No abstract

Two related families of transposons were isolated from Schizosaccharomyces pombe, an organism which has been the object of extensive genetic studies which had previously produced no evidence for the existence of such elements. These two classes of repeated DNAs, dubbed Tfl (transposon of fission yeast 1) and TO2 have many properties of retrotransposons. Tfl and Tf2 both possess long terminal repeats and predicted protein sequences that resemble the protease, reverse transcriptase, and integrase domains of retroviruses. The chromosomal locations and total numbers of Tfl and Tf2 differ greatly in various isolates of S. pombe. The Tf elements are expressed in the form of 4.5-kb mRNAs. The complete sequence of Tfl was determined and suggests that a novel mechanism for regulating its gene expression may be used.Transposable elements are thought to be present in all cell types, yet no such element has previously been reported from Schizosaccharomyces pombe. Transposable elements isolated from Saccharomyces cerevisiae have been characterized to develop an understanding of the molecular mechanisms of transposition in eucaryotic cells (3,4,10). We examined the genome of the related yeast S. pombe for transposable elements, since some of its biological properties, including chromosome condensation and centromere structure, have been shown to be more similar to those of higher eucaryotic cells than to those of S. cerevisiae (15, 40). We initially searched unsuccessfully for transposons in the well-known strains S. pombe 972 and 975 studies by Leupold et al. (14) by using a genetic approach similar to those used successfully with brewer's yeast, S. cerevisiae, to identify transposition events (9,29,37). We then initiated a study of repetitive DNAs isolated from S. pombe strains in other laboratories. As a result, we isolated two families of retrotransposons, transposon of fission yeast 1 (Tfl) and Tf2. The sequence of Tfl was determined and found to contain an open reading frame of 1,340 amino acids that has regions of sequence identity to the protease (PR), reverse transcriptase (RT), and integrase (IN) domains of retroviruses. In addition, RNA blot analysis demonstrated that both Tfl and Tf2 produce 4.5-kb transcripts that are of sufficient size to be full-length copies.Isolation and characterization of Tf element clones. The S. pombe strains used in this study and the element copies cloned from them are described in Table 1. The Tf element clones were identified as follows. We found that probes made from the pEE102 plasmid insert that hybridized to multiple S. pombe DNA sequences mapped within a 1.0-kbp DraI fragment. (This fragment was later shown to contain a single Tfl long terminal repeat [LTR]). This probe hybridized intensely to a 1.6-kbp SnaBI fragment in genomic DNAs from a variety of S. pombe strains (data not shown). Such a fragment was cloned directly by eluting appropriatesize DNA from an agarose gel on which SnaBI-digested strain 972 DNA had been run and ligating it into SmaI-* Corresponding author. digested...

In sharp contrast to the single ORF of the Schizosaccharomyces pombe retrotransposon Tf1, retroviruses and most retrotransposons employ two different ORFs to separately encode the Gag and Pol proteins. The different ORFs are thought to allow for overexpression of the Gag protein relative to Pol protein presumed necessary for the assembly of functional retrovirus particles and virus‐like particles (VLPs). The results of in vivo experiments designed to detect the transposition of Tf1 show that Tf1 is indeed active and can insert itself into the host genome via a true retrotransposition process. Thus, a paradox emerged between the lack of any obvious means of overexpressing Tf1 Gag protein and the demonstrated functionality of the element. Epitope tagging experiments described here confirm that the Tf1 large ORF is intact and that there is no translational or transcriptional mechanism used to overexpress the Tf1 Gag protein. In addition, we used sucrose gradients and antisera specific for Tf1 capsid (CA) and integrase (IN) to show that the Tf1 proteins do assemble into uniform populations of macromolecular particles that also cosediment with Tf1 reverse transcription products. This evidence suggests that Tf1 proteins form VLPs without using the previously described mechanisms that retroviruses and retrotransposons require to overexpress Gag proteins.

Persistence is a phenomenon whereby a subpopulation of bacterial cells enters a transient growth-arrested state that confers antibiotic tolerance. While entrance into persistence has been linked to the activities of toxin proteins, the molecular mechanisms by which toxins induce growth arrest and the persistent state remain unclear. Here, we show that overexpression of the protein kinase HipA in Escherichia coli triggers growth arrest by activating synthesis of the alarmone guanosine tetraphosphate (ppGpp) by the enzyme RelA, a signal typically associated with amino acid starvation. We further demonstrate that chemically suppressing ppGpp synthesis with chloramphenicol relieves inhibition of DNA replication initiation and RNA synthesis in HipA-arrested cells and restores vulnerability to ␤-lactam antibiotics. HipA-arrested cells maintain glucose uptake and oxygen consumption and accumulate amino acids as a consequence of translational inhibition. We harness the active metabolism of HipA-arrested cells to provide a bacteriophage-resistant platform for the production of biotechnologically relevant compounds, which may represent an innovative solution to the costly problem of phage contamination in industrial fermentations.

R ecent genomic comparisons of several invertebrates including Caenorhabditis elegans support the view that expansion of the Hox genes to include most or all of the present paralog groups occurred early in bilaterian evolution before the divergence of protostomes and deuterostomes (1). Previous work in several laboratories identified a small loosely linked cluster of four C. elegans Hox genes ( Fig. 1a): the anterior-group homolog ceh-13, two medial-group homologs of the Antp class, lin-39 and mab-5, and the posterior-group homolog egl-5 (2, 3). The expression patterns and postembryonic functions of these genes are consistent with roles in postembryonic specification of regional identities (4, 5). However, only ceh-13 is essential for embryonic development (6), whereas egl-5 is not (7), supporting an earlier suggestion that additional posterior-group genes might remain to be discovered (3).To search genetically for such genes, we have performed screens for embryonic and larval lethal mutations that result in severe posterior morphological abnormalities without disrupting general aspects of development such as cell proliferation and tissue specification. We have found mutations representing five complementation groups that result in such abnormalities, which we have designated the ''No back end'' or Nob phenotype (ref. 8; L.G.E., S. Carr, H. Wang, and W.B.W., unpublished work). We report herein on nob mutations affecting two previously uncharacterized Hox genes, recently also identified by genomic sequencing (9), which belong to the posterior paralog group and seem to be essential for embryonic development and posterior patterning. Materials and MethodsCloning and Characterization of nob-1 and php-3. C. elegans strains were handled by standard methods (10). The nob-1(ct223) mutation was mapped to a locus between spe-6 and pie-1 on the right arm of chromosome III (LGIIIR) by standard three-factor mapping (11). The ct230 and ct351 mutations were identified in screens for posterior patterning genes on LGIII after mutagenesis with trimethylpsoralen (8); complementation tests with ct223 defined these as alleles of nob-1. Representational difference analysis was performed as described (12) with one modification: given the relative simplicity of the C. elegans genome, genomic DNAs were digested with a four-cutter restriction enzyme, Sau3AI, resulting in average fragments of approximately 300 bp. Difference products from the representational difference analysis experiments were cloned into pBluescript SK(ϩ) (Stratagene) and sequenced on an Applied Biosystems sequencer. The nob-1(ct230) lesion was identified by direct sequencing of PCR products generated by a single-worm PCR assay (13) with primers recognizing sequences in exon 1 and intron 2 of nob-1. The endpoints of the ct223 and ct351 deletions were also determined by single-worm PCR with primers recognizing genomic sequence in the regions flanking nob-1 and php-3. nob-1 cDNA clones yk467d4.5 (nob-1A; GenBank accession no. C49108) and yk403d9.5 (nob-1B; C45910) were s...