1.5.5.13 n. ismfifiii ~l ? 'v. . 1.: ‘ i... Al. “ 3...} d. 3.9,: 3B . "burgh .EYIJ ._ )1. ~ 1:. , .9. 3.1.. ‘ , 3. :31 g I. z. i ) I i :n: 2. . Lurinw .13! «5...: i 35...; :0. +194.“ 2 b. I H n 1. .lhl m. ,. . A! . .111 .9: 31a- aQ'».«v). a: 11:0. ‘ \. .tl ‘0'. it! . yo. shuns ...,....... &§q§m mgaumfifimmugafigfiwg L.» UNIVERSITY LIBRARIES a ? -- Illllllll ‘llllllll l l l ll l 1.11 l 3 1293 01588 8526 This is to certify that the thesis entitled PRELIMINARY INVESTIGATION OF THE MOLECULAR COMPONENTS RESPONSIBLE FOR THE SELFZINCOMPATIBILITY RESPONSE OF SWEET CHERRY presented by Thomas S. Brettin has been accepted towards fulfillment of the requirements for M.S. . Horticulture degree in ‘7 MajWéér 07639 MS U i: an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES rain on or before duo duo. DATE DUE DATE DUE DATE DUE filfi MSU Is An Aflirmutivo Action/Equal Opportunity Institution PRELIMINARY INVESTIGATION OF THE MOLECULAR COMPONENTS RESPONSIBLE FOR THE SELF-INCOMPATIBILITY RESPONSE OF SWEET CHERRY By Thomas S. Brettin A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1996 ABSTRACT PRELIMINARY INVESTIGATION OF THE MOLECULAR COMPONENTS RESPONSIBLE FOR THE SELF-INCOMPATIBILITY RESPONSE OF SWEET CHERRY BY Thomas S. Brettin Self incompatibility (SI) in flowering plants prevents self pollination thereby promoting outcrossing. While this may be advantageous for the survival of the plant species, it presents a problem to farmers and breeders. Crops which are dependent on fruit resulting from successful pollination must rely on cross pollination. In order to better understand the molecular components of the self incompatibility response in sweet cherry (Prunus avium L.), stylar proteins were investigated using different electrophoresis protocols. Nucleic acid analysis using PCR and degenerate primers was employed to isolate DNA sequence which might code for an allele of the SI locus, which have been reported to be ribonucleases in both Rosaceae and Solanaceae families. The work presented here describes the characterization of stylar proteins and the isolation of cDNA which codes for a sweet cherry ribonuclease. To my wife Sherry and my daughter Sarah. m ACKNOWLEDGMENTS I would like to take this opportunity to thank all those who made this thesis possible. In particular, Dr. Amy Iezzoni for allowing me to work in an independent environment while providing the necessary support when it was needed. I would also like to thank Wendy Huss for her seemingly endless technical support of the research presented herein. iv TABLE OF CONTENTS LIST OF TABLES ......................................... LIST OF FIGURES ........................................ INTRODUCTION ........................................... LITERATURE REVIEW Gametophytic self incompatibility in Sweet Cherry ............................................ Gametophytic self incompatibility in the Solanaceae family ................................. Gametophytic self incompatibility in other plants. CHAPTER I PROTEIN ANALYSIS Introduction ...................................... Materials and Methods ............................. Results ........................................... Discussion ........................................ CHAPTER 2 ISOLATION OF A RIBONUCLEASE GENE FROM SWEET CHERRY Introduction ...................................... Materials and Methods ............................. Results ........................................... Discussion ........................................ APPENDIX A Protocols for SDS—PAGE ............................ APPENDIX B Protocols for amplification of recombinant DNA.... APPENDIX C Standard hybridization protocols and solutions.... APPENDIX D Yield calculations for cDNA synthesis reactions... vii viii l 25 26 28 3O 34 35 46 61 7I 73 75 77 APPENDIX E Layout for dot blots shown in figure 4A and 48.... 78 APPENDIX E DNA sequence and amino acid translations for p8 and pG clones ..................................... 79 BIBLIOGRAPHY ........................................... 97 Table Table Table Table Table Table Table LIST OF TABLES Absorbance Readings for Stylar RNA Isolations .................................. Nomenclature and Origin of pS Clones ........ Copy Number Indicated by Dot Blot Hybridization of Labeled Stylar cDNA to p8 and p6 Clones ............................ Clones Used as Probes Against Southern Blots of Different S-Genotypes .............. Summary of BLASTN on all Sequenced Clones... BLASTN Search on pSllB Second Pass Sequence Information ........................ BLASTX Search on pSllB Second Pass Sequence Information ........................ Hi 47 49 52 54 58 59 62 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES SDS PAGE of Total Style Protein from the Cultivars Napoleon (8384), Van (8183), and Ulster (S284) .............................. Two Dimensional NEPHGE of Stylar Proteins.. Overview of Methodology .................... PCR Primers Based on RNase Amino Acid Sequence Homology .......................... Amino Acid Alignment of Different S-allele Rnases ..................................... Dot Blot Analysis of p8 and pG Clones ...... Autoradiographs of pG Clones 29, 47, 110, and 115 (A-D respectively) ................. Autoradiographs of pS Clones l, 8, 9, 23, 27, 103, 141, and 149 (A—H respectively)... Nucleotide and Amino Acid Sequence of the Ribonuclease Portion of pSllB .............. Amino Acid Alignment of pS118 with Published Plant Ribonucleases .............. Wfi 29 32 36 4O 41 53 55 56 6O 63 INTRODUCTION Gametophytic self incompatibility (SI) is the most prevalent mechanism preventing self pollination in flowering plants. Gametophytic SI is commonly controlled by a single nuclear gene (S—locus) with multiple alleles. Self fertilization is prevented by inhibition of pollen tubes that have the same incompatibility genotype (S-allele) as that of the pistil on which they have germinated. Recently, molecular investigations have increased our understanding of the stylar component of the S-alleles in plants from the Solanaceae family. Using cDNA clones of the stylar S-alleles from tobacco, it has been shown that the S- allele products are glycoproteins that are secreted by cells of the stylar transmitting tissue (Cornish et al., 1987). The S-allele glycoproteins have been identified as ribonucleases which degrade the pollen tube RNA of pollen with like S-alleles (Gray et a1. ,1991). Amino acid sequence comparison between 6,3, and l S-allele(s) from Nicotiana alata, Petunia inflata, and Solanum tuberosum, reveal a highly conserved region at the amino terminus and three hypervariable regions (Haring et al., 1990). The pollen product of the S—locus is still not known. Sweet cherry (Prunus avium L.) is one of the more important agricultural crops exhibiting gametophytic SI with six S— alleles identified in breeding experiments (Way, 1967). Because cross pollination is required for fruit set, at least two cross compatible cultivars must be planted in alternate rows in orchards and bees provided as pollinators. Frequently, the two complementary cultivars have different maturity dates making harvesting and spraying less efficient. Yields are often reduced due to limited egg viability and poor pollination since bees remain in their hive when the wind velocity is greater than 25 kilometers per hour and/or when the temperature drops below 10°C. Because of the inefficiencies and yield reduction associated with growing SI sweet cherry cultivars, self compatibility is one of the most important objectives in sweet and sour cherry breeding programs. Self compatibility in sweet cherry was obtained by x-ray radiation applied to Napoleon (SbSfl flower buds at the pollen mother cell stage and subsequent selection in Sfih styles (Lewis and Crowe, 1953). Breeding experiments confirmed that there was a mutation at the S4 locus which affects only pollen activity (i.e. pollen with the mutated S4.alleles is able to fertilize plants with a normal S4.allele and not vice versa)(Lewis and Crowe, 1956). Currently sweet cherry breeders must wait until the seedlings flower (3-5 years) before determining if individual seedlings are SI by laborious controlled pollination of bagged flower clusters and subsequent analysis of fruit set or pollen tube growth in fixed styles. Because bloom may last for only 5 days, SI evaluations of large numbers of seedlings is impossible. If the S-alleles could be identified in hybrid seedlings soon after germination, it would reduce the orchard space required for the seedling populations and permit breeders to evaluate SI from a large number of seedlings. SI in sweet cherry is similar to that in the Solanaceae. The initial experiments that associated glycoproteins with stylar S-alleles were done with sweet cherry (Mau et al., 1982; Williams et al., 1982). An antigenic glycoprotein, isolated from Napoleon styles, was able to inhibit in vitro growth of self-pollen. The isolated glycoprotein contained at least two closely related major components which were suggested to correspond to the products of the two 3- alleles. More recently, northern analysis using mRNA from styles of sweet cherry probed with cDNA from a N. alata 8— allele failed to identify a homologous product (A. Clarke, pers. comm.); however, this was not unexpected because of the limited homology between different S-alleles within a species (Anderson et al., 1986). The objective of this study was to identify the glycoproteins and cDNAs corresponding to the S4.and/or S3 alleles from the sweet cherry cultivar Emperor Francis. Two approaches were used: (1) identify glycoproteins which segregated with known S-alleles [Chapter 1], and (2) design primers to conserved regions of known S-alleles and use a PCR based method to obtain RNases from both genomic DNA and stylar cDNA [Chapter 2]. LITERATURE REVIEW' Gametophytic Self Incompatibility in Sweet Cherry Commercial cultivars of Prunus avium L., sweet cherry, are unable to self pollinate (Crane and Brown, 1937). In addition to self incompatibility, many cultivars are cross incompatible. Commercially important cultivars Napoleon, Bing, Lambert, and Emperor Francis are cross incompatible with each other. Based on these cross incompatible observations, different incompatibility groups were constructed. A comprehensive listing of cultivars and their associated incompatibility group was assembled by Knight (1969). A more recent study by Tehrani and Lay (1991) investigated incompatibility grouping of sweet cherry cultivars from Vineland, Ontario. Lansari and Iezzoni (1990) have extended this analysis to the related sour cherry species Prunus cerasus. The genetic basis of self and cross incompatibilities was described by Crane and Lawrence (1929) and Crane and Brown (1937). Their findings showed that cherry incompatibility is due to a single gene (called S-gene) which prevents 6 normal pollen tube growth,thereby preventing pollination. The incompatibility response was seen when the haploid pollen carries a particular S-gene which is present in the somatic tissue of the female pistil. Self compatibility has been achieved in sweet cherry by applying x—ray radiation to Napoleon flower buds at the pollen mother cell stage and subsequent selection in Sfih styles (Lewis and Crowe 1953). Breeding experiments by Lewis and Crowe (1956) confirmed that there was a mutation in the S4 allele which affects only pollen activity (i.e. pollen with the mutated S} allele is able to escape the incompatibility mechanism when presented on a S4lallele containing pistil). The resulting self-compatible cultivar, Stella, has not lived up to commercial expectations and hence does not appear in commercial orchards. In the early 1980's, the investigation of self incompatibility in sweet cherry turned to the molecular level. As a first step, Raff and Clarke (1981) and Raff et a1. (1981) demonstrated the presence of components in the Sfih styles which were associated with this S-allele group. Both of these components were shown to be unique to the Sfih group immunologically using antiserum raised against style 7 extracts. Mau et al. (1982) began isolating and characterizing components of sweet cherry styles which might be associated with a self-incompatibility genotype using this antigenic information. Their findings consisted of two antigenic glycoproteins, one which was style specific to all Prunus species tested (Antigen P),and one which was associated with the self-incompatibility genotype (Antigen 8). The isolated glycoproteins had a substantial carbohydrate content (Antigen P 17.2%; Antigen S 16.3%),and had apparent molecular weights of 32,000 Da (Antigen P) and 37,000-39,000 Da (Antigen S). Antigen S was found to be secreted into the medium of suspension cells raised from both leaf and stem of Prunus avium. Two dimensional electrophoresis of I125 labeled Antigen S showed two components of similar molecular weight (~37,000 Da) on the alkali side of the gel. The same experiment with Antigen P indicated a single acidic component. It was eluded to that the two components of Antigen P could be alleles of the S— locus. Further studies by Williams et al. (1982) demonstrated the effect of Antigen P and Antigen S on in vitro pollen tube growth. Their findings supported the role of Antigen S in the incompatibility response. Antigen S displayed the 8 ability to inhibit pollen tube growth by 65% at a concentration of 20 ug/ml. None of the other style components tested were effective pollen tube growth inhibitors including Antigen P. Investigators of this group then turned their attention away from Prunus and began to focus on a more favorable biological system, Nicotiana alata. This marked the end of active research into the molecular components of sweet cherry self incompatibility until present day. Gametophytic Self Incompatibility in the Solanaceae Family Investigations into the molecular mechanism of gametophytic SI turned to the Solanaceae family. Different species were investigated with the most effort going into Nicotiana alata by the lab of Adrienne Clark and Petunia hybridia by the labs of Jan Vendrig and Teh-hui Kao. The following review of the Solanaceae family will consider the findings of these two species separately, although much information was obtained concurrently for these two species. Finally, I will summarize some of the smaller projects ongoing in different species in the Solanaceae family. News of progress ceased from 1982 (Williams et al.) on the Sweet cherry work until a major article emerged in Nature. Anderson et a1. (1986) reported the first successful cDNA cloning of a stylar glycoprotein associated with the self— incompatibility phenotype of Nicotiana alata. They set forth as criteria for positive identification of an S-allele that, first, its presence in reproductive tissue must be consistently associated with the corresponding breeding behavior of the plant, and second, that it should show specific biological activity under an appropriate bioassay system (for example, it should inhibit pollen tubes bearing the same S—allele, but not tubes bearing a different S- allele). In their work, they demonstrated genotypic specificity of the S-allele protein by two dimensional electrophoresis of 53 plants resulting from reciprocal crosses involving two genotypically different plants. Complete co-segregation of the S-allele protein with the observed phenotype was shown. The resulting S-allele proteins were characterized as having a molecular weight of about 32,000 Da and an isoelectric point of greater than 9.5. Subsequent amino acid sequencing of the N-terminus of the isolated protein provided degenerate DNA sequence information, which was used to identify a cDNA clone. EXpression analysis of this clone showed both temporal and 10 spatial expression patterns consistent with the timing and location of the incompatibility response. Continued pursuit of the identification of more S-alleles in N. alata lead to the identification of new alleles (Anderson et al. 1989, Jahen et al 1989a, Jahen et al. 1989b). Amino acid sequence comparison of the three existing sequences showed regions of high similarity and regions of high variability. Southern analysis showed co-segregation of the identified cDNAs with the observed phenotype. The approach to the identification of new cDNAs was essentially the same, whereas highly conserved N-terminal amino acid sequence information was used to identify the candidate cDNA clone. Jahen et al. (1989a) employed a new protein purification technique, fast liquid protein chromatography (FPLC), on stylar extracts to prepare protein for N-terminal sequencing. Biological activity was demonstrated by the development and use of an in vitro pollen tube growth assay. Although this pollen tube assay had significant improvements compared to the assay used in the early 1980’s on sweet cherry pollen, there was still some cross reactivity which was inhibiting heterologous pollen (i.e. 83 glycoprotein inhibited not only 55 pollen tubes but also 89 and 5% pollen ll tubes). Inhibition was measured in the presence of 300 to 600 ug/ml of the appropriate S—glycoprotein. At this time, interest was mounting on the possibility that the N—linked glycans on the S—glycoproteins could be the variable component that conferred allele specificity. The methods were in place to isolate S-glycoproteins in pure form (Jahen et al. 1989b). Woodward et al. (1989) underwent investigations to characterize the glycan chains of five different N. alata S-glycoproteins. In summary, their results were indicative of heterogeneity in the structure of the glycan chains. The authors go on to recognize that if allelic specificity were to reside in the fine structure of the glycan chains attached to the protein, then an additional controlling factor could play a role in determining this fine structure, and for genetic reasons, would have to be tightly linked to the S-locus. Alternatively, if the allele specificity of the S— glycoprotein resides in the position of the glycan chains relative to the protein, the order and number of glycosolation sites within the protein would be the determining factor. 12 Progress on the biochemical mechanism of self incompatibility took a large step forward with the discovery of a ribonuclease activity present in the S-glycoproteins. Sequence analysis had shown that three alleles of the S—gene of N. alata (Anderson et al. 1986, Anderson et a1. 1989) encode style glycoproteins with regions of defined homology. Two of those homologous regions displayed precise homology with RNase T2 and RNase Rh. McClure et al. (1989) showed that five known S—alleles (Eh, Eb, 85, Se, and S7) not only contained the region of homology to RNase T2 and RNase Rh, but also displayed ribonuclease activity when assayed as described by Brown and Ho (1986). Soon after the reports that isolated S-alleles had an associated ribonuclease activity, McClure et al. (1990) hypothesized ribonuclease activity as the biochemical process by which pollen tube growth is arrested. This work was based on in Vivo observations that P32 labeled rRNA isolated from styles was degraded when incompatible pollen was present and the same labeled rRNA was not degraded when compatible pollen was present. These observations lead to the proposal of a model in which the gametophytic self incompatibility system in N. alata acts through a cytotoxic mechanism directed against pollen RNA. 13 The action of the ribonuclease activity of isolated S- proteins on pollen tube growth was investigated by Gray et al. (1991). Their results indicated that the selectivity of the degradation was due to selective uptake of S-protein by the pollen tube and not due to selective degradation of RNA. S—proteins inhibited in vitro translation of pollen tube RNA in a non-specific manner in wheat germ cell free extracts. A particularly interesting finding of this study, and perhaps unexpected, was that heat treating Eb-RNase largely destroyed the ribonuclease activity of the protein, but did not reduce the inhibitory effect on in vitro pollen tube growth. Heat treated SQ—RNase accumulation on the outer surface of the pollen grains was greatly increased compared to non-heat treated SQ—RNase. It was suggested that this increased accumulation was responsible for in vitro pollen tube growth inhibition, and that the in vitro and in Vivo systems needed to be compared with caution. This concern was certainly valid since earlier in vitro experiments had shown cross reactivity of some S—proteins. Early attempts to create a transgenic model focused on transforming Nicotiana tabacum with both N. alata S} cDNA and genomic DNA (Murfett et a1. 1992). The hope was that N. tabaccum would express the transgene in both pollen and 14 pistil, hence creating an incompatibility mechanism in an otherwise self-compatible plant. Transgene expression was obtained in the pistil before, during, and after anthesis, and transgene expression was detected in anthers prior to anthesis. Expression levels in the transgenic plant were significantly lower than expression levels in transgene donor (approximately 100 fold lower). There was no observed change in the compatibility type of the transgenic N. tabaccum. This result was presumably due to the inability to reach wildtype expression of the S} allele in N. tabaccum. This summarizes the vast progress made by Adrianne Clark and her associates in understanding the stylar gametophytic SI gene in N. alata. Work done by these investigators opened the door to molecular SI investigations in other members of the Solanacea family such as Petunia inflata, Lycopersicon peruvanium, and Solanum tuberosum. In 1990, publications from the lab of Teh-hui Kao reported the isolation of previously unidentified S-alleles of N. alata. Kheyr—Pour et al. (1990) identified four alleles, Eh, 33,.3n1, and Sz.and compared the deduced amino acid sequence of these and three previously identified S- 15 proteins. Their findings revealed 53.8% homology among the seven proteins. There were 60 conserved residues, including 9 cystines, and 144 variable residues. Of the 144 variable residues, 50 were identified as hypervariable based on their Similarity Indices. The conserved and hypervariable residues could be clustered into five regions. It was this observation that led to the hypothesis that, since the hypervariable regions accounted for most of the inter- sequence variability, these hypervariable regions could be responsible for the allelic specificity. A year earlier, Broothaerts et al. (1989) identified the first S-proteins of P. inflata (Sh Eb, and SS). The proteins were isolated by a combination of ConA—Sepharose and cation exchange FPLC. These proteins were then checked by co-segregation analysis. The N—terminal amino acids were determined and compared to those of S-proteins from N. alata. Homology in this region led to the hypothesis of a common cellular membrane transport peptide for N. alata and P. inflata. These new Petunia S-proteins differed slightly in molecular weights (Si 27000, 32 33000, and S3 30000 Da) and isoelectric points (8.7, 8.9, and 9.3 respectively). The distribution of these proteins in the pistil showed the proteins were present in high concentration in the style and 16 the uppermost part of the stigma. Time course analysis showed an increase in the amount of the proteins during flower maturation. These observations were consistent with earlier findings in N. alata. Finally, these S—proteins were shown to have ribonuclease activity (Broothaerts et al., 1991) and that this ribonuclease activity was still present after deglycosylation of the S-protein with the enzyme peptide—N—glucosidase F (PNGaseF). This suggested that the role of the glycan moieties was other than the ribonuclease function. Identification of the cDNA clones representing the SI, Sm and S3ealleles of P. inflata (Ai et al., 1990) was accomplished using the 53 cDNA clone from N. alata as a probe against stylar cDNA libraries of P. inflata. This was the first successful cross species hybridization experiment. Spatial and temporal expression was consistent with previous studies. Ai et al. (1992) approached the question of self incompatibility by investigating a self-compatible cultivar Of P. hybridia. They cloned two S-allele-like cDNAs which displayed all the previously mentioned characteristics of an S-allele. Homology in the protein N-terminus with previous l7 S-protiens was identified. These S—proteins had ribonuclease activity, and spatial and temporal expression was consistent. The investigators elude to the disruption of the self recognition mechanism as the cause of the self compatible phenotype. Genomic sequences of the flanking regions of two P. inflata S—alleles (Coleman and Kao, 1992) were cloned and shown to contain heterogeneous and repetitive sequences. They reported that the sequence diversity in the flanking region increased to the point that interallelic homology completely disappears. This lack of homology lead Coleman and Kao to conclude that the two alleles have evolved independently as the result of an unknown mechanism which suppresses recombination in this region of the chromosome. Much of the discussion up to now was about the isolation and characterization of S-proteins and their corresponding cDNAs. These experiments were all based on co-segregation analysis of the protein or cDNA with the corresponding phenotype. This type of analysis does not exclude linkage disequilibrium, and thus does not provide definitive proof for the phenotypic effect of these proteins. 18 In the largest breakthrough since the first cloning of a S- allele cDNA, Lee et al. (1994) showed definitively that 3- proteins control rejection of incompatible pollen in P. inflata. Experiments using both loss-of—function and gain— of—function approaches to ascertain whether previously identified P. inflata S-proteins were responsible for self incompatibility interactions between the pistil and pollen showed in fact that this was the case. Loss-of—function was observed when S3 antisense constructs were used to transform plants of the Sfiiggenotype. Subsequent pollination of the transgenic plants showed that 5% pollen was no longer rejected. This breakdown in the incompatibility response was concordant with the disappearance of'Eb mRNA and the S3 glycoprotein. The gain-of—function experiments were based on the transformation of an Sfih plant with a S3cmmstruct and subsequent pollination tests to ascertain the viability of 53 pollen on the transgenic pistils. As expected, the transgenic plant rejected S3gxfl1en. When self pollination was attempted at the immature bud stage, these transgenic plants set fruit. Thus, their failure to set fruits when pollinated with S“ Eb, and 53pmfllen was a true self- 19 incompatible response, and not due to female sterility resulting from tissue culture manipulation. In conclusion, the results of the transgenic experiments provided direct evidence in Vivo that the S-proteins of P. inflata were necessary and sufficient for the pistil to reject self pollen. This discussion has focused on the work done in N. alata and P. inflata. The former provided the scientific community with the first cDNA clone of a S-allele, the later provided the definitive proof that these S-alleles were responsible for self incompatibility. Work in other species of the Solanacea family includes cloning of S-alleles in Solanum chacoense (Xu et al., 1990), cloning of a S-allele in LYCOpersicon esculentum (Chung et al., 1993), and investigations of a self compatible mutation in Solanum tuberosum (Thompson et al., 1991). Gametophytic Self Incompatibility in Other Plants POppy, Papaver rhoeas, displays classical genetic gametophytic self incompatibility. Franklin-Tong et al. (1991) examined poppy stylar proteins and were unable to find any evidence for the involvement of a ribonuclease in 20 the self incompatibility response. Their conclusions were based on two major experiments. The first was a developmental expression analysis of the pistil for ribonuclease activity during the maturation of the pistil. As noted earlier, the level of expression of ribonuclease activity increases with the maturation of the pistil in N. alata and P. inflata, reaching a maximum when pollination occurs naturally. This was not the case with P. rhoeas. Franklin-Tong et al. (1991) found no significant difference in the level of expression during pistil maturation. The second argument against ribonuclease involvement was based on pollen inhibition assays. They tested protein fractions from the pistil known to contain biologically active inhibitors of in vitro pollen tube growth for ribonuclease activity. Those fractions which displayed the greatest ability to inhibit pollen tube growth in vitro contained no significant amounts of ribonuclease activity. The authors point out that one notable difference between P. rhoeas and N. alata is the site of pollen inhibition. In P. rhoeas, the site of inhibition is on the stigma, while in N. alata, the site of inhibition is the style. Hence, while genetically similar to the gametophytic incompatibility system of N. alata, this system resembles the sporophytic 21 incompatibility system of Brassica oleracea in that the site of inhibition is on the stigma. Self-incompatibility related ribonucleases have been identified in the styles of Pyrus serotina (Japanese Pear) by Sassa et al. (1992). In their work, isoelectric focusing of style protein extracts on polyacrylamide gels, followed by ribonuclease activity staining of the gel, revealed basic ribonuclease isoforms which correlated with the known S- genotype. These basic RNase isoforms had a pI between 9.7 and 10.6. In a self-compatible mutant, no ribonuclease activity was observed in the basic region of the gel, indicating further support for the role of ribonucleases in self incompatibility. Work in NBlus domestica, another species which displays gametophytic self incompatibility, has demonstrated the specific inhibition of pollen germination and tube growth can be achieved in vitro using fractionated style protein extracts. Speranza and Calzoni (1990) showed that two style protein fractions obtained by ConA—Sepharose 48 column fractionation could introduce specific inhibition to the in vitro bioassay. These two fractions differed in that only one fraction displayed specific inhibition of pollen 22 germination while both fractions conferred specific inhibition of pollen tube growth. Identification of self-incompatibility related glycoproteins (5%, Sb, SC,.&y EL, and Sf) in Malus domestica was done using two dimensional electrophoresis. Sassa et al. (1994) showed that style glycoproteins associated with self incompatibility could be resolved on a two dimensional polyacrylamide gel. These glycoproteins also cross reacted with antiserum raised against the S} glycoprotein of Pyrus serotina. Molecular weight determinations were estimated between 27 and 30 kD. Isolation of cDNA clones for Malus domestica self- incompatibility alleles produced clones for the Sgand S3 alleles of the cultivar Golden Delicious (Broothaerts et al., 1995). Genomic DNA fragments representing part of the S—gene were amplified by PCR using degenerate primers. These degenerate primers were based on amino acid alignments of the known S—alleles of the Solanacea family. The amplified genomic fragments were used to screen a pistil specific cDNA library. Two pistil specific clones were identified, and analysis of their occurrence in a series of 23 apple varieties with defined S-phenotypes led to the conclusion that they corresponded to the Sgand.S3 alleles. Database searching now reveals that additional.Malus domestica S-alleles have been isolated. The S5,.Sn Eb, and a S-like sequences have been submitted to GenBank (Broothaerts et al., In press 1997). Finally, this discussion should not be completely limited to self incompatible plant species. Stigmatic ribonucleases have been identified in the self compatible species Prunus persica (Roiz and Shoseyov, 1995). Experiments showed that the stigmatic ribonuclease was developmentally regulated, reaching maximum activity at the beginning of anthesis. Both the stigmatic ribonuclease and pancreatic ribonuclease A significantly inhibited pollen tube growth in an in vitro bioassay. The inhibitory effect of these ribonucleases could be eliminated by the addition of RNA as a competitive inhibitor in the bioassay. The significance of these findings is unclear, although it appears that the stigmatic ribonuclease plays a role in pollination. In the self-compatible species Arabadopsis thaliana, three genes with homology to S-ribonucleases have been identified (Taylor and Green, 1991). These ribonucleases showed significant homology with known S-alleles. The functional 24 role of these Rnases in Arabadopsis organism is yet unknown, but their presence defines a broader class of plant ribonucleases. Chapter 1 ANALYSIS OF SWEET CHERRY STYLAR PROTEINS Introduction Glycoproteins associated with S—alleles (S-glycoproteins) were first reported in sweet cherry (Mau et al., 1982; Williams et al., 1982). These isolated glycoproteins were shown by two dimensional electrophoresis to contain two components which were suggested to be the products of the two S-alleles. These proteins had approximate isoelectric points of 10.6. S-allele proteins with similar properties were isolated from Solanaceous crops Nicotiana alata, Petunia inflata, Solanum chacoense, and Lypcopersicon peruvanium and were shown to encode ribonucleases (McClure et al., 1989, Singh et al., 1991, Xu et al., 1990, Chung et al., 1993). In the Rosaceae, identification of six S—proteins by two dimensional electrophoresis in.Malus domestica (Sassa et al., 1994) showed that these S-proteins had physical properties similiar to the S-proteins of Solanaceous crops 25 26 (M.W. 5 30,000 Da and basic pI). In Japanese pear, S-allele related basic RNases were identified in styles by isoelectric focusing (IEF) and staining for RNase activity (Sassa et al., 1992), again showing similiar physical properties to the Solanaceous species. The objective was to determine if proteins with similar characteristics as the S-allele associated proteins could be identified in sweet cherry styles using genotypes differing in their S-alleles. If S-allele associated proteins were identified, a long term goal would be to obtain N-terminal sequence information for comparison with other S-allele sequences. Materials and Methods Plant material: Sweet cherry cultivars representing three different S-allele genotypes were used: Napoleon or Emperor Francis (EbS4), Ulster (5&84), and Van (£hS3). Protein extraction: Styles were collected from field grown trees when the flowers were in the balloon stage (one day prior to anthesis). This involved removing the ovary with a razor blade. PAGE analyses were performed on concentrated soluble stylar proteins. Approximately 20 styles per 27 cultivar were ground in liquid nitrogen. Then, 2 mls of 0.1 M Tris, 0.005 M EDTA , 0.014M B-mercaptoethnaol, and 2%(w/v) PVPP was added and the mixture was centrifuged at 30,000 g for 20 minutes. Proteins were then precipitated from the supernatant by the addition of acetone to a final concentration of 80%. The protein was resuspended in sample buffer (0.0675M tris-HCl pH 6.8, 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol) and stored at -80° C or used immediately for electrophoresis. Protein quantification: The amount of protein needed for PAGE analysis was determined empirically by loading and running varying amounts of protein extract on a PAGE gel followed by staining with 1% coomassie blue R-250. SDS-PAGE: SDS—PAGE was performed as described by Laemmli (1970). The proteins were detected by staining with Coomassie blue. SDS-PAGE was subsequently performed according to Blank et al. (1982) using proteins where beta- mercaptoethanol was omitted from the extraction buffer and sample loading buffer as experiments (data not shown) showed that B-mercaptoethanol had an inhibitory effect on ribonuclease activity in the staining assays. The gels were stained for RNase activity using the procedure of Blank et 28 al. (1982) with the addition of 2 uM ZnClz to the gel wash buffer (Christy Howard, per. comm.). For a detailed Rnase staining procedure, see Appendix A. NEPHGE: Two-dimensional nonequilibrium pH gradient electrophoresis (NEPHGE) was performed on the pistil proteins of the cultivar Napoleon using the extraction procedure described above, except that the acetone precipitated proteins were resuspended in IEF loading buffer (O'Farrel et al., 1977). Gels were stained using a silver staining kit (Sigma Chemical Co., St. Louis, MO) and then dried. Results SDS-PAGE of stylar proteins of three cultivars are shown in Figure 1.1. The most abundant protein had a size of 29 kDa. The broad size of the 29 kDa band suggests that it may represent more than one protein with slightly different mobilites. Napoleon and Ulster, which share the S4.allele, appear to have a component of the ‘29 kD band' that migrates faster than Van, which does not contain the S4.allele. RNase staining of Emperor Francis stylar proteins separated by SDS-PAGE identified 4 different regions of Rnase activity. The different areas resolved after different 29 V (Y) Leaf RNA cDNA synthesis 1 Style RNA Genomic DNA PCR amplification Leaf cDNA Style cDNA 1 Library PCR Construction Amplification with RNase Primers Clone Products Immobilize recombinants Southern Analysis on Membrane using different I genotypes +\- screening using style and leaf cDNA as probes % Identify style specific clones -—>Sequence analysis Figure 2.1 Overview of Methodology. 37 freeze dried and stored at -80° C for DNA isolations, or stored under liquid nitrogen without freeze drying for RNA extraction. Styles from EF were collected from branches that had been cut from field-grown trees one to two days prior to anthesis. Whole styles and stigmas (subsequently referred to as styles) were separated with a razor blade from the ovary of those flowers which were in the late balloon stage. The styles were immediately placed in liquid nitrogen until RNA extraction was performed. The different sweet cherry genotypes used for the Southern analyses were the cultivars Van (Sfi%), Ranier (Sfih), Napoleon (Sfifl), Emperor Francis (Sfih) and Schmidt (Sfi%). Leaves from these cultivars were collected in the spring from field grown trees. Leaves were collected, transported to the laboratory on dry ice, freeze dried, and stored at -20° C until DNA extraction was performed. Nucleic Acid Isolations: DNA was isolated from the freeze dried leaves using the method of Stockinger et a1. (1996) and checked on a 1% agarose gel in 0.5x TBE for degradation. RNA was isolated from approximately 1 gm of leaf and stylar tissue by the method of Manning (1991) with the following modifications. For stylar tissue, four phenol chloroform 38 isoamylalcohol (25:24:l) extractions were performed due to the high ribonuclease levels in the styles. For both leaf and stylar extractions, the [Na+] in the first butoxyethanol precipitation was adjusted to 100 mM. An extra differential precipitation was performed to further reduce the absorbance at 230 nm. RNA quality was checked by electrophoresis in a 1% agarose gel in 0.5x TBE. Purity of the RNA and DNA preparations were checked by absorbance readings at 230 nm, 260 nm, 280 nm and 310 nm. cDNA synthesis: PCR amplified leaf and style cDNA was prepared according to Jepson et al. (1991) using a cDNA synthesis kit (Boehringer Mannheim, Indianapolis, IN). Two mg of total RNA was used in a cDNA reaction with dTls primers. After second strand cDNA synthesis, EcoRI linkers were ligated to the size-selected cDNA (greater than 500bp), unligated linkers were removed using Microcon concentrators (Amicon, Beverly, MA). The cDNA was then subjected to 30 cycles of PCR using primers specific to the linkers. Leaf cDNA was used as a probe on dot blots, while stylar cDNA was used as a probe on dot blots, as template in PCR amplification with RNase primers, and library construction. 39 Stylar cDNA library: Ligated stylar cDNA in lambda gth was packaged, transformed, and plated using the Packagene in vitro packaging system (Promega, Madison, WI) following the manufacture's protocol. The quality of the library was checked by hybridizing pRE12, a ribosomal clone which spans both the 18S and 258 rRNA coding regions (Delseny et al., 1983), to plague lifts from three plates containing approximately 4,500 plaques per plate. Labeling of the probe was done with the Radprime labeling kit (Gibco BRL, Gaithersburg, MD) and hybridization conditions were as described in Appendix C. This library was intended to be used to obtain a full length clone of putative RNases identified following PCR amplification. RNase primers: Primers AI-l and AI-2 (Figure 2.2) were designed based on S-allele and RNases amino acid sequence homology (Figure 2.3) (T.H. Kao personal communication). The AI primers were synthesized at the MSU Macromolecular Structure Facility. A second set of primers, PG-30 and PG- 31, also designed to amplify RNases, were obtained from P. Green (Taylor and Green, 1991)(Figure 2.2). For both primer sets, the approximate number of bases between and including the two primer pairs according to the sequence alignment in Figure 2 is 180 bp. The AI and PG primers were used to 40 Primer AI-la V’ I H G L ‘W P 5'- ATN CAT GGN CTN TGG CC -3' C T Primer AI—2b S C C T G H K 5" CA ACA NGT NCC ATG TTT -3' GT G G C Primer PG—30C H G L ‘W P D 5'- GAATTCAT GGN TTN TGG CCN GA -3’ EcorI Primer PG-31 Y T G H K ‘W’ E 5’- CTCGAGT NCC ATG TTT TTT ATA TTC ~3’ XhOI G C AAC GC C G C i5 Lambda 2 High <5 3 p5085 Low <5 Lambda 3 High <5 pleB Low <5 1 Lambda 4 High <5 psll6 Low <5 1 pg29 Low <5 p8118 Low <5 pg47 Low <5 psl23 Medium <5 pgllO Low >5 p5134 Low <5 pg115 High <5 psl40 Low <5 1 pg293 High >5 psl4l ? <5 psOOl Low <5 psl42 Low <5 1 psOOB Medium <5 psl43 Low <5 1 psOO9 Low <5 psl45 Low <5 psOlB Medium <5 psl49 Medium >5 psO23 Low <5 pslSO Low <5 1 p8027 Low <5 p5155 Low <5 1 p5028 Low <5 psl6l Low <5 1 psO33 Low <5 psl64 Medium >5 psO41 Low <5 psl68 Low <5 a - Copy number is based on signal intensity on the membrane. b — Group refers to those clones which gave identical hybridization patterns. SS A . B . HindIII EcoRI EcoRI HindIII m cn a: m m m m m U: m m m m m m m U) m m m m to w l—‘ w H M Lin) H w l-‘ N I-‘ LL) N H N H (A OJ N H N U) U) U) U) U) U) U) (I) U) U) U) (I) U) U) U) U) U) U) U) U] U) U) A L») d:- A U1 A I») pl) .5 U1 .5 b A U) (A .5 b .5 .1) LA) h) A I. 4...: ,. , ‘3 {Nusmumtt 47:1“ y - 42* ._‘.:“j "*1“ "1W; ‘; ' ‘ . . ‘ m ; 1.4 H 9959““ ‘. u] ‘ J. as m "at ‘ i ‘1 mu ; Ml”: * 74’?) C D HindIII EcoRI HindIII EcoRI U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) (A I-‘ (a) l—| F.) U l—' h) l—‘I N (A) I—‘ (J I—-‘ l\) OJ H (a) H N mmwmmmmmmm mmmmmmmwmm uh (.0 .11 uh :5 La) A :5 L]! A (A) A A (I) H3 (A) .5 sh (II was we)» .~ ~ r... 1,, 99,, if". «my. tar W ~‘ :1.» {R T.» .5: M“ Audit ital-:3- (Hi In,» 33;... “' Iii)? in?” iii? iii . . mm rug)" marl: warm artiste) Figure 2.5 — Autoradiographs of pG Clones 29, 47, 110, and 115 (A—D respectively). Arrows on C (pG110) indicate the S3 specific band. 56 EcoRI HindIII EcoRI HindIII Mmmm mHMA mwM¢ mHmw mwmA mmmw MHM¢ mum» mumm mmwn mmmm mHm» mum» wwmw mwma mmmm MHmA mum» mHmw mum» ‘tmln‘ my 113;)“ uws. fir Ci: ‘. . . mi 5») y. Mitt),=¢ lit) 4 EcoRI HindIII D EcoRI indIII H mmmm mwm» mwmn MHmw mwmb mmmm mHmn mwmb mHmw mwmb 9, 23, 27, (A-H respectively - see next page for E— 8, 6 - Autoradiographs of pS Clones 1, 2. 141, Figure 103, H). and 149 57 I F HindIII EcoRI HindIII EcoRI m m m m m m m m m w w m m m m m m m m m (A) H w H N b) H H N (.40 H LA) H N w H LA.) H N m m m m m m m m m m m m m m m m m m m m .5 w .5 «a U1 a w a: .5: U1 J: u A .5 uu .> L.) A A w grim !‘ g f #- v u «a 5 7““ “it fir-u If”. ww1mW-Wfi‘mfiWmfl‘hfl,mflyfiw “a #3 w v” m ;. , ‘ WWW“ fiflfiflm . H . HindIII EcoRI HindIII EcoRI U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) (A) H (A) H N (A) H H N (A) b) H N (A) H U H N U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) U) (I) U) as w A A u: a: w h .5 Ln .5 w A b Ln .1: w .5 A U1 Figure 2.6 - Autoradiographs of pS Clones 1, 8, 9, 23, 27, 103, 141, and 149 (A-H respectively). 58 Sequencing of the p8 and p6 clones: Five of the six pG clones and four of the low copy pS clones were sequenced to see if they had homology to known ribonucleases. pGllO was not sequenced due to the size of the insert and the inability to reproduce the Southern analysis results. Only two of the 10 clones sequenced had significant homology with sequences in the non—redundant Genbank sequence database when searching using BLASTN (Table 2.5). pGll5 likely contains an 18S rDNA sequence. This is in agreement with the results from the Southern analysis that pGllS is Table 2.5 — Summary of BLASTN on all sequenced clones. Clone Sequence producing Highest-scoring Segment Pairsd P(N)L pSlleT Transcribed sequence-clone YAPSOl; A. thaliana 7.8 e-05 Ribonuclease (RN81) mRNA, A. thaliana 9.8 e-05 S—like RNase; Malus domestica 0.00044 Wounding-induced ribonuclease; Zinnia elegans 0.0038 Ribonuclease mRNA; Zinnia elegans 0.099 pG115r Mitochondrial 188 rRNA; Wheat 4.1 e-70 Mitochondrial 188 rRNA; Maize 4.2 e—70 Mitochondrion fMET, 185, SS repeat; T. aestivum 6.0 e-70 185 rRNA; Soybean 6.7 e-70 58 and 188 rRNA; L. luteus 1.2 e-69 pG115f Mitochondrial 18S rRNA & 58 rRNA; O. berteriana 1.1 e-06 SS and 188 rRNA; L. luteus 0.00012 Mitochondrial atp6, 5' region; Nicotiana tabacum 0.00086 Mitochondrial atpA, 5' region; 0. berteriana 0.001 188 rRNA gene; Soybean 0.0017 pG207r Ig germline kappa b5 chain, JC-region; Rabbit 0.48 a - Databases searched were Non—redundant GenBank, EMBL, DDBJ, and PDB. b - The first 5 hits in the database which had a Smallest Sum Probability of less than 0.5 are listed. c - The suffix f or r on the clone name refers to the forward or reverse sequencing reaction. 59 represented in high copy in the cherry genome (Table 2.4). pS118 had significant homology to known RNase sequences which prompted us to resequence the clone in both the forward and reverse directions. Table 2.6 shows the new P— values and database hits for pS118 based on 420 and 475 bases of good sequence data in two separate forward sequencing reactions and 410 bases of good sequence data in a reverse sequencing reaction. Table 2.6 — BLASTN search on p8118 second pass sequence information. Sequences producing High-scoring Segment Pairs) P(N)b Lycopersicon esculentum mRNA for ribonuclease le 1.5 e—14 Zinnia elegans ribonuclease mRNA 1.1 e-13 Nicotiana alata RNase NE mRNA 1.8 e-ll Malus domestica S—like RNase gene 0.00056 Arabidopsis thaliana ribonuclease (RN51) mRNA 0.013 Zinnia elegans wounding induced ribonuclease mRNA 0.088 a - Databases searched were Non-redundant GenBank, EMBL, DDBJ, and PDB sequences. b — Hits with P(N) > 0.05 are not shown. Identification of the AI primer sequences indicated that pSllB was chimeric. The sequence for the AI—l primer started at position 316 with one base missing. The bases (AAGCACGGGACT) immediately before the AI-l primer sequence represent complementary sequence to the primer AI—Z. Therefore, pSllB resulted from the ligation of two insert DNAs prior to ligation into the vector. The first base of the AI-l primer and the 5' end of the AI-Z primer is not 60 present due to treatment of the PCR products with T4 DNA polymerase prior to ligation. The pSllB region of nucleotide homology with known RNases extended from position 321 of the insert to position 414 (for complete sequence of the chimeric insert of pSllB, see Appendix F). Full sequence information for the RNase sequence of the insert is presented in Figure 2.7. The missing cytosine in the AI primer sequence is identified. Internal primer sequences were identified which could be used to amplify to RNase portion of pSllS for future experiments. V’ l 5'GCATGGTTGTGGCCTAATTATAAGGATGGCTCCTACCCATCTAACTGTGATCCCGATAGT A W L W P N Y K D G S Y P S N C D P D S H G 61 CTCTTCGACAAATCTGAGATCTCAGAGCTAATGAGCAACCTGGAAAAGAACTGGCCGTCA L F D K S E I S E L M S N L E K N W P S 121 CTAAGCTGCCCAAGCAGCAATGGGTTCAGGTTCTGGTCCCATGRATGGGAAAAGCACGGA L S C P S S N G F R F W S H E W E K H G 181 ACATGC 3' T C Figure 2.7 — Nucleotide and amino acid sequence of the ribonuclease portion of pSllB. Bold sequence represents degenerate AI-l and AI-2 primer sequences. Underlined sequence represents internal primers to amplify pS118 DNA for probes in Southern and Northern experiments. Asterisks represent internal conserved amino—acid sequence with 22 known S-alleles and 2 fungal ribonucleases. The'V'narks the spot where a cytosine residue is missing when compared to primer sequence. 61 The BLASTX search of amino acid homology with the pSllB supported the conclusion that pSll8 contains an RNase sequence (Table 2.7). The results presented in Table 2.7 were obtained without correcting for the missing base in the AI—l primer region. The missing base caused two conserved amino acids (H and G) to be shifted out of reading frame; therefore, these two conserved amino acids did not contribute to the P(N) values. Amino acid sequence alingment with known plant ribonucleases revealed 13 conserved amino acid residues (Figure 2.8) when the sequence is corrected for the missing base in the AI-l primer region. Discussion Isolation of intact mRNA was successful from sweet cherry leaf tissue as demonstrated by first stand leaf cDNA synthesis. However, RNA isolation from stylar tissue was problematic presumably due to the abundance of RNases present in the style (see Chapter 1). It is likely that extremely high quality RNA was not obtained from stylar tissue in the course of the project. It seems that the 2- butoxyethanol precipitations are required for the removal of polysaccarides from nucleic acids. The use of a guanidium based extraction buffer to eliminate RNase activity during the course of RNA isolation (Manning, 1991) was not 62 Table 2.7 - BLASTX Search on p8118 Second Pass Sequence Information. Sequences producing High—scoring Segment Pairs3 P(N)b Ribonuclease LE (RNase LE); starvation induced; tomato 4.6 e-27 Ribonuclease LX (RNase LX); starvation induced; tomato 5.5 e-27 Ribonuclease 3 precursor; Arabadopsis thaliana 1.2 e—26 Ribonuclease; Solanum lycopersicum 1.5 e—26 Ribonuclease; Lyc0persicon esculentum 1.7 e—26 S-like RNase; Malus domestica 1.7 e-26 RNase NE; Nicotiana alata 1.9 e-25 Ribonuclease 1 precursor; Arabadopsis thaliana 2.6 e-25 Ribonuclease; Zinnia elegans 1.4 e—23 Ribonuclease; wounding induced; Zinnia elegans 1.5 e-23 Extracellular ribonuclease; Arabidopsis thaliana 3.6 e-18 S-like ribonuclease; Arabidopsis thaliana 5.6 e—18 Ribonuclease 2 precursor; Arabidopsis thaliana 1.2 e-17 Storage protein; Nelumbo nucifera 8.0 e-13 Ribonuclease TRV; Trichoderma viride 1.3 e-09 Ribonuclease T2; Aspergillus oryzae 2.0 e-09 Ribonuclease M; Aspergillus phoenicis 5.0 e—09 Ribonuclease; Momordica charantia 1.5 e-08 Ribonuclease; Physarum polycephalum 1.8 e—08 Ribonuclease; Oyster 1.0 e—07 Ribonuclease X25; Drosophila melanogaster 3.9 e—06 RNase Rh precursor; Rhizopus niveus 5.9 e—06 SI glycoprotein (allele S6); Lycopersicon peruvianum 5.6 e-05 S—allele-associated protein So precursor; Petunis hybrida 6.2 e-05 Ribonuclease LE2; Lentinus edodes 0.00018 S-13 RNase; Lycopersicon peruvianum 0.0011 Self-incompatibility ribonuclease; Solanum carolinense 0.0028 SI glycoprotein (allele S7); Lycopersicon peruvianum 0.0063 S-RNase; Physalis crassifolia 0.0071 Self-incompatibility gene 83; Nicotiana alata 0.014 S7~RNase; Malus domestica 0.029 SB-RNase precursor; Malus domestica 0.03 S2—Rnase precursor; Malus domestica 0.046 Slla-Rnase; Lycopersicon esculentum 0.087 Ribonuclease; Luffa cylindrica 0.096 SZ-protein; Solanum chacoense 0.14 Ribonuclease; Lycopersicon esculentum 0.15 Self-incompatibility ribonuclease; Solanum carolinense 0.15 S9-RNase; Malus domestica 0.16 a - Databases searched were non-redundant GenBank translations, SwissProt, SwissProt update, PDB, and PIR databases. b — Hits with probabilities greater than 0.2 are not listed for brevity. 63 Pa RN51 HGLWPNYKDGSYPSNCDPDSVFDKSEISELMSNLEKNWPSLSC--PSS—NGFR--FWS Md 52 hg1wpsnmnrse1fncsssnvtyakiq—nirtqlemiwpnvfnrknhl ------ gfwn Md S3 hglwpsnvngsdpkkcktti1npqti-tn1taqleiiwpnvlnrkaha ------ rfwr Md SS hglwpsnfngpdpenckvkptasqtidts1kpqleiiwpnvfnradhe ------ squ Md 57 hglwpsdsnghdpvncskstvdaqkl-gn1ttqleiiwpnvynrtdhi ------ sfwd Md S9 hglwpsnssgndpiycknttmnstki-anltarleiiwpnvldrtdhi ------ tfwn Ph 51 hglwpdsisvimn—ncdptktfatiteikqite1ekrwpeltttaqfaltsq—-sfwr Lp 5n hngpdhtsfvmy-dcdplkkyktiddtni1te1darwpq1tstkiiglqfq-—rfwe 5p 57 hngpdhtdyimy-dcnpnkefkkiydkhllnklesrwpqltsheyaglndq—-tfwk RNASE LE hglwpnnndgtyp sncdpnspydqsqisdlissmqqnwptlac——psg- sgst-— fws RNSLX hglwpnykdgkwpqncdressldesefsdlistmeknwpslac—-pss-dglk-—fws At RN51 hglwpnykdgtypsncdaskpfdsstisdl1tsmkkswptlac—-psg-sgea--fwe At RN52 hglwpdyndgswpsccyr-sdfkekeistlmdglekywpslscgspsscnggkgsfwg At RN53 hglwpnyktgngqncnpdsrfddlrvsdlmsdlqrewptlsc--psn-dgmk-—fwt Ph 50 hglwpdsvsvmmy-ncdpptrfnkiretnikne1ekrwpe1tstaqfalksq—-sfwk Conserved HG*WP**********C*********************WP****************FW* Pa RN51 HEWEKHGTC Md S2 rewnkhgac Md 53 kqwrkhgtc Md 5S kqwdkhgtc Md S7 kqvnkhgtc Md S9 kqwnkhgsc Ph 51 yqyekhgtc LP 513 yeyrkhgtc Sp 57 yeyekhglc RNASE LE hewekhgtc RNSLX hewlkhgtc At RN51 hewekhgtc At RN52 hewekhgtc At RN53 hewekhgtc Ph 50 yqyekhgtc Conserved ****KHG*C Md 2 MBJUS'dbmastjca; Ph 2 Perunja hybrjaia,ILp, LE, and LX = lycopersjcon 9301/1917 tum, S p = 52:71.31? um pen! Via/7 am. a n d A t = Arabadhpsjs tbajjana. Figure 2.8 - Amino Acid Alignment of pSllB with Published Plant Ribonucleases. DNA sequence is corrected for missing base in the primer sequence. 64 investigated because of concern about reduced yield. Yield was an important consideration because styles could only be collected once a year and because of the number of styles required to obtain a sufficient quantity of RNA. Plaque lifts done on the PCR amplified stylar cDNA library indicated the presence of ribosomal and chloroplast sequences. Apparently, these sequences were not completely removed prior to cDNA synthesis. DNase treatment prior to cDNA synthesis should have been done to eliminate contaminating chloroplast and nuclear DNA. The ribosomal sequences may have resulted from self priming of ribosomal RNA during the cDNA synthesis, which is a possiblity due to the nature of rRNA secondary structure, coupled with partial degredation or nicking of the rRNA (M. Thomashaw, pers. com.). Due to the limitations in the quantity of stylar RNA and the resulting cDNA, the cDNA was amplified by PCR following the addition of linker sequences. Although this work and other published reports (Akowitz and Manuelidis, 1989, Belyavsky et al., 1989, Jepson et al., 1991) used this technique with total RNA, this step contributed to the presence of ribosomal sequences in the cDNA. If possible, purified mRNA 65 would be preferred. A lambda gth cDNA library was constructed for the purpose of identifying full-length cDNA S-allele clones. This of course, has not yet been done, but it was useful to construct the library at the same time that cDNA was being generated for use in other experiments. Dot blot experiments on the plasmid clones containing PCR generated inserts using the degenerate primers produced two classes of clones. The presence of a strong hybridization signal would indicate that the insert cloned in the plasmid exists in a high proportion in the cDNA population. This could be accounted for by three reasons: a) The sequence represents an abundant mRNA in the tissue from which the cDNA was made. b) The sequence represents a contaminating sequence which was present in high proportions prior to PCR amplification of the cDNA. c) The sequence represents some sequence that was preferentially amplified during PCR amplification of the cDNA. The dot blot experiments hinged on the successful isolation of quality mRNA. This manifests in the reliability of the dot blot experiments in which tissue specific expression of 66 the p6 and p8 clones were assessed (because PCR was used to generate the pS clones, it is imaginable that all one would need is a few good mRNAs leading to a few good cDNAs, hence, high quality RNA may not have been so critical in the generation of the pS clones). The ramification of this may be that the dot blot experiments may not be valid even though the pS clones could contain expressed sequences. Southern analysis using the p6 and p8 clones, on the other hand, was not dependent on any questions of RNA quality. Insight to the nature of the cloned DNA, such as copy number and polymorphism type, supports using the degenerate primers on genomic DNA as a viable strategy. Of particular interest was pGllO, because hybridization patterns on different incompatibility genotypes showed that this probe detected a band that was unique to three cultivars, all of which show the S3 phenotype. Unfortunately, this probe hybridized to more than one region This probe could be chimeric, a combination of two different inserts, which ligated together before ligation into the plasmid, as was the case with pSll8. Ongoing work will result in the necessary sequence information to determine if pGllO contains ribonuclease sequence. 67 Investigation of pS clones by Southern hybridization experiments indicated that most of the clones represent low copy sequences. Since these clones were generated from the amplified style cDNA, we can be fairly confident that there exists a number of single copy sequences in the style cDNA pool. This was also demonstrated by the dot blot experiments in that only 4 out of the 81 pS clones tested in Figure 2.4 indicate a copy number other than low. While the dot blots were successful in indicating copy number with respect to the style cDNA pool, the Southern hybridization experiments gave an indication of uniqueness of each clone with respect to the population of pS clones obtained. The data collected and displayed in Table 2.4 show that many of the clones are unique. There does exist a group of clones which all give the same hybridization pattern, although these clones also show low copy within the cDNA pool. None of the pS clones tested showed S—genotype specificity. While it is possible that the enzymes used do not give a polymorphism at the incompatibility locus, I believe this to be unlikely in light of the nature of the locus, itself being highly polymorphic. The number of clones generated in the pS cloning experiment (172) made Southern analysis unfeasable as a method to test all clones. 68 Sequencing generates by far the most data about each individual Clone. The reasoning behind choosing which clones to sequence was an attempt to select unique clones representing different cloning experiments. Database searching provided convincing evidence that pGllS contains 188 rDNA and that p8118 contains RNase sequence. Further evidence is available for both clones which support the database search results, mainly that Southern analysis shows that, based on signal intensity, pGllS is present with high copy number in the Cherry genome and that p8118 is present with low copy number. It was unfortunate that p8118 Southern analysis experiments did not yield sufficient resolution to determine if there was genotype specificity. Recent work by D.C. Wang (Unpublished data) has shown by Southern hybridization that the RNase portion of p8118 does not display genotype specificity and is single COpy in the cherry genome. Sequencing results show that p8118 is a chimeric clone. This was most likely the result of the insert-to-vector ratio in the ligation reaction (3:1 insert to vector). Sequence data also show that the T4 DNA polymerase treatment of the PCR products removed a few bases at the end of the PCR product. 69 This is an acceptable approach because there are only a couple of bases removed. The missing base in the AI-l primer sequence is quite the mystery, as other sequenced clones show the entire AI~1 primer sequence (see Appendix 5). There may be errors in the sequencing data due to secondary structure although the sequence information at the AI—l primer location is three deep. Another explanation could be hypothesized based on the action of T4 DNA polymerase fidelity. Both of the mentioned hypothesis are without any support and are offered only to provoke thought. The amino acid sequence is convincing evidence in support of the function of the cloned sequence in p8118. What remains to be determined is the expression patterns of the sequence in p8118. Northern hybridization data is incomplete at this point due to the limitations on obtaining stylar RNA. Primers to amplify the ribonuclease region of the clone have been designed to help in answering the question of the origin of the RNase sequence and further Northern hybridization experiments will help answer some of these unfinished questions. 70 The work presented here was clearly successful in the attempt to isolate a clone which could code for a ribonuclease. APPENDICES APPENDIX A APPENDIX.A PROTOCOLS FOR SDS-PAGE The following protocol is commonly known as the Lammeli system and is given for an acrylamide concentration of 10%T, 2.7%C. When SDS—PAGE was followed by RNase activity staining, beta-mercaptoethanol is ommitted in all steps and the resolving gel contains 2 mg/ml yeast RNA. All buffers, glassware, and equipment were Rnase free when Rnase activity staining was to be performed. Final Concentrations Resolving Gel Stacking Gel Tank Buffer Acrylamide conc. 102T, 2.7tC 4aT, 2.7%C Tris HCL 0.375 M 0.125 M Tris Glycine 0.02 M tris base 0.192 M glycine pH 8.8 6.8 8.3 SDS 0.1% 0.1% 0.1% Amoniumpersulfate 0.1% (w/v) 0.05% (w/v) TEMED 0.07% (v/v) 0.05% (v/v) Sample Treatment To prepare the sample for electrophoresis, combine the dry protein with an equal volume of water and 2x treatment buffer (0.125 M tris—HCl pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol). If the protein is already in solution, ommit the water. Put the protein sample in boiling water for 90 seconds, then chill on ice until you are ready to use it. This treated sample can be stored frozen for future runs. Electrophoretic Conditions Generally, with a 1.5 mm thick gel, 10 to 50 ul of sample is added to each lane. Electrophoresis is done at a constant current of 30 mA for about 3.5 hours. It is important not to let the dye front fun off the bottom of the gel as this will distort the resolution of the proteins on the gel. 7] 72 APPENDIX.A Coomassie Blue R—250 Staining The gel is stained with commassie blue (0.125% coomassie blue R-250, 50% methanol, 10% acetic acid) with gentle shaking for 4 hours. It is then destained for 1 hour with destaining solution 1 (50% methanol, 10% acetic acid) and for six hours in destaining solution 2 ( %acetic acid, 5% methanol). Rnase Activity Staining 1. After electrophoresis (no 2-mercptoethanol in any solutions), wash the gel in 25% isopropanol in 0.01 M tris- HCl pH 7.0 twice for 10 minutes each with gentle shaking. This removes SDS from the gel. 2. Next, wash the gel in 2 uM ZnClz, 0.01 M tris—HCl pH 7.0 twice for 10 minutes each. This removes the isopropanol from the gel. 3. Incubate the gel in 0.01 M tris-HCl pH 7.0 at 51 C for 30 minutes to 1 hour. This time can vary as a function of the specific activity of the Rnases on the gel that you wish to detect. 4. Stain the gel in 0.2% toluidine blue in 0.01 M tris pH 7.0 for 10 minutes. 5. Destain the gel in 0.01 M tris—HCl for 1 x 10 minutes and 2 x 20 minutes. 6. Zones of Rnase activity will appear as clear bands on a blue background. APPENDIX B APPENDIX B PROTOCOLS FOR.AMPLIFICATION OF RECOMBINANT DNA PCR Amplification of pUC 19 Inserts Master mix (keep on ice): Component Final conc. Water 10X Buffer 1x dNTPs 200 mM each Forward primer 100 nM Reverse primer 100 nM Taq polymerase 1.25 U Total 48.5 1. Add 48.5 ml of master mix to each 500 ml labeled tube. 2. Add 1.5 ml (15 ng) of template DNA (stock conc. = 10 ng/ml) as a droplet to the side of the tube. 3. Spin down all samples and add approximately 50 ml (or 2 drops) of sterile light mineral oil (Sigma). 4. Add a drop of mineral oil to each well in the Perkin Elmer 480 machine and load samples. Template DNA can come from either a miniprep or, more directly, from the glycerol stock itself using the following procedure: take 1 ml of the glycerol stock and add it to 20 ml water; boil in heat block for 5 min. Use 1 ml of this as template DNA. Amplification Conditions: 94°/4 min followed by 30 cycles of 94°/1 min, 60°/1 min, 72°/2 min. 73 74 APPENDIX B Primer Sequences Forward primer: 5'—CGCCAGGGTTTTCCCAGTCACGAC-3' Reverse primer: 5'-TCACACAGGAAACAGCTATGAC-3' (Primers are available from Promega, but were made by the MSU Macromolecular Structure Facility. They will amplify anything within the multiple cloning site, adding approximately 120 bp to the insert.) PCR.Amplification of lambda gt10 inserts. The protocol is essentially the same for amplification of lambda gt10 inserts with the following modifications: Primers for amplification of lambda gth inserts: Forward: 5’ CTTTTGAGCAAGTTCAGCCTGGGTAAG 3’ Reverse: 5’ GAGGTGGCTTATGAGTATTTCTTCCAGGGTA 3’ PCR reaction mix: 1.5 mM MgClz 500 nM each primer Amplification Conditions: 25 cycles of 94°/1min, 50°/lmin, 72°/1min. In this protocol, template can come from a plaque. Pick the plaque with a toothpick and then transfer the toothpick to 20 microliters of water, swirl the toothpick to dislodge the phage into the water, then use 1-2 microliters of this as the DNA source in the PCR reaction. APPENDIX C APPENDIX C STANDARD HYBRIDIZATION PROTOCOLS AND SOLUTIONS Pre-hybridization Solution (100 ml) 2 -42 ml sterile diH2O 8 -20 ml 25% dextran sulfate (from 500,000 MW dextran) 4 -25 ml 20X 88C 5 -5 ml 1 M Tris, pH 8 1 -2 ml 0.5 M EDTA 0. 0 0 O O 5 }_a -4 ml 50X Denhardts solution -1 ml 20% SDS (MoBio. grade) —1 ml 10 mg/ml salmon sperm DNA, sheared and denatured [\JNCI‘JoD-OOOA 50X Denhardts (200 ml) -2 g bovine serum albumin (fraction V, Sigma) -2 g Ficol (type 400, Pharmacia) —2 g PVP (360,000 MW) -dissolve in this order, one at a time, heating slightly if necessary -sterile diHZO to 200 ml -store in aliquots at —20C Salmon Sperm DNA (sheared/denatured) (or just buy from Boer. Mann.) -dissolve to 10 mg/ml in water (overnight) ~add 5M NaCl to 0.1 M final -extract with phenolzchloroform, then chloroform:isoamyl -shear DNA by passing through a 17 gauge needle 10 times fast -precipitate by adding 2 volumes ice—cold EtOH —wash, dry down, redissolve to 10 mg/ml (use spec. or fluor.) ~boil for 10 min. and quick cool -store at —20 in 1 ml aliquots 75 76 APPENDIX C 2X Wash Buffer (1 liter) -100 ml 20X 88C -850 m1 diH2O —50 ml 20% SDS 0.2x Wash Buffer (1 liter) —10 ml SSC ~965 ml diH2O -25 ml 20% SDS Procedure: 1. Wet membrane in 2x SSC. 2. Place the membrane in a hybridization tube with the DNA side facing in. 3. Add 10 to 20 mls of pre-hybridization solution and prehybridize for a minimum of 4hrs. at 60 to 65 C. 4. Add 20ng of labeled probe to the hybridization tube and hybridize a minimum of 6hrs at 60 to 65C. Varying the hybridization temperature will affect the stringency of the hybridization. For more stringent conditions use 65 C. 5. Pour out the pre-hybridization solution and probe into the radioactive waste container. 6. Add 25 mls of 2X wash buffer and wash for 30 minutes at 60C. 7. Dump wash buffer into the radioactive waste and repeat the 2X wash. 8. Wash twice for 30 min with 0.2x wash buffer at 60 C. 9. Remove membrane from the hybridization tube and pat dry between two pieces of Whatman filter paper. Wrap the membrane in saran wrap and place in a cassette with film and develop overnight. APPENDIX D APPENDIX.D CALCULATIONS OF YIELD FOR cDNA SYNTHESIS REACTIONS = total counts per minute (cpm) in first strand reaction. incorporated cpm in first strand reaction. moles of dCTP in the reaction. A B C Calculation of yield in grams: Yield [g] = B/A x C x 4(nucleotides) x 330 (average M.W. of a nucleotide). Calculation of percent yield: Y yield as calculated above. D = amount of input RNA. % Yield = Y/D x 100. 77 APPENDIX E HONHUOU APPENDIX E LAYOUT FOR DOT BLOTS SHOWN IN FIGURE 4A AND 43 1 2 3 4 5 6 7 8 9 10 11 12 p896 98 p899 pSlOO pSlOl p8102 p8103 p5104 pSlOS p8106 p8107 p5108 p8109 pSllO pSlll pSllZ p8113 pSll4 pSllS pSll6 pSll7 p8118 p8119 p8120 p5122 p5123 p8124 pSlZS p8126 p3127 p8128 pS129 pSl3O p5131 p8132 pSl33 p8134 pSl35 p8136 pSl37 pSl38 pSl39 pSl40 p8141 pSl42 pSl43 p8144 p8145 pSl46 pSl47 pSl48 pSl49 p8150 pSlSl pS152 p8153 p5154 p8155 p5156 p8157 p8158 p8159 pSl6O p816l p5162 pSl63 p8164 p8165 pSl66 p8167 pSl68 p5169 pSl70 p840 p539 p841 p842 p851 p852 p853 p854 p629 p647 pGllO pGllS pGZO7 pGZ93 empty pBS (—) tubulin pUC118 TE empty empty empty empty 78 APPENDIX F APPENDIX.F DNA SEQUENCE AND AMEND ACID TRANSLATIONS FOR.pG AND pS CLONES The suffix f or r after the clone name indicates forward or reverse sequencing primer. The p8 clones were short enough to sequence through to the vector on the far side. The vector sequence was identified and removed. There may be about 20 bases of vector sequence in the p8 clones on the near side which was not removed because the vector sequence could not be unambiguously identified here. Where primer sequence could be identified, the primer sequence is marked in bold underline (note: not all primer matches are perfect matches). 79 80 APPENDIX F pG115f Cloning vector pBS minus, cloning site EcorV I H G L Y P E K V C K R S L Y N Y K V S N S W S I P G E G V * T V T L * L * G I E F M V Y T R R R C V N G H S I T I R Y 5'GAATTCATGGTCTATACCCGGAGAAGGTGTGTAAACGGTCACTCTATAACTATAAGGTAT 10 20 30 40 50 60 3'CTTAAGTACCAGATATGGGCCTCTTCCACACATTTGCCAGTGAGATATTGATATTCCATA F E H D I G P S P T Y V T V R Y S Y P I I * P R Y G S F T H L R D S * L * L T D N M T * V R L L H T F P * E I V I L Y * I S T S G L V E * L D L D S L * L Q S L N L H E W V S * V T R L R L S L I T I T Q S P R V G * L S D * T * T L F D Y N H CAATCTCCACGAGTGGGTTAGTTGAGTGACTAGACTTAGACTCTCTTTGATTACAATCAC 7O 80 90 100 110 120 GTTAGAGGTGCTCACCCAATCAACTCACTGATCTGAATCTGAGAGAAACTAATGTTAGTG L R W S H T L Q T V L S L S E K I V I V I E V L P N T S H S S K S E R Q N C D S D G R T P * N L S * V * V R K S * L * K F * K K * S S K N H K R S K V H R R L G F L K K V E * Q K S * E K Q G P Q K V G F S K K S R V A K I I R E A R S T E G W TTTTCTAAAAAAAGTAGAGTAGCAAAAATCATAAGAGAAGCAAGGTCCACAGAAGGTTGG 130 140 150 160 170 180 AAAAGATTTTTTTCATCTCATCGTTTTTAGTATTCTCTTCGTTCCAGGTGTCTTCCAACC K R F F T S Y C F D Y S F C P G C F T P K * F F Y L L L F * L L L L T W L L N P E L F L L T A F I M L S A L D V S P Q S V V S P V H Q V Y H C G P S Y S R G C P S S K P G S P G L P L R A I I L K R L S E * * A R F T R S T T A G H H T Q E V V GAGTAGTAAGCCCGGTTCACCAGGTCTACCACTGCGGGCCATCATACTCAAGAGGTTGTC 190 200 210 220 230 240 CTCATCATTCGGGCCAAGTGGTCCAGATGGTGACGCCCGGTAGTATGAGTTCTCCAACAG L L L G P E G P R G S R A M M S L L N D T T L G T * W T * W Q P G D Y E L P Q G Y Y A R N V L D V V A P W * V * S T T R G S E A * L Y A Y Q C S R R V Q V A G R W L R G V A I C L P M Q S T C S G G R K L A Q R R S Y M L T N A V D V F R W Q E CTGGCTCAGAGGCGTAGCTATATGCTTACCAATGCAGTCGACGTGTTCAGGTGGCAGGAA 250 260 270 280 290 300 GACCGAGTCTCCGCATCGATATACGAATGGTTACGTCAGCTGCACAAGTCCACCGTCCTT Q S L P T A I H K G I C D V H E P P L F P E S A Y S Y A * W H L R R T * T A P L A * L R L * I S V L A T S T N L H C S P 81 APPENDIX F L C * C S G A A L L V F R C G S A S V P V H GGCTCTGCTAGTGTTCCGGTGCAT 3' 3 l O 32 O CCGAGACGATCACAAGGCCACGTA 5 ' A R S T N R H M S Q * H E P A E A L T G T C 82 APPENDIX F pG115R Cloning vector pBS minus, cloning site EcorV R V P C L F H S T K E L Y K R H C P S S S S P V L V P L D E R A L Q A A L P F F L E S R A C S T R R K S F T S G I A L L 5'CTCGAGTCCCGTGCTTGTTCCACTCGACGAAAGAGCTTTACAAGCGGCATTGCCCTTCTT 10 20 3O 4O 50 6O 3'GAGCTCAGGGCACGAACAAGGTGAGCTGCTTTCTCGAAATGTTCGCCGTAACGGGAAGAA E L G T S T G S S S L A K C A A N G K K R T G H K N W E V F S S * L R C Q G E E S D R A Q E V R R F L K V L P M A R R * L T R Y C W I G L S P I V Q D S P L L P T H A I L L D R A F A H C P R F P T A A H S R D I A G S G F R P L S K I P H C C CACTCACGCGATATTGCTGGATCGGGCTTTCGCCCATTGTCCAAGATTCCCCACTGCTGC 70 80 90 100 110 120 GTGAGTGCGCTATAACGACCTAGCCCGAAAGCGGGTAACAGGTTCTAAGGGGTGACGACG V * A I N S S R A K A W Q G L N G V A A S V R Y Q Q I P S E G M T W S E G S S G E R S I A P D P K R G N D L I G W Q Q G P R G E S G P S L S P S V A D H P K D P P S W G V R A E S Q S Q C G * S S E R P P L V G S P G R V S V P V W L I I R K T CCCCTCGTGGGGAGTCCGGGCCGAGTCTCAGTCCCAGTGTGGCTGATCATCCGAAAGACC 130 140 150 160 170 180 GGGGAGCACCCCTCAGGCCCGGCTCAGAGTCAGGGTCACACCGACTAGTAGGCTTTCTGG G E H P T R A S D * D W H P Q D D S L G G R P S D P G L R L G L T A S * G F S G R T P L G P R T E T G T H S I M R F V W A K Q S L A W S A F T * P T T * Y Y E A S * A V I G L V S L Y L T N Y L I L R G Q L S S H W L G Q P L P D Q L P N T T R CAGCTAAGCAGTCATTGGCTTGGTCAGCCTTTACCTGACCAACTACCTAATACTACGAGG 190 200 210 220 230 240 GTCGATTCGTCAGTAACCGAACCAGTCGGAAATGGACTGGTTGATGGATTATGATGCTCC L * A T M P K T L R * R V L * R I S R P A L C D N A Q D A K V Q G V V * Y * S A S L L * Q S P * G K G S W S G L V V L S H Q Q R L A S O D W P T V A V P R Y D R S S T A F S F S G L A D C R S S T V R P L I N S V * L L R I G R L S Q F H G T T CTCATCAACAGCGTTTAGCTTCTCAGGATTGGCCGACTGTCGCAGTTCCACGGTACGACC 250 260 270 280 290 300 GAGTAGTTGTCGCAAATCGAAGAGTCCTAACCGGCTGACAGCGTCAAGGTGCCATGCTGG E D V A N L K E P N A S Q R L E V T R G * * C R K A. E * S Q G V T A T G R Y S R M L L T * S R L I P R S D C N W P V V T 83 APPENDIX F R H C L L S L C G S T V A S L E V L S P L S T V T L R Q Y C G F S R S A V A I V Y C H S A A V L W L L S K C * GTCGCCATTGTCTACTGTCACTCTGCGGCAGTACTGTGGCTTCTCTCGAAGTGCTGA 3' 310 320 330 340 350 CAGCGGTAACAGATGACAGTGAGACGCCGTCATGACACCGAAGAGAGCTTCACGACT 5' D G N D V T V R R C Y Q P K E R L A S R W Q R S D S Q P L V T A E R S T S A M T * Q * E A A T S H S R E F H Q 84 APPENDIX F pGZ9F Cloning vector pBS minus, cloning site EcorV R V P C L S Y S D D Q D R T A R N N G R S S A V F I I L G * P G S Y G T K Q W E L E C R V Y H T R M T R I V R H E T M G 5'CTCGAGTGCCGTGTTTATCATACTCGGATGACCAGGATCGTACGGCACGAAACAATGGGA 10 20 3O 40 50 60 3'GAGCTCACGGCACAAATAGTATGAGCCTACTGGTCCTAGCATGCCGTGCTTTGTTACCCT E L A T N I M S P H G P D Y P V F C H S R T G H K D Y E S S W S R V A R F L P L S H R T * * V R I V L I T R C S V I P S T F C L N I G S I * K Q M T V P K * S R N F L P Q H R E H L E T N D S S Q M K P E L S A S T * G A F R N K * Q F P N E A GAACTTTCTGCCTCAACATAGGGAGCATTTAGAAACAAATGACAGTTCCCAAATGAAGCC 7O 80 90 100 110 120 CTTGAAAGACGGAGTTGTATCCCTCGTAAATCTTTGTTTACTGTCAAGGGTTTACTTCGG F K R G * C L S C K S V F S L E W I F G V K Q R L M P L M * F C I V T G L H L R S E A E V Y P A N L F L H C N G F S A S I L P F H S R R G S T H R G I V N V H N N T P I S F * E R O Y P S W Y C * C P Q E Y S H F I L G E A V P I V V L L M S T GAATACTCCCATTTCATTCTAGGAGAGGCAGTACCCATCGTGGTATTGTTAATGTCCACA 130 140 150 160 170 180 CTTATGAGGGTAAAGTAAGATCCTCTCCGTCATGGGTAGCACCATAACAATTACAGGTGT F V G M E N * S L C Y G D H Y Q * H G C I S G N * E L L P L V W R P I T L T W L Y E W K M R P S A T G M T T N N I D V V Q H I Y K E * A P D V M S V L T L S R S P T Y L * G I G S * R D V S T D S Q Q I T N I F I R N R L L T * C Q Y * L S A D ACCAACATATTTATAAGGAATAGGCTCCTGACGTGATGTCAGTACTGACTCTCAGCAGAT 190 200 210 220 230 240 TGGTTGTATAAATATTCCTTATCCGAGGACTGCACTACAGTCATGACTGAGAGTCGTCTA G V Y K Y P I P E Q R S T L V S E * C I W C I * L S Y A G S T I D T S V R L L D L M N I L F L S R V H H * Y Q S E A S * S I H Q CATCAG 3' GTAGTC 5' M L D * 85 APPENDIX F pG29R Cloning vector pBS minus, cloning site EcorV I H G F C L E T V T L T S T S G S L L C N S W I L P R D S Y L D I H V R E P I V E F M D S A * R Q L P * H P R Q G A Y C 5'GAATTCATGGATTCTGCCTAGAGACAGTTACCTTGACATCCACGTCAGGGAGCCTATTGT 10 20 3O 4O 5O 6O 3'CTTAAGTACCTAAGACGGATCTCTGTCAATGGAACTGTAGGTGCAGTCCCTCGGATAACA F E H I R G L S L * R S M W T L S G I T I * P N Q R S V T V K V D V D P L R N H N M S E A * L C N G Q C G R * P A * Q A L Q K Y C V W L * H N N T T M V L P L L P S K I L C L V V T * Q Y H D G T A S A A F K N I V F G C D I T I P R W Y C L C GCCTTCAAAAATATTGTGTTTGGTTGTGACATAACAATACCACGATGGTACTGCCTCTGC 7O 80 90 100 110 120 CGGAAGTTTTTATAACACAAACCAACACTGTATTGTTATGGTGCTACCATGACGGAGACG G E F I N H K T T V Y C Y W S P V A E A R * F Y Q T Q N H C L L V V I T S G R S K L F I T N P Q S M V I G R H Y Q R Q O K * M S I G F I D F I V S I S Y V G R V E M N E Y R L H * L H C L N L L C G Q S * N E * V S A S L T S L S Q S P M W A E TGAAATGAATGAGTATCGGCTTCATTGACTTCATTGTCTCAATCTCCTATGTGGGCAGAG 130 140 150 160 170 180 ACTTTACTTACTCATAGCCGAAGTAACTGAAGTAACAGAGTTAGAGGATACACCCGTCTC S I F S Y R S * Q S * Q R L R R H P C L F H I L I P K M S K M T E I E * T P L T F S H T D A E N V E N D * D G I H A S D S M S H V R H L H V S R P A S P C L T S G I TCTCCATGTCTCACGTCCGGCATC 3' 190 200 AGAGGTACAGAGTGCAGGCCGTAG 5' R W T E R G A D E M D * T R C G H R V D P M 86 APPENDIX F pG29f Cloning vector pBS minus, cloning site EcorV R V P C L S Y S D D Q D R T A R N N G R S S A V F I I L G * P G S Y G T K Q W E L E C R V Y H T R M T R I V R H E T M G 5'CTCGAGTGCCGTGTTTATCATACTCGGATGACCAGGATCGTACGGCACGAAACAATGGGA 10 20 30 40 50 6O 3'GAGCTCACGGCACAAATAGTATGAGCCTACTGGTCCTAGCATGCCGTGCTTTGTTACCCT E L A. T N I M S P H G P D Y P V F C H S R T G H K D Y E S S W S R V A R F L P L S H R T * * V R I V L I T R C S V I P S T F C L N I G S I * K Q M T V P K * S R N F L P Q H R E H L E T N D S S Q M K P E L S A S T * G A F R N K * Q F P N E A GAACTTTCTGCCTCAACATAGGGAGCATTTAGAAACAAATGACAGTTCCCAAATGAAGCC 7O 80 90 100 110 120 CTTGAAAGACGGAGTTGTATCCCTCGTAAATCTTTGTTTACTGTCAAGGGTTTACTTCGG F K R G * C L S C K S V F S L E W I F G V K Q R L M P L M * F C I V T G L H L R S E A E V Y P A N L F L H C N G F S A S I L P F H S R R G S T H R G I V N V H N N T P I S F * E R Q Y P S W Y C * C P Q E Y S H F I L G E A V P I V V L L M S T GAATACTCCCATTTCATTCTAGGAGAGGCAGTACCCATCGTGGTATTGTTAATGTCCACA 130 140 150 160 170 180 CTTATGAGGGTAAAGTAAGATCCTCTCCGTCATGGGTAGCACCATAACAATTACAGGTGT F V G M E N * S L C Y G D H Y Q * H G C I S G N * E L L P L V W R P I T L T W L Y E W K M R P S A T G M T T N N I D V V Q H I Y K E * A P D V M S V L T L S R S P T Y L * G I G S * R D V S T D S Q Q I T N I F I R N R L L T * C Q Y * L S A D ACCAACATATTTATAAGGAATAGGCTCCTGACGTGATGTCAGTACTGACTCTCAGCAGAT 190 200 210 220 230 240 TGGTTGTATAAATATTCCTTATCCGAGGACTGCACTACAGTCATGACTGAGAGTCGTCTA G V Y K Y P I P E Q R S T L V S E * C I W C I * L S Y A G S T I D T S V R L L D L M N I L F L S R V H H * Y Q S E A S * S I H Q CATCAG 3' GTAGTC 5' M L D Jr 87 APPENDIX F pG293f Cloning vector pBS minus, cloning site EcorV I H G L C R K I R T R L V Y L K D I L I N S R T V P E D * N P L S L S K R Y S D E F T D C A G R L E P A * F I * K I F * 5'GAATTCACGGACTGTGCCGGAAGATTAGAACCCGCTTAGTTTATCTAAAAGATATTCTGA 10 20 30 4O 50 6O 3'CTTAAGTGCCTGACACGGCCTTCTAATCTTGGGCGAATCAAATAGATTTTCTATAAGACT F E R V T G S S * F G S L K D L L Y E S I * P S H R F I L V R K T * R F S I R I N V S Q A P L N S G A * N I * F I N Q Y F W R N T I F E D K Q T L F L * K E I S I L E E Y H F R G * A D S V F I K R D K Y F G G I P F S R I S R L C F Y K K R * TATTTTGGAGGAATACCATTTTCGAGGATAAGCAGACTCTGTTTTTATAAAAAGAGATAA 70 80 90 100 110 120 ATAAAACCTCCTTATGGTAAAAGCTCCTATTCGTCTGAGACAAAAATATTTTTCTCTATT I K S S Y W K R P Y A S E T K I F L S L N Q L F V M K S S L C V R N K Y F S I L K P P I G N E L I L L S Q K * L F L Y A G K H E T A K S * S H H L Q D S R P F R R E T * N C Q K L E P P S P R L Q T F Q A G N M K L P K A R A T I S K T P D L S GCGGGAAACATGAAACTGCCAAAAGCTAGAGCCACCATCTCCAAGACTCCAGACCTTTCA 130 140 150 160 170 180 CGCCCTTTGTACTTTGACGGTTTTCGATCTCGGTGGTAGAGGTTCTGAGGTCTGGAAAGT R S V H F Q W F S S G G D G L S W V K * P F C S V A L L * L W W R W S E L G K L P F M F S G F A L A V M E L V G S R E S A G E V G V C * D G L G S A I V P S P G S R * G W S V L R R T R I S Y R T V A W E Q V R L E C V K T D S D Q L S Y R R L GAGCAGGTGAGGTTGGAGTGTGTTAAGACGGACTCGGATCAGCTATCGTACCGTCGCCTG 190 200 210 220 230 240 CTCGTCCACTCCAACCTCACACAATTCTGCCTGAGCCTAGTCGATAGCATGGCAGCGGAC L L H P Q L T N L R V R I L * R V T A Q A P S T P T H * S P S P D A I T G D G P C T L N S H T L V S E S * S D Y R R R S G A R G E G GAGGGGCC 3' CTCCCCGG 5' L P P A P G 88 APPENDIX F pG207R Cloning vector pBS minus, cloning site EcorV I H G L W P E L D R E A V P H Y * P R A N S W F M A G A * P R S S A S L L T T G E F M V Y G R S L T E K Q C L I T D H G 5'GAATTCATGGTTTATGGCCGGAGCTTGACCGAGAAGCAGTGCCTCATTACTGACCACGGG 10 20 3O 4O 50 6O 3'CTTAAGTACCAAATACCGGCCTCGAACTGGCTCTTCGTCACGGAGTAATGACTGGTGCCC F E H N I A P A Q G L L L A E N S V V P I * P K H G S S S R S A T G * * Q G R A N M T * P R L K V S F C H R M V S W P S A S T I * S A V A S S G A T I E K I L S C F H Y L I S C G F V W S H N * E N I I L L P L S D Q L W L R L E P Q L R K Y Y CTGCTTCCACTATCTGATCAGCTGTGGCTTCGTCTGGAGCCACAATTGAGAAAATATTAT 70 80 90 100 110 120 GACGAAGGTGATAGACTAGTCGACACCGAAGCAGACCTCGGTGTTAACTCTTTTATAATA Q K W * R I L Q P K T Q L W L Q S F I I A E V I Q D A T A E D P A V I S F I N D S G S D S * S H S R R S G C N L F Y * R F T V D I F T S P K V D Q A F F Y S * Y F Y L T K S R P S L L L Q L I F L P H Q K S T K P S CTTTTACAGTTGATATTTTTACCTCACCAAAAGTCGACCAAGCCTTCC 3' 130 140 150 160 GAAAATGTCAACTATAAAAATGGAGTGGTTTTCAGCTGGTTCGGAAGG 5' K * L Q Y K * R V L L R G L R G K V T S I K V E G F T S W A K K C N I N K G * W F D V L G E I 89 APPENDIX F pG47f Cloning vector pBS minus, cloning site EcorV I H G L W P E L D R E A V P H Y * P R A N S W F M A G A * P R S S A S L L T T G E F M V Y G R S L T E K Q C L I T D H G 5'GAATTCATGGTTTATGGCCGGAGCTTGACCGAGAAGCAGTGCCTCATTACTGACCACGGG 10 20 3O 4O 50 6O 3'CTTAAGTACCAAATACCGGCCTCGAACTGGCTCTTCGTCACGGAGTAATGACTGGTGCCC F E H N I A P A Q G L L L A E N S V V P I * P K H G S S S R S A T G * * Q G R A N M T * P R L K V S F C H R M V S W P S A F H Y L I S C G F V W S H N * E N I I C F P L S D Q L W L R L E P Q L R K Y Y L L S T I * S A V A S S G A T I E K I L CTGCTTTCCACTATCTGATCAGCTGTGGCTTCGTCTGGAGCCACAATTGAGAAAATATTA 70 80 90 100 110 120 GACGAAAGGTGATAGACTAGTCGACACCGAAGCAGACCTCGGTGTTAACTCTTTTATAAT Q K G S D S * S H S R R S G C N L F Y * A K W * R I L Q P K T Q L W L Q S F I I S E V I Q D A T A E D P A V I S F I N D F Y M * Y F Y L T K S R P S L P Q I I F L L H V I F L P H O K S T K P S A D N F S F T C D I F T S P K V D Q A F R R * F TCTTTTACATGTGATATTTTTACCTCACCAAAAGTCGACCAAGCCTTCCGCAGATAATTT 130 140 150 160 170 180 AGAAAATGTACACTATAAAAATGGAGTGGTTTTCAGCTGGTTCGGAAGGCGTCTATTAAA R K C T I N K G * W F D V L G E A S L K K * M H Y K * R V L L R G L R G C I I K K V H S I K V E G F T S W A K R L Y N K Y C P N I R D F * S N N T P H Q L D P S L L P Q Y * G F L K Q * Y T P P V G P Q F I A P I L G I F E A I I H P T S W T P TTTATTGCCCCAATATTAGGGATTTTTGAAGCAATAATACACCCCACCAGTTGGACCCCA 190 200 210 220 230 240 AAATAACGGGGTTATAATCCCTAAAAACTTCGTTATTATGTGGGGTGGTCAACCTGGGGT K N G W Y * P N K F C Y Y V G G T P G W * Q G L I L S K Q L L L V G W W N S G L I A G I N P I K S A I I C G V L Q V G T I D T S E V K R F L Q M X D * F V C S Y * Y * * G Q E I P P D G G L V R X * V L I L V R S R D S S R W X I S S X V V GTATTGATACTAGTGAGGTCAAGAGATTCCTCCAGATGGNGGATTAGTTCGTNTGTAGTA 3' 250 260 270 280 290 300 CATAACTATGATCACTCCAGTTCTCTAAGGAGGTCTACCNCCTAATCAAGCANACATCAT 5' Y Q Y * H P * S I G G S P P N T R X Y Y I S V L S T L L N R W I X S * N T X L N I S T L D L S E E L H X I L E X T T 90 APPENDIX F pG293r Cloning vector pBS minus, cloning site EcorV R V P C L T H S T C F C K S S G V L E M S S S V L N T L H L L L Q K L W S L G D L E F R A * H T P P A S A K A L E S W R 5'CTCGAGTTCCGTGCTTAACACACTCCACCTGCTTCTGCAAAAGCTCTGGAGTCTTGGAGA 10 20 30 40 50 6O 3'GAGCTCAAGGCACGAATTGTGTGAGGTGGACGAAGACGTTTTCGAGACCTCAGAACCTCT E L E T S L V S W R S R C F S Q L R P S R T G H K V C E V Q K Q L L E P T K S I S N R A * C V G G A E A F A R S D Q L H A G S Q L L G Q F H V S R L S L F L I K G W L S A F G T V S C F P L I S L F N K W L A L S F W D S F M F P A Y L S F * * TGGCTGGCTCTCAGCTTTTGGGACAGTTTCATGTTTCCCGCTTATCTCTCTTTTTAATAA 7O 80 90 100 110 120 ACCGACCGAGAGTCGAAAACCCTGTCAAAGTACAAAGGGCGAATAGAGAGAAAAATTATT P Q S E A K P V T E H K G S I E R K L L A P E * S K P C N * T E R K D R K K I F S A R L K O S L K M N G A * R E K * Y F T E S L I L E M V F L Q N I R I S F * R N R V S Y P R N G I P P K Y Q N I F L K K Q S L L S S K W Y S S K I S E Y L S E AAACAGAGTCTCTTATCCTCGAAATGGTATTCCTCCAAAATATCAGAATATCTTTCTGAA 130 140 150 160 170 180 TTTGTCTCAGAGAATAGGAGCTTTACCATAAGGAGGTTTTATAGTCTTATAGAAAGACTT F L T E * G R F P I G G F Y * F I K R F V S D R I R S I T N R W F I L I D K Q L C L R K D E F H Y E E L I D S Y R E S S D K L S GATAAACTAAGCG 3' 190 CTATTTGATTCGC 5' I F * A Y V L R L S L 91 APPENDIX F pS85f Cloning vector pUC118, cloning site HincIII X Q V X A X L D P I I T Q I F F Q X E A S A G X C X X G S H H N T N F F P X G S I X R F X P X W I P S * H K F F S K X K 5'ATCNGCAGGTTTNTGCCANNTTGGATCCCATCATAACACAAATTTTTTTCCAAANGGAAG 10 20 3O 4O 50 6O 3'TAGNCGTCCAAANACGGTNNAACCTAGGGTAGTATTGTGTTTAAAAAAAGGTTTNCCTTC D A P K X W X P D W * L V F K K G F P L X C T X A X X S G M M V C I K K W X S A X L N X G X Q I G D Y C L N K E L X F R N W F S A E A A V V E N I * L E V L K G E L V * R R G R G R R E Y L V G G A E G R I G L A P R P R S * R I S S W R C * R CGAATTGGTTTAGCGCCGAGGCCGCGGTCGTAGAGAATATCTAGTTGGAGGTGCTGAAGG 70 80 90 100 110 120 GCTTAACCAAATCGCGGCTCCGGCGCCAGCATCTCTTATAGATCAACCTCCACGACTTCC S N T * R R P R P R L S Y R T P P A S P F Q N L A S A A T T S F I * N S T S F P I P K A G L G R D Y L I D L Q L H Q L P L M W G S R P M V L T Q R A G C V X W V A D V G X T A D G S N T K S W M R X L G G * C G X H G R W F * H K E L D A * X G GGCTGATGTGGGGNTCACGGCCGATGGTTCTAACACAAAGAGCTGGATGCGTGANTTGGG 130 140 150 160 170 180 CCGACTACACCCCNAGTGCCGGCTACCAAGATTGTGTTTCTCGACCTACGCACTNAACCC A S T P X V A S P E L V F L Q I R S X P S I H P X R G I T R V C L A P H T X Q T O H P X * P R H N * C L S S S A H X P H Q S G G X S E L Y A N L H E D I L R * R A I R G X L * I I R Q S P * R Y S P L K C N P G G A L N Y T P I S M K I F S V E TGCAATCCGGGGGGNGCTCTGAATTATACGCCAATCTCCATGAAGATATTCTCCGTTGAA 190 200 210 220 230 240 ACGTTAGGCCCCCCNCGAGACTTAATATGCGGTTAGAGGTACTTCTATAAGAGGCAACTT A I R P X S Q I I R W D G H L Y E G N F C D P P X E S N Y A L R W S S I R R Q L L G P P A R F * V G I E M F I N E T S S I G L A G I L L S Q V N I Q S C P L * R N R T C W D T L I P S K H S I L S F I E E * D L L G Y S Y P K * T F N L V L Y R GAATAGGACTTGCTGGGATACTCTTATCCCAAGTAAACATTCAATCTTGTCCTTTATAGA 250 260 270 280 290 300 CTTATCCTGAACGACCCTATGAGAATAGGGTTCATTTGTAAGTTAGAACAGGAAATATCT F L V Q Q S V R I G L L C E I K D K I S I P S A P I S K D W T F M * D Q G K Y L Y S K S P Y E * G L Y V N L R T R * L P 92 APPENDIX F P Q T V H A T N R A G H K P C GGCCACAAACCGTGCAT 3 1 0 CCGGTGTTTGGCACGTA A V F R A G C V T C W L G H M 93 APPENDIX E pSlO3f Cloning vector pUC118, cloning site HincIII L D A C R S V Q V P C L K D Q E S C H Y L G C L Q V R T G T V L E R S R I L S L A W M P A G P Y R Y R A * K I K N L V I 5'GCTTGGATGCCTGCAGGTCCGTACAGGTACCGTGCTTGAAAGATCAAGAATCTTGTCATT 10 2O 30 4O 50 6O 3'CGAACCTACGGACGTCCAGGCATGTCCATGGCACGAACTTTCTAGTTCTTAGAACAGTAA S P H R C T R V P V T S S L D L I K D N K S A Q L D T C T G H K F S * S D Q * * Q I G A P G Y L Y R A Q F I L F R T M V T L I I I F F F N P Y P D * C Q Y S H S H T H Y H L L L Q S L P G L V P I Q P F T H S L S S S S S I L T R T S A N T A I ACACACTCATTATCATCTTCTTCTTCAATCCTTACCCGGACTAGTGCCAATACAGCCATT 7O 80 90 100 110 120 TGTGTGAGTAATAGTAGAAGAAGAAGTTAGGAATGGGCCTGATCACGGTTATGTCGGTAA C V * * * R R R * D K G P S T G I C G N V S M I M K K K L G * G S * H W Y L W E C E N D D E E E I R V R V L A L V A M R N L P T S T S I P Q Y S S T I L R * S T * L A N I H L N T T I L K Y H S S V E Y L T C Q H P P Q Y H N T Q V P F F G R V CTAACTTGCCAACATCCACCTCAATACCACAATACTCAAGTACCATTCTTCGGTAGAGTA 130 140 150 160 170 180 GATTGAACGGTTGTAGGTGGAGTTATGGTGTTATGAGTTCATGGTAAGAAGCCATCTCAT * S A L M W R L V V I S L Y W E E T S Y L K G V D V E I G C Y E L V M R R Y L V V Q W C G G * Y W L V * T G N K P L T C L G P Q P M T G A T T H H W G H N P * CACTGGGGCCACAACCCATGA 3' 190 200 GTGACCCCGGTGTTGGGTACT 5' V P A V V W S S P G C G M Q P W L G H ‘ .L 94 APPENDIX F pSll6f Cloning vector pUC118, cloning site HincIII X X P A G P S H G L W P Q C T L P K N G X H X C R S I T W V V A P V Y S T E E W X X X L Q V H H M G C G P S V L Y R R M 5'GNTNCATNCCTGCAGGTCCATCACATGGGTTGTGGCCCCAGTGTACTCTACCGAAGAATG 10 20 3O 40 50 60 3'CNANGTANGGACGTCCAGGTAGTGTACCCAACACCGGGGTCACATGAGATGGCTTCTTAC X X X Q L D M V H T T A G T Y E V S S H X M G A P G D C P N H G W H V R G F F P X X R C T W * M P Q P G L T S * R L I T T * V L W Y * G G C W Q V R M A V L A L Y L S I V V L R W M L A S X N G C I G T V L E Y C G I E V D V G K X E W L Y W H GTACTTGAGTATTGTGGTATTGAGGTGGATGTTGGCAAGTNAGAATGGCTGTATTGGCAC 70 8O 90 100 110 120 CATGAACTCATAACACCATAACTCCACCTACAACCGTTCANTCTTACCGACATAACCGTG Y K L I T T N L H I N A L X F P Q I P V V Q T N H Y Q P P H Q C T L I A T N A S S S Y Q P I S T S T P L X S H S Y Q C * V R V R I E E E D D N X C V M T R F L I S P G K D * R R R * * X V C N D K I L D * S G * G L K K K M I X S V * * Q D S * TAGTCCGGGTAAGGATTGAAGAAGAAGATGATAATNAGTGTGTAATGACAAGATTCTTGA 130 140 150 160 170 180 ATCAGGCCCATTCCTAACTTCTTCTTCTACTATTANTCACACATTACTGTTCTAAGAACT L G P L S Q L L L H Y X T H L S L I R S T R T L I S S S S S L X H T I V L N K I D P Y P N F F F I I I L T Y H C S E Q D TCT AGA 95 APPENDIX F p8118 Cloning vector pUC118, cloning site HincIII C M P A G R T C A V L Q V D Y R Y T * * L H A C R S N M C R A S S G L Q I Y I I L A C L Q V E H V P C F K W T T D I H N 5'CTTGCATGCCTGCAGGTCGAACATGTGCCGTGCTTCAAGTGGACTACAGATATACATAAT 10 20 30 40 50 6O 3'GAACGTACGGACGTCCAGCTTGTACACGGCACGAAGTTCACCTGATGTCTATATGTATTA K C A Q L D F M H R A E L P S C I Y M I Q M G A P R V H A T S * T S * L Y V Y Y A H R C T S C T G H K L H V V S I C L L M G R * * K L C G P C V L V A C E A L S N G E I I K A L R P M R V S G V * S I E K W G D N K S S A A H A C * W R V K H * AAATGGGGAGATAATAAAAGCTCTGCGGCCCATGCGTGTTAGTGGCGTGTGAAGCATTGA 70 80 90 100 110 120 TTTACCCCTCTATTATTTTCGAGACGCCGGGTACGCACAATCACCGCACACTTCGTAACT F P S I I F A R R G M R T L P T H L M S I P L Y Y F S Q P G H T N T A H S A N L H P S L L L E A A W A H * H R T F C Q T S C Q Y D T S * S I I I L P Q F L R Y L F L P V * Y K L E H Y N T S P I P E V P V L A S M I Q V R A L * Y F P N S * G T GTTCTTGCCAGTATGATACAAGTTAGAGCATTATAATACTTCCCCAATTCCTGAGGTACC 130 140 150 160 170 180 CAAGAACGGTCATACTATGTTCAATCTCGTAATATTATGAAGGGGTTAAGGACTCCATGG N K G T H Y L N S C * L V E G I G S T G E Q W Y S V L * L M I I S G W N R L Y R R A L I I C T L A N Y Y K G L E Q P V * I T * * A C T D F G R K A R A S * R Y V N N I V G M H G F W P K S * S Q L K V R * * H S R H A R I L A E K L E P A E G T TAATAACATAGTAGGCATGCACGGATTTTGGCCGAAAAGCTAGAGCCAGCTGAAGGTACG 190 200 210 220 230 240 ATTATTGTATCATCCGTACGTGCCTAAAACCGGCTTTTCGATCTCGGTCGACTTCCATGC L L M T P M C P N Q G F L * L W S F T R I V Y Y A H V S K P R F A L A L Q L Y T Y C L L C A R I K A S F S S G A S P V Y V K T S M H V L H L N D S D R M V G T S S Q N K H A C F T P K * L * Q N G G H I * S K Q A C M F Y T * M T L T E W W A H TAGTCAAAACAAGCATGCATGTTTTACACCTAAATGACTCTGACAGAATGGTGGGCACAT 250 260 270 280 290 300 ATCAGTTTTGTTCGTACGTACAAAATGTGGATTTACTGAGACTGTCTTACCACCCGTGTA L * F L C A H K V G L H S Q C F P P C M T L V L M C T K C R F S E S L I T P V D D F C A H M N * V * I V R V S H H A C * 96 APPENDIX F R S T G L H G C G L I I R M A P T H L T T K H G T A W L W P N Y K D G S Y P S N H E A R D C M V V A * L * G W L L P I * CACGAAGCACGGGACTGCATGGTTGTGGCCTAATTATAAGGATGGCTCCTACCCATCTAA 310 320 330 340 350 360 GTGCTTCGTGCCCTGACGTACCAACACCGGATTAATATTCCTACCGAGGATGGGTAGATT V F C P V A H N H G L * L S P E * G D L R L V P S C P Q P R I I L I A G V W R V S A R S Q M T T A * N Y P H S R G M * S V I P I V S S T N L R S Q S * * A T W K C D P D S L F D K S E I S E L M S N L E L * S R * S L R Q I * D L R A N E Q P G CTGTGATCCCGATAGTCTCTTCGACAAATCTGAGATCTCAGAGCTAATGAGCAACCTGGA 370 380 390 400 410 420 GACACTAGGGCTATCAGAGAAGCTGTTTAGACTCTAGAGTCTCGATTACTCGTTGGACCT Q S G S L R K S L D S I E S S I L L R S T I G I T E E V F R L D * L * H A V Q F H D R Y D R R C I Q S R L A L S C G P F R T G R H * A A Q A A M G S G S G P M N K N W P S L S C P S S N G F R F W S H E K E L A V T K L P K Q Q W V Q V L V P * AAAGAACTGGCCGTCACTAAGCTGCCCAAGCAGCAATGGGTTCAGGTTCTGGTCCCATGA 430 440 450 460 470 480 TTTCTTGACCGGCAGTGATTCGACGGGTTCGTCGTTACCCAAGTCCAAGACCAGGGTACT F F Q G D S L Q G L L L P N L N Q D W S L V P R * * A A W A A I P E P E P G M F S S A T V L S G L C C H T * T R T G H I G K S T E H A W E K H G T C M G K A R N M L ATGGGAAAAGCACGGAACATGCTC 3' 490 500 TACCCTTTTCGTGCCTTGTACGAG 5' H S F C P V H E P F L V S C A P F A R F M S BIBLIOGRAPHY Ai Y, Tsai D, and Kao T (1992) Cloning and sequencing of cDNAs encoding two 8 proteins of a self-compatible cultivar of Petunia hybrida. 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