Poliovirion [32P]RNA, after digestion with RNase T2, yields mononucleotides and a labeled compound "X," which is not negatively charged at pH 5. X contains, relative to the label in virion RNA, one to two phosphates and is partially acid insoluble. It can be labeled with tritiated amino acids 3 hr after infection, is insoluble in chloroform/methanol, and can be digested with Pronase. These observations suggest that X is a protein. The protein cannot be removed from the polio genome when the RNA is (i) sedimented through a sucrose gradient in 0.5 M NaCl, (ii) heated to 100 degrees in the presence of sodium dodecyl sulfate followed by sedimentation through a sucrose gradient in 80% dimethylsulfoxide, or (iii) banded in 4 M cesium trichloroacetate. Digestion of the 32P-labeled protein with Pronase yields one major 32P-labeled product, which contains pUp. The protein migrates faster than capsid protein VP4 in a polyacrylamide gel. Our data show that the genome of poliovirus, but not poliovirus mRNA [A. Nomoto, Y. F. Lee, and E. Wimmer (1976) Proc. Natl. Acad. Sci. USA 73, 375-380], is covalently attached to a small virus-coded protein (molecular weight less than 7000), which we call VPg. VPg is probably linked to the 5' end of the polio genome. Possible functions of VPg in viral replication are discussed.

TUP1 encodes a transcriptional repressor that negatively controls filamentous growth in Candida albicans. Using subtractive hybridization, we identified six genes, termed repressed by TUP1 (RBT), whose expression is regulated by TUP1. One of the genes (HWP1) has previously been characterized, and a seventh TUP1-repressed gene (WAP1) was recovered due to its high similarity to RBT5. These genes all encode secreted or cell surface proteins, and four out of the seven (HWP1, RBT1, RBT5, and WAP1) encode putatively GPI-modified cell wall proteins. The remaining three, RBT2, RBT4, and RBT7, encode, respectively, an apparent ferric reductase, a plant pathogenesis-related protein (PR-1), and a putative secreted RNase T2. The expression of RBT1, RBT4, RBT5, HWP1, and WAP1 was induced in wild-type cells during the switch from the yeast form to filamentous growth, indicating the importance of TUP1 in regulating this process and implicating the RBTs in hyphal-specific functions. We produced knockout strains in C. albicans for RBT1, RBT2, RBT4, RBT5, and WAP1 and detected no phenotypes on several laboratory media. However, two animal models for C. albicans infection, a rabbit cornea model and a mouse systemic infection model, revealed that rbt1Delta and rbt4Delta strains had significantly reduced virulence. TUP1 appears, therefore, to regulate many genes in C. albicans, a significant fraction of which are induced during filamentous growth, and some of which participate in pathogenesis.

Poliovirus was grown in HeLa cells in the presence of phosphorus-32 and actinomycin D. Three to four hours after infection, viral mRNA was recovered from polyribosomes and its identity verified by two-dimensional gel electrophoresis of RNase T1 digests. Digestion of the viral [32P]mRNA with RNase T2 and separation of the products by ion exchange chromatography at pH 5 yielded pUp as possible 5' terminus but no "capping group" of the structure m7G(5')ppp(5')Np. Total cytoplasmic [32P]RNA of HeLa cells, on the other hand, was found to contain capping groups. Neither the capping group nor ppNp or pppNp was found in an RNase T2 digest of poliovirion [32P]RNA, in agreement with previous results [Wimmer, E. (1972) J. Mol. Biol. 68, 537-540]. The data indicate that 5'-terminal m7G(5')ppp(5')Np is absent from poliovirus RNAs and, therefore, is not involved in poliovirus protein synthesis.

Neutralizing activity against T2 bacteriophage appeared in cultures of lymph node cells from normal rats in response to their in vitro stimulation with a cell-free filtrate derived from homogenized rat macrophages which had been incubated with T2 bacteriophage. This activity was specifically directed against T2 bacteriophage. It resided in a fraction of the culture fluid which had the salting-out properties of serum globulin. Phage neutralization was inhibited by antibody specific for rat serum gamma globulin. Antibody production against T2 bacteriophage in cultures of lymph node cells from normal animals failed to occur if (a) T2 bacteriophage alone was added, (b) if the incubation period of macrophages and T2 phage was unduly shortened, (c) if the cell-free filtrate was heated at 80-100 degrees C for 15 minutes, (d) if more than an optimal amount of T2 bacteriophage was added to the macrophages. Additional factors which prevented the formation of antibody were the heat inactivation of the lymph node cells or the addition to the culture medium of either streptomycin or ribonuclease. Finally, it was found that macrophages and lymph node cells had to be obtained from animals of one and the same species. All essential findings on the production of antibody to T2 bacteriophage in vitro could be confirmed by substitution of the chick embryo for the tissue culture medium. The results are discussed in terms of a possible mechanism of antibody production in which an RNAse-sensitive substance resulting from the interaction of macrophages and antigen is capable of stimulating antibody synthesis in lymphocytic cells.

The cellular response to stress conditions involves a decision between survival or cell death when damage is severe. A conserved stress response in eukaryotes involves endonucleolytic cleavage of transfer RNAs (tRNAs). The mechanism and significance of such tRNA cleavage is unknown. We show that in yeast, tRNAs are cleaved by the RNase T2 family member Rny1p, which is released from the vacuole into the cytosol during oxidative stress. Rny1p modulates yeast cell survival during oxidative stress independently of its catalytic ability. This suggests that upon release to the cytosol, Rny1p promotes cell death by direct interactions with downstream components. Thus, detection of Rny1p, and possibly its orthologues, in the cytosol may be a conserved mechanism for assessing cellular damage and determining cell survival, analogous to the role of cytochrome c as a marker for mitochondrial damage.

Poliovirus RNA that has been derivatized at the 3'-end with NaIO(4)-NaB(3)H(4) yields, after hydrolysis with alkali or RNase T2, predominantly labeled residues of modified adenosine; no labeled nucleoside derivative is produced by digestion with RNase A or RNase T1. The 3'-terminal bases of the RNA are, therefore,...ApA(OH). Hydrolyzates of poliovirus [(32)P]RNA, after exhaustive digestion with RNase T1 or RNase A, contain, besides internal oligonucleotides, polynucleotides resistant to further action of ribonucleases T1 and A, respectively; these polynucleotides were isolated by membrane-filter binding or ion-exchange chromatography. The sequence of the T1-resistant polynucleotide was determined to be (Ap)(n)A(OH), that of the RNase A-resistant polynucleotide was GpGp(Ap)(n)A(OH). The chain length (n) of the polyadenylic acid, as analyzed by different methods, averages 89 nucleotides. Gel electrophoresis revealed heterogeneity of the size of poly(A). Poliovirus RNA, when labeled in vitro at the 3'-end, contains [3'-(3)H]poly(A); when labeled in vivo with [(3)H]A, it contains [(3)H](Ap)(n)A(OH). The data establish that... YpGpGp(Ap)([unk])A(OH) is the 3'-terminal sequence of poliovirus RNA, Type 1 (Mahoney). Since this mammalian virus reproduces in the cell cytoplasm, these observations may modify prior interpretations of the function of polyadenylate ends on messenger RNAs.

Sequence analysis of 5'-[32P] labeled tRNA and eukaryotic mRNA using an adaptation of a method recently described by Donis-Keller, Maxam and Gilbert for mapping guanines, adenines and pyrimidines from the 5'-end of an RNA is described. In addition, a technique utilizing two-dimensional polyacrylamide gel electrophoresis for identification of pyrimidines within a sequence is described. 5'-[32P] Labeled rabbit beta-globin mRNA and N. crassa mitochondrial initiator tRNA were partially digested with T1- RNase for cleavage at G residues, with U2-RNase for cleavage at A residues, with an extracellular RNase from B. cereus for cleavage at pyrimidine residues and with T2-RNase or with alkali for cleavage at all four residues. The 5'-[32P] labeled partial digestion products were separated according to their size, by electrophoresis in adjacent lanes of a polyacrylamide slab gel and the location of G's, A's and of pyrimidines extending 60-80 nucleotides from the 5'-end of the RNA determined. Two-dimensional polyacrylamide gel electrophoresis was used to separate the 5'-[32P] labeled fragments present in partial alkali digests of a 5'-[32P] labeled mRNA. The mobility shifts corresponding to the difference of a C residue were distinct from those corresponding to a U residue and this formed the basis of a method for distinguishing between the pyrimidines.

Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming.

The poly A-containing mRNA of cultured hamster (BHK-21) cells has been examined with regard to methylation status. Steady state-labeled mRNA was obtained by incubating cells for 20-22h in the presence of [methyl-3H]-methionine and 32Pi. The degree of methylation of this RNA was 1.8 methyl groups per 1000 nucleotides, or 4-5 methyl groups on the average per molecule. The nature of the methylated residues was determined by paper chromatography and electrophoresis of acid and alkaline hydrolysates, by DEAE cellulose chromatography of alkaline hydrolysates and of T2 RNase digests, and by examining the effect of subjecting samples to "beta-elimination." Approx. half of the methyl groups occurred in standard ("internal") linkage, 10% as m5Cp and 40% as m6Ap residues. The remainder occurred at least for the most part in "blocked" 5'-termini with the presumptive structure m7G(5')ppp(Nm)p.., where Nm was Gm, m6Am, Um, or Cm.

The MutS, MutL, and MutH proteins play major roles in several DNA repair pathways. We previously reported that the cellular amounts of MutS and MutH decreased by as much as 10-fold in stationary-phase cultures. Consequently, we tested whether the amounts of MutS, MutL, and MutH were regulated by two global regulators, RpoS (sigma38) and Hfq (HF-I [putative RNA chaperone]), which are involved in stationary-phase transition. We report here that mutations in hfq and rpoS reversed the stationary-phase down-regulation of the amounts of MutS and MutH. hfq regulation of the amount of MutS in stationary-phase cultures was mediated by RpoS-dependent and -independent mechanisms, whereas hfq regulation of the amount of MutH was mediated only through RpoS. Consistent with this interpretation, the amount of MutS but not MutH was regulated by Hfq, but not RpoS, in exponentially growing cells. The amount of MutL remained unchanged in rpoS, hfq-1, and rpoS+, hfq+ strains in exponentially growing and stationary-phase cultures and served as a control. The beta-galactosidase activities of single-copy mutS-lacZ operon and gene fusions suggested that hfq regulates mutS posttranscriptionally in exponentially growing cultures. RNase T2 protection assays revealed increased amounts of mutS transcript that are attributed to increased mutS transcript stability in hfq-1 mutants. Lack of Hfq also increased the amounts and stabilities of transcripts initiated from P(miaA) and P1hfqHS, two of the promoters for hfq, suggesting autoregulation, but did not change the half-life of bulk mRNA. These results suggest that the amounts of MutS and MutH may be adjusted in cells subjected to different stress conditions by an RpoS-dependent mechanism. In addition, Hfq directly or indirectly regulates several genes, including mutS, hfq, and miaA, by an RpoS-independent mechanism that destabilizes transcripts.

T2-type RNases are responsible for self-pollen recognition and rejection in three distantly related families of flowering plants-the Solanaceae, Scrophulariaceae, and Rosaceae. We used phylogenetic analyses of 67 T2-type RNases together with information on intron number and position to determine whether the use of RNases for self-incompatibility in these families is homologous or convergent. All methods of phylogenetic reconstruction as well as patterns of variation in intron structure find that all self-incompatibility RNases along with non-S genes from only two taxa form a monophyletic clade. Several lines of evidence suggest that the best interpretation of this pattern is homology of self-incompatibility RNases from the Scrophulariaceae, Solanaceae, and Rosaceae. Because the most recent common ancestor of these three families is the ancestor of approximately 75% of dicot families, our results indicate that RNase-based self-incompatibility was the ancestral state in the majority of dicots.

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed. In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site A0. The sites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.

A single capped oligonucleotide is released from Trypanosoma brucei poly(A)+ RNA upon digestion with RNase T2. This observation supports the hypothesis that all T. brucei mRNAs share a common leader sequence. Digestion of the T2-resistant species with nucleotide pyrophosphatase shows that the capping nucleotide is 7-methylguanosine 5'-monophosphate (pm7G). Additional characterization of the T2-resistant fragment indicates that modifications are present on the first four transcribed nucleotides; the 5' termini of T. brucei mRNAs can, therefore, be described as "cap 4" structures. Identical 5'-cap structures are found on the T. brucei spliced leader (SL) RNA; an observation compatible with the hypothesis that the small SL RNA acts as a donor of the SL for the mRNA. However, we find that within a population of purified SL RNAs are species that are capped but incompletely modified. The presence of these unmodified and partially modified species allowed us to analyze the 5' sequence of the SL RNA transcript. The results indicate that transcription begins four nucleotides upstream of the reported 5' end. Therefore, the T. brucei SL transcript is actually 39 rather than 35 nucleotides long. We have also analyzed the capped oligonucleotide of a distantly related Trypanosomatid, Leptomonas collosoma and find it to be identical to that of T. brucei. The potential significance of these results is discussed in light of observations of trypanosome gene expression.

The MutL, MutS, and MutH proteins mediate methyl-directed mismatch (MDM) repair and help to maintain chromosome stability in Escherichia coli. We determined the amounts of the MDM repair proteins in exponentially growing, stationary-phase, and nutrient-starved bacteria by quantitative Western immunoblotting. Extracts of null mutants containing various amounts of purified MDM repair proteins were used as quantitation standards. In bacteria growing exponentially in enriched minimal salts-glucose medium, about 113 MutL dimers, 186 MutS dimers, and 135 MutH monomers were present per cell. Calculations with the in vitro dissociation constants of MutS binding to different mismatches suggested that MutS is not present in excess, and may be nearly limiting in some cases, for MDM repair in exponentially growing cells. Remarkably, when bacteria entered late stationary phase or were deprived of a utilizable carbon source for several days, the cellular amount of MutS dropped at least 10-fold and became barely detectable by the methods used. In contrast, the amount of MutH dropped only about threefold and the amount of MutL remained essentially constant in late-stationary-phase and carbon-starved cells compared with those in exponentially growing bacteria. RNase T2 protection assays showed that the amounts of mutS, mutH, and mutL, but not miaA, transcripts decreased to undetectable levels in late-stationary-phase cells. These results suggested that depletion of MutS in nutritionally stressed cells was possibly caused by the relative instability of MutS compared with MutL and MutH. Our findings suggest that the MDM repair capacity is repressed in nutritionally stressed bacteria and correlate with conclusions from recent studies of adaptive mutagenesis. On the other hand, we did not detect induction of MutS or MutL in cells containing stable mismatches in multicopy single-stranded DNA encoded by bacterial retrons.

The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has been of considerable value in a variety of applications. However, Pasquinelli et al. [RNA (1995) 1:957-967] found that one-third to one-half of the caps are incorporated in the reverse orientation, that is, with the m7G moiety of m7GpppG linked by a 3'-5' phosphodiester bond to the first nucleotide residue of the RNA chain. Such reverse caps are unlikely to be recognized by eIF4E, based on previous studies, and thus complicate any comparison of the translational efficiencies of in vitro-synthesized mRNAs. We therefore designed two novel cap analogs, P(1)-3'-deoxy-7-methyguanosine-5' P3-guanosine-5' triphosphate and P(1)-3'-O,7-dimethylguanosine-5' P3-guanosine-5' triphosphate, that are, theoretically, incapable of being incorporated in the reverse orientation. The key reactions of pyrophosphate bond formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts. Structures were proven by 1H NMR. Transcripts produced with SP6 polymerase using "anti-reverse" cap analogs (ARCAs) were of the predicted length and indistinguishable in size and homogeneity from those produced with m7GpppG or GpppG. Analysis of the transcripts with RNase T2 and tobacco acid pyrophosphatase indicated that reverse caps were formed with m7GpppG but not with ARCAs. Both of the ARCAs inhibited cell-free translation with a K(I) similar to that of m7GpppG. Finally, the translational efficiency of ARCA-capped transcripts in a rabbit reticulocyte lysate was 2.3- to 2.6-fold higher than that of m7GpppG-capped transcripts. This suggests the presence of reverse caps in conventional in vitro-synthesized mRNAs reduces their translational efficiency.

OBJECTIVE

The RNASE III endonuclease Dicer is one of the key enzymes of microRNA biogenesis. The influence of Dicer-expression in tumour cells on the prognosis of patients with several cancers has been studied with controversial results among different cancer types. To date no one has examined the effect of this biomarker on survival in colorectal carcinoma. Thus, we aimed to study the influence of Dicer expression on survival in colorectal cancer.

METHODS

We performed immunohistochemical analyses on formalin-fixed paraffin embedded (FFPE) cancer tissue with an antibody against the Dicer protein. Tumour material from 237 cases was available from patients with colorectal adeonocarcinomas with moderate differentiation (G2) and without evidence of lymph-node (N0) or distant metastasis (M0). Sixty-four cases were in T2 and 173 in T3 stages. A tissue microarray (TMA) was constructed with each tumour in triplicate. Each tumour was assigned to a scoring scale of 0-3, depending on the cytoplasmatic expression of Dicer. A Kaplan-Maier analysis was performed and the log-rank test was used for significance levels by using SPSS v.17 software.

RESULTS

The expression of Dicer in colorectal carcinoma shows a strong association with poor survival (cancer specific survival=CSS, p<0,001) as well as with reduced progression free survival (PFS, p<0,001). In the group with no Dicer staining there was no recorded relapse (0/15) compared with 10/18 relapses in the group with the strongest staining of Dicer.

CONCLUSIONS

Strong expression of the central microRNA biosynthesis enzyme Dicer predicts poor prognosis in patients with colorectal cancer. This is in line with investigations on prostate cancer. Contradictory, in breast, lung and ovary cancer Dicer has been shown to be a marker of good prognosis. Further studies on the cellular functions of Dicer need to address these issues.

A repeating structure of cytoplasmic poly(A)-ribonucleoprotein is revealed by digestion with T2 RNase. A pattern of fragments that are multiples of about 27 residues is obtained. The repeating structure is readily reconstituted from purified poly(A) and cytoplasmic factors. Reconstitution is specific for poly(A), as shown by the lack of competition by poly(G), poly(C), poly(dA), and tRNA. The repeating structure is absent from the nucleus, and so appears to be formed upon transport to the cytoplasm.

tRNAs, like other RNAs, are subject to quality control steps during and after biosynthesis. We previously described a rapid tRNA degradation (RTD) pathway in which the 5'-3' exonucleases Rat1 and Xrn1 degrade mature tRNA(Val(AAC)) in yeast mutants lacking m(7)G and m(5)C, and mature tRNA(Ser(CGA)) in mutants lacking Um and ac(4)C. To understand how the RTD pathway selects substrate tRNAs among different tRNAs lacking the same modifications, we used a genetic screen to examine tRNA(Ser(CGA)) variants. Our results suggest that RTD substrate recognition in vivo depends primarily on the stability of the acceptor and T-stems, and not the anti-codon stem, and does not necessarily depend on modifications, since fully modified tRNAs are subject to RTD if appropriately destabilized. We found that weaker predicted stability of the acceptor and T-stems of tRNAs is strongly correlated with RTD sensitivity, increased RNase T2 sensitivity of this region of the tRNA in vitro, and increased exposure of the 5' end to phosphatase. We also found that purified Xrn1 selectively degrades RTD substrate tRNAs in vitro under conditions in which nonsubstrates are immune. These results suggest that tRNAs have evolved not only for accurate translation, but for resistance to attack by RTD.

A rapid, simple, and highly sensitive method for sequence analysis of RNA was developed, which consists of the following steps: (i) controlled hydrolysis of the RNA by brief heating in water; (ii) (32P)-labeling of 5'-hydroxyl groups of the fragments produced in (i); (iii) resolution of labeled fragments by size on polyacrylamide gels giving the familiar "ladder"; (iv) contact transfer ("print") of the ladder from the gel to a PEI-cellulose thin layer; (v) in situ treatment of the ladder with RNase T2 resulting in the release of 5'-(32P)-labeled nucleoside-3',5' diphosphates; (vi) contact transfer and thin-layer separation of (32P)-labeled nucleotides on PEI-cellulose in ammonium sulfate and ammonium formate solvents; (vii) autoradiography. The chromatographic behavior of the 4 major and 18 modified nucleotides was determined. The positions of major and modified nucleotides in the sequence can be read directly from the separation patterns displayed on X-ray film. As this is the only sequencing method presently available that allows one to display and identify directly the positions in the RNA chain of major and modified nucleotides, no additional procedures are required to analyze the latter.

The S-like ribonucleases (RNases) RNS1 and RNS2 of Arabidopsis are members of the widespread T2 ribonuclease family, whose members also include the S-RNases, involved in gametophytic self-incompatibility in plants. Both RNS1 and RNS2 mRNAs have been shown previously to be induced by inorganic phosphate (Pi) starvation. In our study we examined this regulation at the protein level and determined the effects of diminishing RNS1 and RNS2 expression using antisense techniques. The Pi-starvation control of RNS1 and RNS2 was confirmed using antibodies specific for each protein. These specific antibodies also demonstrated that RNS1 is secreted, whereas RNS2 is intracellular. By introducing antisense constructs, mRNA accumulation was inhibited by up to 90% for RNS1 and up to 65% for RNS2. These plants contained abnormally high levels of anthocyanins, the production of which is often associated with several forms of stress, including Pi starvation. This effect demonstrates that diminishing the amounts of either RNS1 or RNS2 leads to effects that cannot be compensated for by the actions of other RNases, even though Arabidopsis contains a large number of different RNase activities. These results, together with the differential localization of the proteins, imply that RNS1 and RNS2 have distinct functions in the plant.

(1) N2,N2,7-Trimethylguanosine, not previously detected as a component of plant RNA, is shown to be present in the RNA which is isotopically labelled when dry wheat embryos imbibe water in a medium that contains[methyl-3H]methionine. (2) N2,N2,7-Trimethylguanosine and 7-methylguanosine are released as part of "capped" oligonucleotides when the isotopically labelled RNA from imbibing wheat embryos is subjected to hydrolysis by RNase T2. (3) By way of contrast with the "capped" RNA of animal cells, 5'-terminal "cap" structures (m7Gppp- and m32,2,7 Gppp-) in the "capped" RNA from the higher plant organism are not bonded to pneultimate O2'-methylnucleoside constituents. (4) In an allied study, it has been found that recovery of poly (A)-rich RNA from dry wheat embryos depends on the inclusion of sodium dodecyl sulphate (SDS) in phosphate-buffered (pH 6.8) phenolic emulsions. By way of contrast, recovery of poly (A)-rich RNA from dry wheat embryos does not depend on the inclusion of SDS in Tris (hydroxymethyl) aminomethane buffered (pH 9.0) phenolic emulsions.

Stylar ribonucleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus x domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.

The miaA tRNA modification gene was cloned and located by insertion mutagenesis and DNA sequence analysis. The miaA gene product, tRNA delta 2-isopentenylpyrophosphate (IPP) transferase, catalyzes the first step in the biosynthesis of 2-methylthio-N6-(delta 2-isopentenyl)-adenosine (ms2i6A) adjacent to the anticodon of several tRNA species. The translation start of miaA was deduced by comparison with mod5, which encodes a homologous enzyme in yeasts. Minicell experiments showed that Escherichia coli IPP transferase has a molecular mass of 33.5 kilodaltons (kDa). Transcriptional fusions, plasmid and chromosomal cassette insertion mutations, and RNase T2 mapping of in vivo miaA transcription were used to examine the relationship between miaA and mutL, which encodes a polypeptide necessary for methyl-directed mismatch repair. The combined results showed that miaA, mutL, and a gene that encodes a 47-kDa polypeptide occur very close together, are transcribed in the same direction in the order 47-kDa polypeptide gene-mutL-miaA, and likely form a complex operon containing a weak internal promoter. Three additional relationships were demonstrated between mutagenesis and the miaA gene or ms2i6A tRNA modification. First, miaA transcription was induced by 2-aminopurine. Second, chromosomal miaA insertion mutations increased the spontaneous mutation frequency with a spectrum distinct from mutL mutations. Third, limitation of miaA+ bacteria for iron, which causes tRNA undermodification from ms2i6A to i6A, also increased spontaneous mutation frequency. These results support the notion that complex operons organize metabolically related genes whose primary functions appear to be completely different. In addition, the results are consistent with the idea that mechanisms exist to increase spontaneous mutation frequency when cells need to adapt to environmental stress.

cDNAs encoding three S-RNases of almond (Prunus dulcis), which belongs to the family Rosaceae, were cloned and sequenced. The comparison of amino acid sequences between the S-RNases of almond and those of other rosaceous species showed that the amino acid sequences of the rosaceous S-RNases are highly divergent, and intra-subfamilial similarities are higher than inter-subfamilial similarities. Twelve amino acid sequences of the rosaceous S-RNases were aligned to characterize their primary structural features. In spite of their high level of diversification, the rosaceous S-RNases were found to have five conserved regions, C1, C2, C3, C5, and RC4 which is Rosaceae-specific conserved region. Many variable sites fall into one region, named RHV. RHV is located at a similar position to that of the hypervariable region a (HVa) of the solanaceous S-RNases, and is assumed to be involved in recognizing S-specificity of pollen. On the other hand, the region corresponding to another solanaceous hypervariable region (HVb) was not variable in the rosaceous S-RNases. In the phylogenetic tree of the T2/S type RNase, the rosaceous S-RNase fall into two subfamily-specific groups (Amygdaloideae and Maloideae). The results of sequence comparisons and phylogenetic analysis imply that the present S-RNases of Rosaceae have diverged again relatively recently, after the divergence of subfamilies.