Trypanosoma brucei mRNA is discontinuously synthesized via the 5' addition of a "mini-exon" sequence. The mini-exon-specific cap structure was purified from a complete RNase T2 and phosphatase digest of in vivo 32P-labeled poly(A)+RNA. The purified cap structure was sequenced by a series of partial and complete enzymatic digests by nuclease P1 and venom phosphodiesterase. This approach demonstrated that the T. brucei mini-exon cap structure consists of N7-methylguanosine linked in a conventional 5'-5' triphosphate bond to five nucleotides, in the sequence A*A*C(2'-O)mU*A (asterisks denote modifications that were not fully characterized in this work). 2'-O-methylations and other modifications appear to be present in this novel cap structure, which could have a functional role in the metabolism of the mini-exon.

RNA splicing in archaea requires at least an endonuclease and a ligase, as is the case for the splicing of eukaryal nuclear tRNAs. Splicing endonucleases from archaea and eukarya are homologous, although they differ in subunit composition and substrate recognition properties. However, they all produce 2',3' cyclic phosphate and 5'-hydroxyl termini. An in vitro-transcribed, partial intron-deleted Haloferax volcanii elongator tRNA(Met) has been used to study splicing by H. volcanii cell extracts. Substrates and products were analyzed by nearest neighbor analyses using nuclease P1 and RNase T2, and fingerprinting analyses using acid-urea gels in the first dimension and gradient thin layer chromatography in the second dimension. The results suggest that 2',3' cyclic phosphate at the 3' end of the 5' exon is converted into the splice junction phosphate forming a 3',5'-phosphodiester linkage. This resembles the animal cell type systems where the junction phosphate preexists in the transcript, and differs from yeast type systems, where GTP is the source of junction phosphate.

The T(2) family of nonspecific endoribonucleases (EC ) is a widespread family of RNases found in every organism examined thus far. Most T(2) enzymes are secretory RNases and therefore are found extracellularly or in compartments of the endomembrane system that would minimize their contact with cellular RNA. Although the biological functions of various T(2) RNases have been postulated on the basis of enzyme location or gene expression patterns, the cellular roles of these enzymes are generally unknown. In the present work, we characterized Rny1, the only T(2) RNase in Saccharomyces cerevisiae. Rny1 was found to be an active, secreted RNase whose gene expression is controlled by heat shock and osmotic stress. Inactivation of RNY1 leads to unusually large cells that are temperature-sensitive for growth. These phenotypes can be complemented not only by RNY1 but also by both structurally related and unrelated secretory RNases. Additionally, the complementation depends on RNase activity. When coupled with a recent report on the effect of specific RNAs on membrane permeability [Khvorova, A., Kwak, Y-G., Tamkun, M., Majerfeld, I. & Yarus, M. (1999) Proc. Natl. Acad. Sci. USA 96, 10649-10654], our work suggests an unexpected role for Rny1 and possibly other secretory RNases. These enzymes may regulate membrane permeability or stability, a hypothesis that could present an alternative perspective for understanding their functions.

BACKGROUND

Human RNASET2 is a T2-RNase glycoprotein encoded by the RNASET2 gene, which is located on chromosome 6 (6q27). Deletion in 6q27 is associated with several human malignancies.

METHODS

A synthetic RNASET2 gene that was optimized for expression in the yeast Pichia pastoris was designed according to the cDNA sequence and was cloned under the control of the methanol-induced promoter fused to the alpha-mating secretion peptide. The recombinant protein was purified from the culture supernatant of transformed P. pastoris through an affinity Sepharose-concanavalin A column. Actin-binding activity was examined by membrane blotting using monoclonal mouse antiactin immunoglobulin M and by cross-linking in solution to G-actin using 1-[3-(dimethylamino)propyl]-3-ethyl-carboimide methiodide. The antiangiogenic activity of RNASET2 (from 0.5 microM to 10 microM) was assessed by a human umbilical vein endothelial (HUVE) cell assay in the presence of 1 microg/mL angiogenin, basic fibroblast growth factor (bFGF), or recombinant human vascular endothelial growth factor (VEGF). Cell colony formation was examined in human colon HT2T2 or the enzymatic-inactivated RNASET2 (EI-RNASET2) (1 microM each). In an athymic mouse xenograft model, LS174T human cancer cells were injected subcutaneously. When tumors were palpable, the mice were treated for 3 weeks with RNASET2 (1 mg/kg), paclitaxel (10 mg/kg or 15 mg/kg), or a combination of the 2 drugs.

RESULTS

The recombinant RNASET2 was identified as a 27-kilodalton glycoprotein that possessed the ability to bind actin in vitro. RNASET2 significantly inhibited clonogenicity in HT2T2 produced a similar effect, suggesting that its antitumorigenic activity is unrelated to its RNase activity. In HUVE cells, RNASET2 inhibited angiogenin-, bFGF-, and VEGF-induced tube formation in a dose-dependent manner. In athymic mice, RNASET2 inhibited the development of an LS174T-derived xenograft by 40%. A synergistic effect was obtained with combined RNASET2 and paclitaxel treatments.

CONCLUSIONS

The current results suggested that RNASET2 represents a new class of antitumorigenic and antiangiogenic drugs, and the findings of this study emphasize the advantage of using agents like RNASET2 in combined therapy.

The backbone dynamics of the uniformly 15N-labeled ribonuclease H (RNase H) domain of human immunodeficiency virus (HIV-1) reverse transcriptase have been investigated using two-dimensional inverse-detected heteronuclear 15N-1HNMR spectroscopy. 15N T1, T2, and nuclear Overhauser enhancement (NOE) data were obtained for 107 out of a total of 134 backbone amide groups. The overall rotational correlation time (tau R) for the protein at 26 degrees C is 10.4 ns. The backbone N-H vectors for all the measurable residues exhibit very fast motions on a time scale of less than or equal to 20 ps. The 15N relaxation data for only 14 residues can be explained by this single internal motion alone. A further 39 residues display a second motion on a time scale ranging from 28.8 ps to 3.9 ns, while another 15 residues are characterized by an additional motion on the 170-ns to 2.25-ms time scale resulting in 15N T2 exchange line broadening. There are 39 residues that exhibit both the additional 15N T2 exchange line broadening and the slow (28.8 ps-3.9 ns) internal motion. Thus, the RNase H domain experiences extensive mobility throughout its structure as evidenced by the 93 residues which exhibit multiple modes of motion. Distinctly mobile regions of the protein are identified by large decreases in the overall order parameter (S2) and correspond to the C-terminal residues and the loop regions between beta-strands beta 1 and beta 2 and between alpha-helix alpha B and beta-strand beta 4.(ABSTRACT TRUNCATED AT 250 WORDS)

A subcellular system is described which is capable of in vitro synthesis of large nuclear RNA and the formation of both cap I [m7G(5')pppXmpYp] and capII [m7G(5')-pppXmpYmpZp] structures. This system, which consists of partially purified intact nuclei and residual cytoplasmic tags, carries out both guanosine addition, utilizing GTP, and the appropriate methylation reactions, utilizing S-adenosylmethionine as the methyl donor. The general structure of the caps was verified by analyses of methylated derivatives recovered after RNase T2 hydrolysis and after digestion with P1 nuclease, bacterial alkaline phosphatase,and nucleotide pyrophosphatase. Cap formation in large nuclear RNA species was found to be closely associated with transcription, as indicated by alpha-manitin sensitivity and a requirement for the presence of all four nucleoside triphosphates. Recovery of a class of cap II structures, in which only the methyl group at position Y is labeled, as well as capII structures in which all methylated constituents are labeled, indicates the presence of at least two independent methylation events in the in vitro system.

A procedure is described for the synthesis of the title compounds via phosphotriester intermediates. The 2-cyanoethyl group is used to protect the P-SH function during the course of the synthesis. Resolution of the phosphorus diastereomers is accomplished at the phosphotriester stage. Removal of the 2-cyanoethyl group without racemization, followed by removal of the other protective groups, affords the optically pure diastereomers of 5'-O-adenosyl 3'-O-uridyl phosphorothioate. Their designation as Rp and Sp follows from the stereospecificity in the hydrolysis catalyzed by RNase A. These diastereomers are useful for the investigation of the stereospecificity as well as of the stereochemical course of action of nucleases. Snake venom exonuclease hydrolyses only the Rp diastereomer, whereas both diastereomers are substrates for RNases A and T2. The results with the latter indicate that RNase T2 also operates by an in-line mechanism.

Although present in different organisms and conserved in their protein sequence, the biological functions of T2 ribonucleases (RNase) are generally unknown. Tomato (Lycopersicon esculentum) LX is a T2/S-like RNase and its expression is known to be associated with phosphate starvation, ethylene responses, and senescence and programmed cell death. In this study, LX function was investigated using antisense tomato plants in which the LX protein level was reduced. LX protein levels normally become elevated when leaves senesce and antisense inhibition of LX retarded the progression of senescence. Moreover, we observed a marked delay of leaf abscission in LX-deficient plants. This correlated with specific induction of LX protein in the tomato mature abscission zone tissue. LX RNase gene regulation and the consequences of antisense inhibition indicate that LX has an important functional role in both abscission and senescence.

We previously identified a novel regulator of the exotoxin A gene (toxA) in Pseudomonas aeruginosa, PtxR, that belongs to the LysR family of prokaryotic regulatory proteins. Preliminary data also suggest that PtxR affects the expression of siderophores in P. aeruginosa. Because toxA expression and siderophore production in this organism are coordinately regulated by the ferric uptake regulator (Fur) and the Fur-regulated alternative sigma factor PvdS, regulation of ptxR itself in the context of these regulators was examined. RNase protection analyses of ptxR transcription revealed that there are two independent transcription initiation sites (T1 and T2). While transcription from the promoter of T1 is constitutive throughout the growth cycle of PAO1, transcription from the second promoter (P2) is negatively affected by iron. Transcription from the P2 promoter is constitutive in a fur mutant under microaerobic conditions but still iron regulated during aerobic growth. High concentrations (>100 nM) of the ferric uptake regulatory protein (Fur) failed to bind to either of the promoter regions of ptxR in either gel mobility shift assays or DNase I footprint experiments. These results indicate that Fur indirectly regulates the iron-dependent expression of ptxR. Iron-regulated transcription of ptxR from the P2 promoter, but not constitutive expression from the P1 promoter, was dependent on the Fur-regulated alternative sigma factor gene pvdS, even under aerobic conditions. Consequently, there are two levels of iron-regulated expression of ptxR. The iron-regulated expression of ptxR under microaerobic conditions from the P2 promoter of ptxR is mediated indirectly by Fur through the iron-regulated expression of pvdS. In contrast, pvdS-mediated iron regulation of ptxR under aerobic conditions is Fur independent.

To investigate the role of 5' noncoding leader sequence of herpes simplex virus type 1 (HSV-1) mRNA in infected cells, the promoter for the 65,000-dalton virion stimulatory protein (VSP), a beta-gamma polypeptide, was introduced into plasmids bearing the chloramphenicol acetyltransferase (cat) gene together with various lengths of adjacent viral leader sequences. Plasmids containing longer lengths of leader sequence gave rise to significantly higher levels of CAT enzyme in transfected cells superinfected with HSV-1. RNase T2 protection assays of CAT mRNA showed that transcription was initiated from an authentic viral cap site in all VSP-CAT constructs and that CAT mRNA levels corresponded to CAT enzyme levels. Use of cis-linked simian virus 40 enhancer sequences demonstrated that the effect was virus specific. Constructs containing 12 and 48 base pairs of the VSP mRNA leader gave HSV infection-induced CAT activities intermediate between those of the leaderless construct and the VSP-(+77)-CAT construct. Actinomycin D chase experiments demonstrated that the longest leader sequences increased hybrid CAT mRNA stability at least twofold in infected cells. Cotransfection experiments with a cosmid bearing four virus-specified transcription factors (ICP4, ICP0, ICP27, and VSP-65K) showed that sequences from -3 to +77, with respect to the viral mRNA cap site, also contained signals responsive to transcriptional activation.

We have isolated and sequenced cDNAs for S2- and S3-alleles of the self-incompatibility locus (S-locus) in Solanum chacoense Bitt., a wild potato species displaying gametophytic self-incompatibility. The S2- and S3-alleles encode pistil-specific proteins of 30 kDa and 31 kDa, respectively, which were previously identified based on cosegregation with their respective alleles in genetic crosses. The amino acid sequence homology between the S2- and S3-proteins is 41.5%. This high degree of sequence variability between alleles is a distinctive feature of the S-gene system. Of the 31 amino acid residues which were previously found to be conserved among three Nicotiana alata S-proteins (S2, S3, and S6) and two fungal ribonucleases (RNase T2 and RNase Rh), 27 are also conserved in the S2- and S3-proteins of S. chacoense. These residues include two histidines implicated in the active site of the RNase T2, six cysteines, four of which form disulfide bonds in RNase T2, and hydrophobic residues which might form the core structure of the protein. The finding that these residues are conserved among S-proteins with very divergent sequences suggests a functional role for the ribonuclease activity of the S-protein in gametophytic self-incompatibility.

BACKGROUND

ACTIBIND is an Aspergillus niger extracellular ribonuclease (T2-ribonuclease [RNase]) that possesses actin-binding activity. In plants, ACTIBIND inhibits the elongation and alters the orientation of pollen tubes by interfering with the intracellular actin network. The question rose whether ACTIBIND can also affect mammalian cancer development.

METHODS

Cell colony formation was performed in human colon (HT-29, Caco-2, RSB), breast (ZR-75-1), and ovarian (2780) cancer cells in the presence or absence of 1 muM ACTIBIND. In HT-29 and ZR-75-1 cells, the effect of ACTIBIND on cell migration was studied by microscopic observations and by invasion assay through Matrigel. Tube formation was assessed in human umbilical vein endothelial cells (HUVEC) in the presence of angiogenin or basic fibroblast growth factor (bFGF) (1 microg/mL each) following overnight incubation with 1 or 10 microM ACTIBIND. In an athymic mouse xenograft model, HT-29 cells were injected subcutaneously, followed by subcutaneous (0.4-8 mg/mouse/injection) or intraperitoneal (0.001-1 mg/mouse/injection) injections of ACTIBIND. In a rat dimethylhydrazine (DMH)-colorectal carcinogenesis model, ACTIBIND was released directly into the colon via osmotic micropumps (250 microg/rat/day) or given orally via microcapsules (1.6 mg/rat/day). Aberrant crypt foci, tumors in the distal colon, and tumor blood vessels were examined.

RESULTS

ACTIBIND had an anticlonogenic effect unrelated to its ribonuclease activity. It also inhibited angiogenin-induced HUVEC tube formation in a dose-responsive manner. ACTIBIND was found to bind actin in vitro. It also bound to cancer cell surfaces, leading to disruption of the internal actin network and inhibiting cell motility and invasiveness through Matrigel-coated filters. In mice, ACTIBIND inhibited HT-29 xenograft tumor development, given either as a subcutaneous or intraperitoneal treatment. In rats, ACTIBIND exerted preventive and therapeutic effects on developing colonic tumors induced by DMH. It also reduced the degree of tumor observation.

CONCLUSIONS

This study indicated that ACTIBIND is an effective antiangiogenic and anticarcinogenic factor.

Digestion with ribonuclease T2 has been used to study the size of poly(U) protected by ribosome binding. Several different preparations of ribosomes all appear to cover 49 nucleotides of message; however, there are two partially accessible internal nuclease cleavage sites, which yield, ultimately, fragments 20, 16 and 13 nucleotides in length. Curiously, the site between fragments of length 20 and 16 is accessible to RNase T2 but not to the several much smaller RNases. Arguments based on the quantitative pattern of cleavage and comparisons with previous studies lead to the conclusion that the 20-mer is the 5' fragment, while 13-mer (which is lost the moment it is cleaved from the 16-mer) is the 3' fragment. Both ribosome-bound tRNAs appear to contact only the 16-mer. The presence of the two internal cleavage sites fits nicely with recent electron microscopic data suggesting that mRNA forms a loop around the 30 S subunit.

32P-labeled, late simian virus 40-specific RNA was isoalted from infected CV1 cells and completely degraded with RNase T2 and bacterial alkaline phosphatase. The RNase-resistant material was fractionated two dimensionally and further characterized with Penicillium nuclease and nucleotide pyrophosphatase. Two major 5' termini were identified in late simian virus 40 RNA, namely, 7-methyl Gppp 2',6-dimethyl ApUp and 7-methyl Gppp 2',6-dimethyl Ap 2'-methyl, UpUp. Both 5' termini are present in unfractionated viral RNA as well as in the separated 16S and 19S species. As both caps differ only in secondary modification, it is possible that they are derived from the same site on the DNA. The relatively higher cap II content of the 16S mRNA may be related to its slower rate of turnover.

The stability of mRNA in Escherichia coli cells changed after phage T4 infection. Stable E. coli mRNAs such as lpp and ompA were drastically destabilized immediately after infection. In contrast, T4 phage soc mRNA that had been unstable before infection became stabilized after infection. The host RNases E and G both contributed to the destabilization of these mRNAs. Accordingly, these RNases may alter their target RNAs before and after infection. An RNA chaperon, Hfq, and polyadenylation at 3' ends of mRNA are known key factors for destabilization of ompA and lpp mRNAs in uninfected cells. However, they had no effect on the destabilization of E. coli mRNAs after infection. On the other hand, T4 infection in the presence of rifampicin or infection of a deletion mutant, Deltatk2, did not destabilize the host mRNAs. These results strongly suggest that a phage-encoded factor is responsible for the destabilization of host mRNAs. Destabilization of host mRNAs was also observed after infection by phages T2 and T7.

Type II protein secretion (T2S) by Legionella pneumophila is required for intracellular infection of host cells, including macrophages and the amoebae Acanthamoeba castellanii and Hartmannella vermiformis. Previous proteomic analysis revealed that T2S by L. pneumophila 130b mediates the export of >25 proteins, including several that appeared to be novel. Following confirmation that they are unlike known proteins, T2S substrates NttA, NttB, and LegP were targeted for mutation. nttA mutants were impaired for intracellular multiplication in A. castellanii but not H. vermiformis or macrophages, suggesting that novel exoproteins which are specific to Legionella are especially important for infection. Because the importance of NttA was host cell dependent, we examined a panel of T2S substrate mutants that had not been tested before in more than one amoeba. As a result, RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all proved to be required for optimal intracellular multiplication in H. vermiformis but not A. castellanii. Further examination of an lspF mutant lacking the T2S apparatus documented that T2S is also critical for infection of the amoeba Naegleria lovaniensis. Mutants lacking SrnA, PlaC, or ProA, but not those deficient for NttA, were defective in N. lovaniensis. Based upon analysis of a double mutant lacking PlaC and ProA, the role of ProA in H. vermiformis was connected to its ability to activate PlaC, whereas in N. lovaniensis, ProA appeared to have multiple functions. Together, these data document that the T2S system exports multiple effectors, including a novel one, which contribute in different ways to the broad host range of L. pneumophila.

Several toxins abolish cellular protein synthesis by attacking specific sites in 28 S RNA. One of these toxins, alpha-sarcin, is an RNase that also cleaves nonspecifically on the 3' side of purines in deproteinized RNA. Several other RNases were injected into Xenopus oocytes, examined for their ability to abolish protein synthesis, and compared with alpha-sarcin and ricin. Surprisingly, pancreatic RNase A or B abolished oocyte protein synthesis at concentrations (approximately 0.03 nM) comparable to, or lower than, the amount of alpha-sarcin (approximately 2 nM) or ricin (approximately 0.07 nM) required to abolish protein synthesis. RNases S and T1 only inhibited oocyte protein synthesis when used at concentrations approximately 10 x higher than RNase A whereas RNases C, T2, U2, and nuclease P1 required concentrations approximately 100 times higher than RNase A to abolish protein synthesis. There was a direct correlation between the degradation of oocyte RNA and the inhibition of protein synthesis. The RNase inhibitors RNasin and Inhibit-Ace injected into the oocyte both prevented RNase A from hydrolyzing oocyte rRNA and abolishing protein synthesis. Enzymatically inactive oxidized RNase A did not inhibit protein synthesis when injected into the oocyte. None of the RNases or alpha-sarcin abolished protein synthesis when added to oocyte extracellular medium. Angiogenin is a human plasma protein that induces blood vessel formation in chick embryos, has 35% amino acid identity with RNase A, and cleaves 18 S and 28 S RNA in rabbit reticulocyte lysates (St. Clair, D. K., Rybak, S. M., Riordan, J. F. & Vallee, B. L. (1988) Biochemistry 27, 7263-7268, and references therein). Recombinant angiogenin injected into oocytes abolished protein synthesis, and this toxic effect was inhibited by RNasin but was not inhibited by Inhibit-Ace. Unlike RNase A and the other nucleases that hydrolyzed cellular rRNA, no cleavage of 18 or 28 S RNA by recombinant angiogenin was seen at concentrations 100 x greater than necessary to abolish protein synthesis. Recombinant angiogenin must selectively attack specific RNA(s) or another target in the cell.

Melanoma is a very aggressive and highly angiogenic tumor in which standard treatments have had only limited success. Patients with advanced disease have a 5-year survival rate of 5%. In search for alternatives, we identified a natural product extracted from the fungus Aspergillus niger, termed ACTIBIND, that inhibits tumor growth and metastasis of melanoma in vivo. ACTIBIND, a T2 RNase, exerts antitumorigenic and antiangiogenic activities by competing with the angiogenic factor angiogenin (itself an RNase homologue). Thus, there was decreased expression and activity of the matrix metalloproteinase 2 in melanoma and vascular endothelial cells, decreased vascularization, and increased tumor cell apoptosis in vivo. ACTIBIND significantly inhibited angiogenesis in an in vivo angiogenesis assay with sponges containing angiogenin. In vitro, ACTIBIND was internalized by both melanoma and human umbilical vein endothelial cells, reached the cell nuclei, and inhibited the activity of angiogenin response elements in a dose-dependent manner. Collectively, our data indicate that ACTIBIND should be tested for its potential as a new antiangiogenic modality for the treatment of melanoma.

The methylated constituents of early adenovirus 2 mRNA were studied. RNA was isolated from polyribosomes of cells double labeled with [methyl-3H]methionine and 32PO4 from 2 to 7 g postinfection in the presence of cycloheximide. Cycloheximide ensures that methylation and processing are performed by preexisting host cell enzymes. RNA was fractionated into polyadenylic [poly(A)]+ and poly(A)- molecules using poly(U)-Sepharose, and undergraded virus-specific RNA was isolated by hybridization to viral DNA in 50% formamide at 37 degrees C. Viral mRNA was digested with RNase T2 and chromatographed on DEAE-Sephadex in 7 M urea. Two 3H-labeled RNase T2-resistant oligonucleotide fractions with charges between -5 and -6 were obtained, consistent with two classes of 5' terminal methyl "cap" structures, m7G(5')ppp(5')NmpNp (cap 1) and m7G(5')ppp(5')NmNmpNp (cap 2) (Nm is a ribose 2'-O-methylation). The putative cap 1 contains all the methylated constituents of cap 1 plus Cm. The molar ratios of m7G to 2'-O-methylnucleosides is about 1.0 for cap 1 and 0.5 for cap 2, consistent with the proposed cap structures. Most significant, compositional analysis indicates four different cap 1 structures and at least three different cap 2 structures. Thus there is a minimum of seven early viral mRNA species with different cap structures, unless each type of mRNA can have more than one 5' terminus. In addition to methylated caps, early mRNA contains internal base methylations, exclusively as m6A, as shown by analyses of the mononucleotide (-2 charge) fraction. m6A was present in the ratio of 1 mol of m6Ap per 450 nucleotides. Thus viral mRNA molecules contain two to three internal m6A residues per methyl cap, since there is on the average 1 cap per 1,250 nucleotides.

Pestiviruses, which belong to the Flaviviridae family of RNA viruses, are important agents of veterinary diseases causing substantial economical losses in animal farming worldwide. Pestivirus particles display three envelope glycoproteins at their surface: E(rns), E1, and E2. We report here the crystal structure of the catalytic domain of E(rns), the ribonucleolytic activity of which is believed to counteract the innate immunity of the host. The structure reveals a three-dimensional fold corresponding to T2 ribonucleases from plants and fungi. Cocrystallization experiments with mono- and oligonucleotides revealed the structural basis for substrate recognition at two binding sites previously identified for T2 RNases. A detailed analysis of poly-U cleavage products using (31)P-NMR and size exclusion chromatography, together with molecular docking studies, provides a comprehensive mechanistic picture of E(rns) activity on its substrates and reveals the presence of at least one additional nucleotide binding site.

A recent body of evidence indicates an active role for stromal (mis)-regulation in the progression of neoplasias. Within this conceptual framework, genes belonging to the growing but still poorly characterized class of tumor antagonizing/malignancy suppressor genes (TAG/MSG) seem to play a crucial role in the regulation of the cross-talk between stromal and epithelial cells by controlling malignant growth in vivo without affecting any cancer-related phenotype in vitro. Here, we have functionally characterized the human RNASET2 gene, which encodes the first human member of the widespread Rh/T2/S family of extracellular RNases and was recently found to be down-regulated at the transcript level in several primary ovarian tumors or cell lines and in melanoma cell lines. Although we could not detect any activity for RNASET2 in several functional in vitro assays, a remarkable control of ovarian tumorigenesis could be detected in vivo. Moreover, the control of ovarian tumorigenesis mediated by this unique tumor suppressor gene occurs through modification of the cellular microenvironment and the induction of immunocompetent cells of the monocyte/macrophage lineage. Taken together, the data presented in this work strongly indicate RNASET2 as a previously unexplored member of the growing family of tumor-antagonizing genes.

d1-1811 is a viable SV40 mutant with a 40 base pair deletion that includes the major wild-type capping site of late mRNA at map position 0.72. The late viral mRNAs induced by d1-1811 have now been further characterized by inversed S1-mapping analysis. The S1-resistant, 32P-labeled RNA fragments derived from the leader region were examined by fingerprinting and by analysis of RNase T2-generated 5'-terminal cap structures. The results show that most if not all of the mutant leader fragments analyzed have their 5' terminus to the left of the d1-1811 deletion site, i.e., closer to the origin of DNA replication. The major altered leader fragment is a continuous transcript from the DNA in the region 0.716 to 0.761 map unit and its 5' terminus has been precisely mapped at nucleotide L290. The observation that the cap structure of the major altered leader is only a minor cap species in wild-type late RNA suggests regulation in the use of different capping sites in SV40.

The hook-basal body (HBB) is a key intermediate structure in the flagellar assembly pathway in Salmonella typhimurium. The FlgM protein inhibits the flagellum-specific transcription factor sigma28 in the absence of the intact HBB structure and is secreted out of the cell following HBB completion. The flk gene encodes a positive regulator of the activity of FlgM at an assembly step just prior to HBB completion: at the point of assembly of the P- and L-rings. FlgM inhibition of sigma28-dependent class 3 flagellar gene transcription was relieved in P- and L-ring assembly mutants (flgA, flgH, and flgI) by introduction of a null mutation in the flk gene (J. E. Karlinsey et al., J. Bacteriol. 179:2389-2400, 1997). In P- and L-ring mutant strains, recessive mutations in flk resulted in a reduction in intracellular FlgM levels to those seen in wild-type (Fla+) strains. The reduction in intracellular FlgM levels by mutations in the flk gene was concomitant with a 10-fold increase in transcription of the flgMN operon compared to that of the isogenic flk+ strain, while transcription of the flgAMN operon was unaffected. This was true for both direct measurement of the flgAMN and flgMN mRNA transcripts by RNase T2 protection assays and for lac operon fusions to either the flgAMN or flgMN promoter. Loss of Flk did not allow secretion of FlgM through basal-body structures lacking the P- and L-rings. Intracellular FlgM was stable to proteolysis, and turnover occurred primarily after export out of the cell. Loss of Flk did not result in increased FlgM turnover in either P- or L-ring mutant strains. With lacZ translational fusions to flgM, a null mutation in flk resulted in a significant reduction of flgM-lacZ mRNA translation, expressed from the class 3 flgMN promoter, in P- and L-ring mutant strains. No reduction in either flgAMN or flgMN mRNA stability was measured in the absence of Flk in Fla+, ring mutant, or HBB deletion strains. We conclude that the reduction in the intracellular FlgM levels by mutation in the flk gene is only at the level of flgM mRNA translation.