the coding and noncoding portions of the genes. Enkephalin-coding sequences as well as 5' flanking sequences appear to be the most highly conserved. The importance and possible function of these sequences are discussed.     

S locus F-box brothers: multiple and pollen-specific F-box genes with S haplotype-specific polymorphisms in apple and Japanese pear

Although recent findings suggest that the F-box genes SFB/SLF control pollen-part S specificity in the S-RNase-based gametophytic self-incompatibility (GSI) system, how these genes operate in the system is unknown, and functional variation of pollen S genes in different species has been reported. Here, we analyzed the S locus of two species of Maloideae: apple (Malus domestica) and Japanese pear (Pyrus pyrifolia). The sequencing of a 317-kb region of the apple S9 haplotype revealed two similar F-box genes. Homologous sequences were isolated from different haplotypes of apple and Japanese pear, and they were found to be polymorphic genes derived from the S locus. Since each S haplotype contains two or three related genes, the genes were named SFBB for S locus F-box brothers. The SFBB genes are specifically expressed in pollen, and variable regions of the SFBB genes are under positive selection. In a style-specific mutant S haplotype of Japanese pear, the SFBB genes are retained. Apart from their multiplicity, SFBB genes meet the expected characteristics of pollen S. The unique multiplicity of SFBB genes as the pollen S candidate is discussed in the context of mechanistic variation in the S-RNase-based GSI system.     

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.