S-gene Product Research Articles

In silico Molecular Characterization and Phylogenetic Analyses of SARS-CoV-2 in Mediterranean Basin

Background: The novel human coronavirus disease COVID-19 has become the fifth reported pandemic since the global spread of 1918 flu and counted as the first documented coronavirus worldwide spread in the history. The COVID-19 was initially considered as a respiratory disease, but SARS-CoV-2 can lead to cause other serious complications.
 Purpose: This study aimed to conduct phylogenetic analyses of the whole genome of SARS-CoV-2 strains isolated from infected humans in Mediterranean basin countries, Orf1ab gene, S gene, M gene, N gene and Orf3a gene sequences. In addition, the products of Orf1ab, S, M and N genes were also phylogenetically analyzed. Changes that occurred on the S-gene product of these SARS-CoV-2 strains were also detected.
 Materials and Methods: The whole genome of SARS-CoV-2 isolates, the genes and the gene products (Accessed July 20, 2020) were recovered in Mediterranean basin countries were retrieved from GenBank Database previously available in National Center for Biotechnology Information (NCBI) using BLAST (Basic Local Alignment Search Tool) system. Analyses of these sequences were carried out using computer program MEGA6 software.
 Results: The Phylogenetic analyses showed that Bat coronavirus RaTG13 isolate is more closely related to SARS-CoV-2 isolates than Pangolin coronaviruse isolates. The S gene product of this virus mediates entry into the host cell and has S1/S2 cleavage site containing multibasic amino acid sequence (PRRAR) which is not detected in other closely related coronaviruses. Many coronavirus strains that deposited in GenBank, showed that they have PRR sequence in Orf1ab gene product. Conclusion: we conclude that part of multibasic S1/S2 motif acquired by recombination or insertion. Theoretically, any coronavirus strain acquired this sequence becomes highly pathogenic to humans. The dominant mutation (79.3%) at S gene product level was 614D→G. The impact of mutations detected in S gene product on virus transmission, diagnosis, pathogenicity and strategies of antiviral therapy, should be rapidly assessed in further studies.

Darwin's foundation for investigating self-incompatibility and the progress toward a physiological model for S-RNase-based SI

Charles Darwin made extensive observations of the pollination biology of a wide variety of plants. He carefully documented the consequences of self-pollination and described species that were self-sterile but that could easily be crossed with other plants of the same species. He believed that compatibility was controlled by the 'mutual action' of pollen and pistil contents. A genetic model for self-sterility was developed in the early 1900 s based on studies of the compatibility relationships among, what are now referred to as, self-incompatible (SI) Nicotiana species. Today, it is believed that SI in these species is controlled by an interaction between S-RNases produced in the pistil and F-box proteins expressed in pollen and, moreover, that this S-RNase-based SI system is shared by a great diversity of other plant species. Current research is aimed at understanding how the mutual actions of these S-gene products function in the physiological context of pollen tube growth.

Molecular mechanism for the recognition reaction in the self-incompatibility of Brassica species

Many higher plants have developed systems to prevent self-fertilization. The self-incompatibility (SI) system has been well known since the time of Darwin. In Brassica, SI is controlled by a single polymorphic locus called S. Discrimination of self and non-self pollen by the pistil should occur on the surface of the stigma. We have investigated the molecular mechanism underlying the SI system and have determined the S-gene products in pistil and pollen. The S-determinant of pollen (SP11) is a small cysteine-rich protein and is the ligand of a receptor complex in papilla cells, which is composed of S-receptor kinase (SRK) and S-locus glycoprotein (SLG). Ligand binding induces the autophosphorylation of SRK, which triggers the signaling cascade that results in the rejection of self-pollen. The ligand-receptor interaction occurs in an S-haplotype specific manner. This is the fundamental reaction of self-recognition system underlying SI in Brassica.

Crystal Structure at 1.5-Å Resolution of Pyrus pyrifolia Pistil Ribonuclease Responsible for Gametophytic Self-incompatibility

The crystal structure of the Pyrus pyrifolia pistil ribonuclease (S(3)-RNase) responsible for gametophytic self-incompatibility was determined at 1.5-A resolution. It consists of eight helices and seven beta-strands, and its folding topology is typical of RNase T(2) family enzymes. Based on a structural comparison of S(3)-RNase with RNase Rh, a fungal RNase T(2) family enzyme, the active site residues of S(3)-RNase assigned were His(33) and His(88) as catalysts and Glu(84) and Lys(87) as stabilizers of an intermediate in the transition state. Moreover, amino acid residues that constitute substrate binding sites of the two RNases could be superimposed geometrically. A hypervariable (HV) region that has an S-allele-specific sequence comprises a long loop and short alpha-helix. This region is far from the active site cleft, exposed on the molecule's surface, and positively charged. Four positively selected (PS) regions, in which the number of nonsynonymous substitutions exceeds that of synonymous ones, are located on either side of the active site cleft, and accessible to solvent. These structural features suggest that the HV or PS regions may interact with a pollen S-gene product(s) to recognize self and non-self pollen.

The 1.55 Å resolution structure of Nicotiana alata SF11-RNase associated with gametophytic self-incompatibility

The crystal structure of Nicotiana alata (ornamental tobacco) S F11-RNase, an S-allelic glycoprotein associated with gametophytic self-incompatibility, was determined by X-ray diffraction at 1.55 Å resolution. The protein has a tertiary structure typical of members of the RNase T 2 family as it consists of a variant of the (α+β) fold and has eight helices and seven strands. A heptasaccharide moiety is also present, and amino acid residues that serve as the catalytic acid and base can be assigned to His32 and His91, respectively. Two “hypervariable” regions, known as HVa and HVb, are the proposed sites of S-allele discrimination during the self-incompatibility reaction, and in the S F11-RNase these are well separated from the active site. HVa and HVb are composed of a long, positively charged loop followed by a part of an α-helix and short, negatively charged α-helix, respectively. The S F11-RNase structure shows both regions are readily accessible to the solvent and hence could participate in the process of self/non-self discrimination between the S-RNase and an unknown pollen S-gene product(s) upon pollination.

483 Identification of S-RNase in Tetraploid Sour Cherry

Most fruit tree species of Prunus exhibit gametophytic self-incompatibility, which is controlled by a single locus with multiple alleles (S-alleles). One interesting aspect of gametophytic self-incompatibility is that it commonly “breaks down” as a result of polyploidy, resulting in self-compatible individuals. This phenomenon is exhibited in the diploid sweet cherry (P. avium) and the tetraploid sour cherry (P. cerasus), in which most cultivars are self-compatible. Recently, S-gene products in pistil of Prunus species were shown to be S-RNases. As sour cherry is one Prunus species, it is likely to possess S-alleles encoding pistil S-RNases. To confirm this, we surveyed stylar extracts of 11 sour cherry cultivars, including six self-compatible and five self-incompatible cultivars, by 2D-PAGE. As expected, all 11 cultivars tested yielded glycoprotein spots similar to S-RNases of other Prunus species in terms of Mr, immunological characteristics, and N-terminal sequences. A cDNA clone encoding one of these glycoproteins was cloned from the cDNA library constructed from styles with stigmas of a self-compatible cultivar, `Erdi Botermo'. Deduced amino acid sequence from the cDNA clone contained two active sites of T2/S type RNases and five conserved regions of rosaceous S-RNases. In order to determine the inheritance of self-incompatibility and S-allele diversity in sour cherry, we conducted genomic DNA blot analysis for sour cherry germplasm collections and mapping populations in MSU using the cDNA as a probe. To date, it appears as if self-compatibility in sour cherry is not simply controlled by a self-fertile allele as demonstrated in other Prunus species.

Determining S-genotypes of Two Sweet Cherry(Prunus avium L.) Cultivars, 'Takasago(Rockport Bigarreau)' and 'Hinode(Early Purple)'.

'Takasago (Rockport Bigarreau)', one of the major early ripening cultivars of sweet cherry in Japan, was reported to be cross-incompatible with 'Hinode (Early Purple)'. Thus, they were assumed to have the same S-alleles, although their S-genotypes were unknown. However, when 'Takasago' was cross-pollinated with 'Hinode' at the Yamagata Prefectural Horticultural Experiment Station, the fruit set normally (unpublished data), which suggests that they are cross-compatible. In this study, we re-evaluated crossability between the two cultivars by controlled pollination tests and determined their S-genotypes by the molecular typing system recently developed for S-alleles of sweet cherry. The cross-pollination tests conducted at two different locations in Japan showed that 'Takasago' and 'Hinode' were self-incompatible but reciprocally cross-compatible. Furthermore, 2D-PAGE (two dimentional-polyacrylamide gel electrophoresis) analysis for stylar extracts revealed that they have none of the common S-RNases, S-gene products in styles of sweet cherry, which suggests that their S-genotypes are completely different. By S-RNase spot patterns in 2D-PAGE profiles and RFLP (restriction fragment length polymorphism) analysis, S-genotypes of 'Takasago' and 'Hinode' were proposed to be S1S6 and S2Sx(Sx is a novel S-allele different from S1 to S6), respectively. This implies that 'Hinode' is cross-compatible with all cultivars of known S-genotypes. The usefulness of biochemical and molecular methods for S-allele typing employed in this study is discussed.

Expression of a fusion protein of Pyrus pyrifolia S-RNase with glutathione-S-transferase in E. coli

S-RNase is a style-specific ribonuclease which is associated with gametophytic self-incompatibility. An expression vector of a fusion protein of Pyrus pyrifolia(Japanese pear) S3-RNase with glutathione-S-transferase (GST) was constructed and transformed into E. coli. Using this system, the fusion protein, GST-S3-RNase, was expressed as an active form and can be used for screening pollen S-gene product(s).

Efficacy of hepatitis B vaccines with and without preS2-region product in Sumo wrestlers in Japan

One hundred and twenty Sumo wrestlers in Japan received 20 μg of a hepatitis B vaccine containing 20% (w/w) preS2-region product, while the other 181 received 10 μg of the vaccine without preS2-region product, four times during 20 months. They were followed for antibody to hepatitis B surface antigen (anti-HBs) by passive hemagglutination up to 32 months after the first vaccination. The seroconversion to anti-HBs occurred more frequently in the wrestlers receiving preS2-plus vaccine than in those receiving preS2-minus vaccine at the end of observation (87% vs. 66%, P<0.001). Among the seroconverters, anti-HBs titers were higher in the wrestlers with preS2-plus than preS2-minus vaccines (geometric mean hemagglutination titer (2 N): 4.0±1.8 vs. 3.1±1.6, P<0.001). In 27, 83 and ten wrestlers of light (≤100 kg), middle (101–150 kg) and heavy (>150 kg) weight who received preS2-plus vaccine, the seroconversion occurred in 96%, 86% and 70%, respectively. In 43, 123 and 15 wrestlers of light, middle and heavy weight receiving preS2-minus vaccine, by contrast, seroconversion occurred in 81%, 60% and 67%, respectively. These results indicate that seroconversion to anti-HBs in Sumo wrestlers would be more efficient with preS2-plus than preS2-minus vaccines, and that the efficacy would be inversely correlated with the body weight. It remains to be seen whether an enhanced efficacy of the preS2-plus vaccine was due to the inclusion of preS2-region product or to the amount of S-gene product that was 1.6 times greater than in the preS2-minus vaccine.

Detection and inheritance of stylar ribonucleases associated with incompatibility alleles in apricot

Stylar proteins were surveyed by non-equilibrium pH gradient electrofocusing to identify S-RNases associated with gametophytic self-incompatibility in nine apricot cultivars. RNase activities associated with the alleles of incompatibility S 1 , S 2 , S 5 , and S 6 and with the allele of compatibility Sc were clearly identified. Two other bands that we considered related to the alleles S 3 and S 4 were unique to cultivars Sunglo and Harcot, respectively. Two generations of 17 seedlings from the cross Moniqui× Pepito and 38 from Gitano × Pepito were used to determine the inheritance of the S-RNases. Inheritance of these RNase bands followed the expected segregation ratios and the band combinations correlated perfectly with the known self-incompatibility status of the seedlings determined after self-pollination and observation of pollen tube growth. All evidence presented in this study strongly suggests that RNases are associated with gametophytic self-incompatibility of apricot and that RNases may be the S-gene products. This is the first report identifying S-RNases and describing the inheritance of these S-RNases in apricot.

A preS mutation isolated from a patient with chronic hepatitis B infection leads to virus retention and misassembly

A preS mutation derived from a patient with chronic hepatitis B virus (HBV) infection who had HBV reinfection with fibrosing cholestatic hepatitis after orthotopic liver transplantation was characterized. Sequence analysis of the HBV genome revealed two deletions and a point mutation in the regulatory CCAAT element of the S promoter. To investigate the particular preS mutation for replication competence and viral assembly in functional experiments, the mutant preS region was introduced into a replication competent HBV plasmid. Functional studies were performed by transfecting this plasmid into hepatoma cells. Analysis of the mutant HBV strain revealed an inverse ratio of S-gene products in comparison to wild-type HBV that leads to intracellular viral retention. An atypical intracellular distribution of HBV proteins and an enhanced nuclear localization of HBV DNA was also detected. Additionally, a major fraction of the extracellular viral particles was malformed. The association of intracellular accumulation of viral proteins with cirrhosis and fibrosing cholestatic hepatitis has been described recently. In this study, we show that the particular preS mutation accounted for the viral retention, which may have contributed to a more progressive form of liver disease found in this HBV-positive patient after liver transplantation. (Gastroenterology 1997 Dec;113(6):1976-82)

Mutations in the envelope gene of hepatitis B virus variants co-occurring with antibody to surface antigen in sera from patients with chronic hepatitis B.

Three clones of hepatitis B virus (HBV) DNA were propagated from sera of each of five patients with chronic hepatitis B who possessed hepatitis B surface antigen (HBsAg) and antibody to HBsAg in their serum. The clones were sequenced within the envelope gene (the preS1, preS2 regions and the S gene). Clones from four patients had various missense mutations involving codons 124-147 of the S-gene which encode amino acids in the loop structures that form the conformational, common antigenic determinant of HBsAg. Clones from three patients had Asn-130 (Gly in the wild-type), which generated a potential N-glycosylation site, Asn-Thr-Ser, spanning amino acids 130-132 of the S-gene product. In addition, clones from one patient had Arg-145 (Gly in the wild-type), which has been reported in escape mutants of HBV. One of the three clones from another patient had Ser-126 in place of lle or Thr in wild-type HBV, but the remaining two had no mutations known to affect expression of the common determinant of HBsAg. The remaining patient possessed HBsAg of subtype adr and anti-HBs specific for the w determinant. Clones from this patient did not reveal any mutations which are known to affect the common antigenic determinant of HBsAg.

The expression of a potato (Solanum tuberosum) S-RNase gene in Nicotiana tabacum pollen

Self-incompatibility in the Solanaceae is controlled by a single multiallelic genetic locus, the S locus. The stylar gene products of the S locus are abundant glycoproteins with ribonuclease activity, secreted in the transmitting tract tissue of the pistil. To investigate the structural and functional integrity and possible phenotypic effects of expression of the S-gene product in the male gametophyte, N. tabacum plants were transformed with a construct containing the genomic S 2 -RNase coding sequence from S. tuberosum under the control of the promoter of the pollen-specific LAT52 gene from tomato. The expression pattern of the S 2 RNase in the male gametophyte at both the protein and RNA level was found to be identical to that already reported for expression of the β-glucuronidase (GUS) gene directed by the LAT52 promoter in transgenic tomato and tobacco. The S 2 -RNase gene fusion led to a tissue-specific and developmentally regulated accumulation of the S 2 polypeptide in pollen of transgenic tobacco plants. The transgenic protein product was of the same size and charge as the potato stylar product, had ribonuclease activity, and was glycosylated. The transgenic plants, however, did not show any morphological variations in their flower organs, and their fertility was not influenced by the accumulation of the S 2 -RNase protein in pollen.

Expression and Inheritance of S-Proteins in Self-Compatible and -Incompatible Japanese Pears.

Expression and inheritance of style specific S2- and S4-allele associated S-proteins reported earlier (Hiratsuka et al., 1995) were studied using progenies of self-compatible 'Osa-Nijisseiki'.The S-protein bands of 'Osa-Nijisseiki', which was generated from self-incompatible 'Nijisseiki' as a mutant, were compared with those of 'Nijisseiki' by isoelectricfocusing polyacrylamide gel electrophoresis (IEF-PAGE). The protein band associated with S4sm-allele in 'Osa-Nijisseiki' was stained less densely with Coomassie Brilliant Blue (CBB) than did the band from 'Nijisseiki'; the RNase activity of the former band was also weaker than that of the latter as reported by Sassa et al. (1992, 1993). However, no difference was observed in the S2-protein band between 'Osa-Nijisseiki' and 'Nijisseiki'. The S-genotypes of the 'Osa-Nijisseiki' x self and 'Osa-Nijisseiki' x 'Nijisseiki' progenies were determined and their styles subjected to S-protein analyses. Progenies with the So4sm-gene from 'Osa-Nijisseiki' possessed a lightly stained S4-protein band, whereas those of 'Nijisseiki' had almost the same density as did 'Nijisseiki'. The S2-protein band was detected consistently in progenies carrying the S2-allele.Style specific S2- and S4-protein bands including a faint S4-band of 'Osa-Nijisseiki' were, thus, inherited with no exception to the progenies carrying respective S-alleles. These results may show that the proteins reported here are the S-gene products of Japanese pears and that of 'Osa-Nijisseiki' is a mutant which represses the S4-gene expression.

Hepatitis B surface antigen particles of subtypes adw and adr, and compound subtype (adwr) in symptom-free carriers in Japan.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p less than 0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p less than 0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p less than 0.001). Sera with HBsAg/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p less than 0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.

Molecular basis of the incompatibility mechanism in Papaver rhoeas L.

In this study we have begun to dissect the molecular mechanism of the self-incompatibility reaction of Papaver rhoeas. In order to gain an insight into the cellular activities, which lead to the inhibition of pollen tube growth following a self-incompatible response, we have been studying the effects of various metabolic inhibitors on pollen-stigmatic extract interactions in vitro. The results indicate that both transcription and glycosylation are required for the full inhibition of pollen tube growth during an incompatible response in P. rhoeas. The ability of actinomycin D to alleviate an incompatible reaction suggests that during the response pollen gene expression is induced; we have evidence that this is indeed the case and have identified novel proteins produced in the pollen which are associated with the incompatibility response. These findings give a clear indication that de novo transcription of pollen genes which are specific to this response, play an important role in the inhibition of pollen tube growth in this species. This provides a significant step towards the elucidation of the mechanism whereby pollen tube growth is arrested following an incompatible reaction in this species. Ribonuclease assays have revealed that, in contrast to the S-linked glycoprotein of Nicotiana alata, there is no detectable ribonuclease activity that correlates with the presence of the functional stigmatic S-gene product in P. rhoeas.

Gametophytic self-incompatibility inPapaver rhoeas L.

We have developed an in vitro system whereby we can reproduce the self-incompatibility (SI) reactions ofP. rhoeas in pollen grown in vitro, using stigmatic extracts. This has enabled us to investigate a number of aspects of SI, which would otherwise be difficult. On the stigma side of the reaction, the in vitro system has enabled us to characterize and partially purify the stigmatic S-component, following S-specific activity. It has also enabled us to establish that, in contrast to the S-linked glycoprotein ofNicotiana alata, no detectable ribonuclease activity correlates with the presence of the functional stigmatic S-gene product in this species. Turning to look at the pollen side, we have used the in vitro system to study the metabolic events occurring in the pollen ofP. rhoeas as a consequence of the SI reaction. We have determined that it requires both de novo glyco-sylation and RNA transcription for full inhibition of pollen-tube growth during the SI reaction. Transcription products of pollen SI response genes, which are produced specificially in an incompatible reaction, have been identified. These pollen response genes have been cloned and are currently being characterized. Since the extracellular pollen-stigma interaction results directly in gene transcription in the pollen, it seems likely that a signal transduction mechanism may be operating in the SI response. The in vitro system has allowed us to begin to investigate this possibility. We have detected rapid and transient phosphorylation of certain pollen proteins, together with changes in phosphatase activity during the SI reaction. These studies provide evidence for a role for signal transduction in the SI reaction. Thus, our in vitro system has enabled us to begin to examine, not only stigma and pollen components, but also the interaction between them in the SI reaction.

Distinct H-2-linked regulation of T-cell responses to the pre-S and S regions of the same hepatitis B surface antigen polypeptide allows circumvention of nonresponsiveness to the S region.

Recently, additional polypeptide components of the surface envelope of hepatitis B virus (HBV) have been identified. The pre-S(1) and pre-S(2) regions of the HBV genome encode NH2-terminal amino acid residues that together with the S-gene product (25 kDa) comprise polypeptides of 33 kDa and 39 kDa. The possible immunopathologic significance of these larger polypeptides and their relevance to vaccine development prompted us to examine the murine immune response to pre-S(2)-encoded determinants as compared to S-encoded determinants on the same polypeptide. Previous work showed that the pre-S(2) region elicits greater antibody production in vivo than does the S region of hepatitis B surface antigen. In this study, we examined immunogenicity of the pre-S(2) region at the T-cell level, H-2- and non-H-2-linked genetic influences on the pre-S(2) response, and the effect of the immune response to one region on the immune response to the other region. The results indicate that (i) the pre-S(2) region is significantly more immunogenic than the S region at the T-cell level; (ii) pre-S(2)-region-specific T-cell activation is regulated by H-2-linked genes and correlates with the H-2 restriction of in vivo antibody production to the pre-S(2) region; (iii) the H-2 restriction of the T-cell response to the pre-S(2) region is distinct from the H-2 restriction of the T-cell response to S-region determinants; (iv) non-H-2-linked and non-Igh-linked genes also influence the humoral immune response to the pre-S(2) region; and (v) immunization of an S-region-nonresponder, pre-S(2)-region T-cell-responder strain with HBV envelope particles containing both the pre-S(2) and S regions can circumvent nonresponsiveness to the S region through pre-S(2)-specific T-cell helper function.

Location and chemical synthesis of a pre-S gene coded immunodominant epitope of hepatitis B virus.

Immunodominant, disulfide-bond independent epitopes recognized by human antibodies to hepatitis B virus (HBV) are located within the 55-residue amino terminal portion (coded for by the pre-S region of HBV DNA) of minor HBV envelope components larger than the major protein constituents encoded by the S gene. A peptide having the sequence of the first 26 amino acids from the amino terminal methionine was synthesized and elicited antibodies (at dilutions of greater than or equal to 1 to 10(5) ) to the HBV envelope. These antibodies can be utilized for diagnostic tests. The immunogenicity of the peptide was substantially increased by covalent attachment to liposomes. The disulfide bond-independent determinants on sequences coded for by the pre-S gene may be more easily mimicked by peptide analogs than "conformational" determinants on the S-gene product.

Overcoming interspecific pollen incompatibility through the use of ionising radiation

Attempts to overcome interspecific incompatibility in Nicotiana by X-irradiation of male parents have shown a difference in response between a self-incompatible (S.I.) and a self-compatible (S.C.) species. Compatibility can be produced in the SI pollen of the former but not in the Sf pollen of the latter The observations suggest that interspecific S-gene determination may be gametophytic for the SI alleles but sporophytic in the case of the Sf allele. This sporophytic determination of the Sf allele occurs very early, possibly at the time of floral induction, or even earlier. Irradiation of floral buds, therefore, does not produce effective mutations, for by then the S-gene product is already laid down in the cytoplasm. By analogy it is suggested that in species with a sporophytically determined intraspecific incompatibility system, the S-gene products may also be laid down very early. This is in accordance with recent cytochemical observations. In SISf hybrids the difference in time and tissue of action between sporophytically active Sf and gametophytically active SI may allow the two alleles to be expressed without interaction.