The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MATa until the detection of MATa DNA took 60 min. When unbudded Gl-phase cells were induced, switching obeyed the rules established for normal homothallic cells; both the induced cell and its daughter switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the Gl phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HMLa and HMRa.Homothallic mating type switching in Saccharomyces cerevisiae is an efficient mitotic gene conversion event by which cells interconvert between a and a mating types (reviewed in references 10, 20, and 25). The process is initiated by a double-strand break at the MAT locus (33) created by the action of the HO endonuclease (21,22). In essence, MAT switching can be thought of as a specialized double-strand break repair event (10,20,25,33,35) in which the donor acts as a template for new DNA synthesis. Repair of the double-strand break at MAT results in the substitution of MAT Ya or Ya sequences with nonhomologous sequences derived from one of two unexpressed mating type loci, HML and HMR. The three mating type loci have regions of homology, X and Zl, which flank the allelespecific Y region; MAT and HML have two regions of additional homology, W and Z2 (2, 34) (see Fig. 2A).A single switching event gives rise to a pair of switched cells, suggesting that this event normally occurs in Gl phase, prior to replication of the chromosomal DNA (12). The observation of Nasmyth (26) that HO expression is limited to the Gl phase lends support to this conclusion. It is not clear, however, if limitation of switching to Gl can be explained solely on the basis of periodic HO expression or whether accessibility of MAT to the HO endonuclease is also restricted in a cell cycle-dependent fashion.The present study was undertaken for two reasons: (i) analysis of mating type switching at the DNA level, from initiation to completion, could be used to determine the kinetics of switching and the time interval during which DNA intermediates should be sought; and (ii) physical monitoring o...

In previous studies, Holliday junctions generated during RecA-mediated strand-exchange reactions were resolved by fractionated Escherichia coli extracts. We now report the specific binding and cleavage of synthetic Holliday junctions (50 base pairs long) by a fraction purified by chromatography on DEAE-cellulose, phosphocellulose, and singlestranded DNA-cellulose. The cleavage reaction provided a sensitive assay with which to screen extracts prepared from recombination/repair-deficient mutants. Cells with mutations in ruvC lack the nuclease activity that cleaves synthetic Holiday junctions in vitro. This deficiency was restored by a multicopy plasmid carrying a ruvC' gene that overexpressed junctionresolving activity. The UV sensitivity and deficiency in recombinational repair of DNA exhibited by ruv mutants lead us to suggest that RuvC resolves Holliday junctions in vivo.Much of our current understanding of the enzymology of genetic recombination in Escherichia coli comes from studies of RecA and RecBCD. In vitro, RecA catalyzes homologous pairing and strand-exchange reactions between DNA molecules to form recombination intermediates, whereas RecBCD is a multifunctional enzyme with DNA helicase and single-and double-strand exonuclease activities (1). However, genetic studies indicate that the products of the recF, recG, recJ, recN, recO, recQ, recR, ruvA, ruvB, and ruvC genes are also required for normal levels of recombination (2). A number of these proteins have now been cloned, overexpressed, and purified; RecF binds single-stranded DNA (ssDNA) (3), RecJ is a ssDNA exonuclease (4), RecQ is a DNA helicase (5), and RuvB is an ATPase (6). Many proteins required for genetic recombination are also needed for the recombinational repair ofDNA damage, and the recA, recN, recQ, ruvA, and ruvB genes are inducible and regulated by LexA protein (2, 7).A vital step in the recombination of DNA is the resolution of recombination intermediates into mature heteroduplex products. To understand the biochemistry of the resolution process in E. coli, a system in which recombination intermediates could be made and resolved in vitro was developed (8). We utilized the homologous-pairing and strand-exchange properties of RecA to produce intermediates in which two duplex molecules were linked by a single crossover, or Holliday junction. These structures were used to detect an activity from fractionated E. coli extracts that resolved the intermediates into recombinant products. Resolution occurred by specific endonucleolytic cleavage at the site of the Holliday junction (9).The presence of a nuclease that is specific for Holliday junctions is confirmed and extended by the present experiments. Using a more purified protein fraction than that reported (9), we show that the resolution activity binds to small synthetic Holliday junctions and cleaves them to produce nicked-duplex products. The development of this sensitive cleavage assay allowed us to screen a series of recombination/repair-deficient cells. We found that the specif...

The trans-splicing of short spliced leader (SL) RNAs onto the 59 ends of mRNAs occurs in a diverse range of taxa. In nematodes, all species so far characterized utilize a characteristic, conserved spliced leader, SL1, as well as variants that are employed in the resolution of operons. Here we report the identification of spliced leader trans-splicing in the basal nematode Trichinella spiralis, and show that this nematode does not possess a canonical SL1, but rather has at least 15 distinct spliced leaders, encoded by at least 19 SL RNA genes. The individual spliced leaders vary in both size and primary sequence, showing a much higher degree of diversity compared to other known trans-spliced leaders. In a survey of T. spiralis mRNAs, individual mRNAs were found to be trans-spliced to a number of different spliced leader sequences. These data provide the first indication that the last common ancestor of the phylum Nematoda utilized spliced leader trans-splicing and that the canonical spliced leader, SL1, found in Caenorhabditis elegans, evolved after the divergence of the major nematode clades. This discovery sheds important light on the nature and evolution of mRNA processing in the Nematoda.

Neospora caninum is an apicomplexan parasite of veterinary importance which invades many different cell types and tissues. N. caninum tachyzoites proliferate intracellularly by endodyogeny. Eventually the massive proliferation of tachyzoites leads to host cell lysis and the newly formed parasites are released and invade neighbouring cells. Tachyzoite cell surface molecules could serve as ligands, mediating host cell adhesion and invasion. Nc-p43 is a recently identified N. caninum tachyzoite surface protein which is functionally involved in the processes leading to host cell invasion in vitro. Affinity-purified antibodies directed against Nc-p43 were used to screen a lambda gt22A-cDNA expression library constructed from N. caninum tachyzoites. The cDNA insert of one immunoreactive clone was subcloned and expressed in E. coli as a poly-histidine fusion protein. The identity of the resulting recombinant antigen termed recNc-p43 was confirmed by immunoblotting, immunofluorescence and electron microscopy using affinity-purified antibodies. The sequence of the cDNA insert encoding recNc-p43 was determined. Analysis of the deduced amino acid sequence revealed that Nc-p43 exhibited similarity to SAG1 (p30) and SAG3 (p43), 2 major surface antigens of Toxoplasma gondii tachyzoites. These similarities were not reflected on the immunochemical level, since no cross-antigenicity between SAG1, SAG3 and Nc-p43 was observed.

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