No abstract

We constructed a transfer system consisting of two compatible multicopy plasmids carrying the transfer regions Tral and Tra2 of the broad-host-range IncP plasmid RP4. In this system, the plasmid containing the Tral region with the origin of transfer (oriT) was transferred, whereas additional functions essential for the conjugative process were provided from the Tra2 plasmid in trans. The Tra2 region, as determined for matings between Escherichia coli cells, maps between coordinates 18.03 and 29.26 kb of the RP4 standard map. The section of Tra2 required for mobilization of the plasmid RSF1010 (IncQ) and the propagation of bacteriophages Pf3 and PRD1 appears to be the same as that needed for RP4 transfer. Tra2 regions of RP4 (IncPa) and R751 (IncPj3) are interchangeable, facilitating mobilization of the plasmid carrying the RP4 Tral region. The transfer frequencies of both systems are similar. Transcription of Tra2 proceeds clockwise relative to the standard map of RP4 and is probably initiated at a promoter region located upstream of trbB (kilB). From this promoter region the trfA operon and the Tra2 operon are likely to be transcribed divergently. A second potential promoter has been located immediately upstream of trbB (kiIB). Plasmids encoding the functional Tra2 region can only be maintained stably in host cells in the presence of the RP4 regulation region carrying the korA-korB operon or part of it. This indicates the involvement of RP4 key regulatory functions that apparently are active not only in the control of replication but also in conjugation.The conjugative transfer system of the promiscuous plasmid RP4 consists of two distinct regions, Tral and Tra2, separated by the Par (partitioning)/Mrs (multimer resolution system) region, the fiwA locus, IS8, and the kanamycin resistance gene (aphA). Tral, containing the origin of transfer (oriT), encodes functions involved in generating the single strand to be transferred and also includes the primase genes (59; for reviews see references 19 and 60). However, functions encoded by Tra2 have not yet been characterized extensively. Genetic approaches by transposon mutagenesis and complementation studies located the Tra2 region between the genes trbB (kiIB) andfiwA (3,4,37,51 acting replication function), encoding the replication protein and the Par/Mrs region, specifying a multimer resolution system (17,41). Plasmids containing the minimal functional Tra2 region could only be maintained stably when the korA-korB operon was present in trans, implying that KorA and KorB are involved in regulating expression of the transfer loci of Tra2. MATERUILS AND METHODSStrains, phages, and plasmids. E. coli HB101 (8) and S17-1 (47) were used as hosts for plasmids, and the nalidixic acid-resistant strain HB101 Nxr was used as a recipient for filter matings. Cells were grown in YT medium (33) buffered with 25 mM 3-(N-morpholino)propanesulfonic acid (sodium salt, pH 8.0) and supplemented with 0.1% glucose and 25 ,ug of thiamine-hydrochloride per ml. When appropriate, antibiotics...

Transfer functions of the conjugative plasmid RP4 (IncPot) are distributed among distinct regions of the genome, designated Tral and Tra2. By deletion analyses, we determined the limits of the Tral region, essential for intraspecific Escherichia coli matings. The Tral core region encompasses approximately 5.8 kb, including the genes traF, -G, -H, -I, -J, and -K as well as the origin of transfer. The traM gene product, however, is not absolutely required for conjugation but significantly increases transfer efficiency. To determine the transfer phenotype of genes encoded by the Tra2 core region, we generated a series of defined Tra2 mutants. This revealed that at least trbB, -C, -E, -G, and -L are essential for RP4 conjugation. To classify these transfer functions as components of the DNA transfer and replication (Dtr) or of the mating pair fonnation (Mpf) system, we analyzed the corresponding derivatives with respect to mobilization of IncQ plasmids and donor-specific phage propagation. We found that all of the Tra2 genes listed above and the traG and traF genes of Tral are required for RSF1010 mobilization. Expression oftraF from Tral in conjunction with the Tra2 core was sufficient for phage propagation. This implies that the TraG protein is not directly involved in pilus formation and potentially connects the relaxosome with proteins enabling the membrane passage of the DNA. The proposed roles of the RP4 transfer gene products are discussed in the context of virulence functions encoded by the evolutionarily related Ti T-DNA transfer system of agrobacteria.The study of conjugative DNA transfer of IncP plasmids gains increasing interest, since transfer of these plasmids to a wide range of remotely related microorganisms (for review, see references 21, 23, 65, and 76), even including yeasts (25), was observed. In addition, several findings provide evidence for a close relationship between RP4-mediated bacterial conjugation and T-DNA transfer from agrobacteria to plant cells (37,39,49,72,81). These similarities are underscored by recent data demonstrating that theAgrobacterium tumefaciens pTi Vir region can mobilize the small non-self-transmissible IncQ plasmid RSF1010 among agrobacteria (7) in a manner resembling the mobilization of these plasmids by IncP plasmids (16,78). However, it is noteworthy that mobilization frequencies obtained by the Ti system are extremely low.Functions responsible for RP4 transfer are encoded by two distinct regions of the genome, designated Tral and Tra2. The Tral region encompasses the origin of transfer (oriT) as well as functions responsible for the cleaving-joining process at the nick site (traI, -J, and -K; for review, see references 23 and 76). The traI and traJ gene products are required for relaxosome formation as well as for site-and strand-specific nicking at oriT, by which Tral becomes covalently associated with the 5' end of the DNA strand destined for transfer to the recipient cell (51). traK and traJ are specificity determinants on the basis of the observation that the...

The sterol 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS [follicular-fluid meiosis-activating sterol]) from human follicular fluid has recently been identified as a compound that induces the resumption of meiosis. FF-MAS and various oxysterols have been reported to transactivate the orphan receptor LXRalpha. The objective was to determine the biological activity of synthetic FF-MAS on the resumption of meiosis and final maturation of mouse oocytes in vitro. In order to evaluate whether LXRalpha might mediate FF-MAS action on the oocyte, we compared the capability of various compounds to activate LXRalpha-dependent transcription and to induce resumption of meiosis in the oocyte assay. Ovaries were isolated from immature mice primed with FSH 48 h before collection. Naked oocytes (NkO) and cumulus enclosed oocytes (CEO) were isolated from follicles. The oocytes were cultured in two groups, NkO and CEO, respectively, in media containing either 3 mM hypoxanthine, 5 microM IBMX, or 0.100 mM dbcAMP to maintain the oocytes in the germinal vesicle stage. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) after 24 h of in vitro culture. FF-MAS overcame the meiotic inhibition by hypoxanthine in both the NkO group and CEO group in a dose-dependent manner within the concentration range 0.07-7 microM. FF-MAS displayed similar potency in all inhibitory agents used. Also, FF-MAS significantly increased the formation of polar bodies in both the CEO and NkO group. The oxysterols 22(R)-hydroxycholesterol (a potent ligand for the LXRalpha receptor), 16-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, as well as cholesterol, were tested without any significant effect on maturation compared to that of controls. Oxysterols and FF-MAS were observed to activate LXRalpha. In conclusion, the results reported here clearly demonstrate that synthetic FF-MAS exclusively is capable of mediating resumption of meiosis in vitro in both NkO and CEO irrespective of the inhibitory substance used. In contrast, the oxysterols and cholesterol had no significant biological activity on this oocyte function, and consequently we found no correlation between LXRalpha activation and meiosis stimulation.

Synthetically produced meiosis-activating sterol, a sterol originally derived from follicular fluid (FF-MAS), induces meiotic maturation of mouse oocytes in vitro. We therefore compared FF-MAS-induced maturation of naked mouse oocytes arrested in prophase I by either hypoxanthine (Hx) or forskolin (Fo) with spontaneous maturation of naked oocytes. FF-MAS-treated oocytes overcame the meiotic block by Hx or Fo, although germinal vesicle breakdown was delayed by 11 h and 7 h, respectively. We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly and by delaying CG release, which is known to contribute to reduced fertilization.