The synaptonemal complex (SC) is a proteinaceous complex that apparently mediates synapsis between homologous chromosomes during meiotic prophase. In Saccharomyces cerevisiae, the Zip1 protein is the integral component of the SC. In the absence of a DNA double-strand break or the SC initiation protein Zip3, Zip1 proteins aggregate to form a polycomplex (PC). In addition, Zip1 is also responsible for DSB-independent nonhomologous centromere coupling at early meiotic prophase. We report here that Zip3 is a SUMO (small ubiquitin-related modifier) E3 ligase and that Zip1 is a binding protein for SUMO-conjugated products. Our results also suggest that at early meiotic prophase, Zip1 interacts with Zip3-independent Smt3 conjugates (e.g., Top2) to promote nonhomologous centromere coupling. At and after mid-prophase, the Zip1 protein begins to associate with Zip3-dependent Smt3 conjugates (e.g., Red1) along meiotic chromosomes in the wild-type cell to form SCs and with Smt3 polymeric chains in the zip3 mutant to form PCs.[Keywords: Meiosis; synaptomenal complex; Zip1; Zip3; SUMO; Ulp2] Supplemental material is available at http://www.genesdev.org.
The synaptonemal complex (SC) is a tripartite protein structure consisting of two parallel axial elements (AEs) and a central region. During meiosis, the SC connects paired homologous chromosomes, promoting interhomologue (IH) recombination. Here, we report that, like the CE component Zip1, Saccharomyces cerevisiae axial-element structural protein, Red1, can bind small ubiquitin-like modifier (SUMO) polymeric chains. The Red1–SUMO chain interaction is dispensable for the initiation of meiotic DNA recombination, but it is essential for Tel1- and Mec1-dependent Hop1 phosphorylation, which ensures IH recombination by preventing the inter-sister chromatid DNA repair pathway. Our results also indicate that Red1 and Zip1 may directly sandwich the SUMO chains to mediate SC assembly. We suggest that Red1 and SUMO chains function together to couple homologous recombination and Mec1–Tel1 kinase activation with chromosome synapsis during yeast meiosis.
Two Escherichia coli isolates were recovered from the blood of two cancer patients and were demonstrated to produce high levels of the AmpC -lactamase with isoelectric points of >9.0. The hypertranscription of ampC RNA was observed by Northern blot hybridization in both isolates. One isolate (isolate EC44) had a point mutation (G3A at position ؊28) and insertion of thymidine between positions ؊20 and ؊19 of the ampC promoter gene (GenBank accession no. AE000487). The single nucleotide insertion of T between positions ؊19 and ؊20 created an optimal distance (17 bp) in the Pribnow box for ampC hyperproduction. The other isolate (isolate EC38) had two point mutations (G3A at position ؊28 and C3T at position ؉58) and a 2-base (GT) insertion between positions ؊14 and ؊15. Although the insertion of GT between positions ؊14 and ؊15 may create a new promoter next to the original promoter, cloning of the ampC region with truncated nucleotides of the original ؊35 region of EC38 failed to verify the hypothesis that a new promoter would be created by such a nucleotide insertion. Instead, multiple start sites for ampC transcription at ؊1, ؉1, ؉2, and ؉3 were observed in an S1 nuclease protection assay. These results suggest that the RNA polymerase is flexible in the selection of a start site in ampC hypertranscription. In conclusion, nucleotide insertions between the ؊35 and ؊10 ampC promoter sequences was the mechanism for the hyperproduction of AmpC -lactamase and resistance to oxyimino-cephalosporins. The failure of the two patients to respond to treatment with oxyiminocephalosporins highlights the important role of such a resistance mechanism in the clinical setting.
Tigecycline (TGC)-resistant extensively drug-resistant Acinetobacter baumannii (XDRAB) is an increasing threat in regard to nosocomial infections. The resistance-nodulation-cell division (RND) efflux pump has played an important role in TGC resistance. In this study, total 81 TGC-resistant XDRAB isolates were analyzed for their responses to the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). We found that NMP could reduce by 4-fold or greater than 4-fold the minimum inhibitory concentration (MIC) of TGC in 45 isolates (55.6 %). After typing with pulsed-field gel electrophoresis (PFGE), group A appeared to be the major cluster with good synergistic response to NMP. Transcripts of the AdeABC efflux pump gene were consistently more correlated with TGC resistance than transcripts of the AdeFGJ or AdeIJK efflux pump genes in these isolates. Of the 81 isolates, the amino acid sequences of AdeR and AdeS were further classified and combined into 31 different codes. Although the dissemination of TGC-resistant XDRAB isolates was genetically diverse in our hospital, their responses to NMP conversion were still strain-dependent. We found that AdeRS combination codes were better than PFGE typing in separating groups of isolates with different sensitivity to NMP conversion.
The synaptonemal complex (SC) is a meiosis-specific tripartite structure that forms between two homologous chromosomes; it consists of a central region and two parallel lateral elements. Lateral elements also are called axial elements prior to synapsis. In Saccharomyces cerevisiae, Red1, Hop1, and Mek1 are structural components of axial/lateral elements. The red1/mek1/hop1 mutants all exhibit reduced levels of interhomolog recombination and produce no viable spores. Red1 is a phosphoprotein. Several earlier reports proposed that phosphorylated Red1 plays important roles in meiosis, including in signaling meiotic DNA damage or in preventing exit from the pachytene chromosomes. We report here that the phosphorylation of Red1 is carried out in CDC28-dependent and CDC28-independent manners. In contrast to previous results, we found Red1 phosphorylation to be independent of meiotic DNA recombination, the Mec1/Tel1 DNA damage checkpoint kinases, and the Mek1 kinase. To functionally validate the phosphorylation of Red1, we mapped the phosphorylation sites on this protein. A red1 14A mutant showing no detectable Red1 phosphorylation did not exhibit decreased sporulation efficiency, defects in viable spore production, or defects in meiotic DNA damage checkpoints. Thus, our results suggest that the phosphorylation of Red1 is not essential for its functions in meiosis.Meiosis is a critical component in the cycle of sexual reproduction, because it reduces the chromosome complement to haploidy in preparation for fertilization. This event is achieved by a single round of premeiotic DNA replication followed by two successive rounds of chromosome segregation to produce four haploid gametes. The first nuclear division (MI) is reductional, separating the newly recombined homologs from one another while leaving sister chromatids attached. The second nuclear division (MII), in which the sister chromatids segregate, is more typical of mitotic division and is called equational division. A prominent feature of meiosis is that pairing and recombination must occur between homologous chromosomes during the meiotic prophase. In contrast, these events rarely happen in mitosis. Meiotic DNA recombination plays a crucial role in meiosis, not only providing a potent source of genetic variation but also playing a mechanical role during MI. Specifically, crossover recombination results in a physical connection (i.e., chiasmata) between homologous chromosomes that allows them to orient properly on the spindle (for a review, see reference 59).Meiotic DNA recombination is initiated by the formation of DNA double-strand breaks (DSBs). Spo11, a meiosis-specific type II topoisomerase, generates DSBs together with several other factors in a cell cycle-programmed manner (26). The Mre11-Rad50-Xrs2 nuclease complex then resects these DSBs to generate 3Ј single-stranded tails that invade the intact DNA duplexes used for DNA repair (35). Most of these events use homologous chromosomes, not sister chromatids, as the templates for DNA repair to yield crossove...
bRecombination and synapsis of homologous chromosomes are hallmarks of meiosis in many organisms. Meiotic recombination is initiated by Spo11-induced DNA double-strand breaks (DSBs), whereas chromosome synapsis is mediated by a tripartite structure named the synaptonemal complex (SC). Previously, we proposed that budding yeast SC is assembled via noncovalent interactions between the axial SC protein Red1, SUMO chains or conjugates, and the central SC protein Zip1. Incomplete synapsis and unrepaired DNA are monitored by Mec1/Tel1-dependent checkpoint responses that prevent exit from the pachytene stage. Here, our results distinguished three distinct modes of Mec1/Tec1 activation during early meiosis that led to phosphorylation of three targets, histone H2A at S129 (␥H2A), Hop1, and Zip1, which are involved, respectively, in DNA replication, the interhomolog recombination and chromosome synapsis checkpoint, and destabilization of homology-independent centromere pairing. ␥H2A phosphorylation is Red1 independent and occurs prior to Spo11-induced DSBs. DSB-and Red1-dependent Hop1 phosphorylation is activated via interaction of the Red1-SUMO chain/conjugate ensemble with the Ddc1-Rad17-Mec3 (9-1-1) checkpoint complex and the Mre11-Rad50-Xrs2 complex. During SC assembly, Zip1 outcompetes 9-1-1 from the Red1-SUMO chain ensemble to attenuate Hop1 phosphorylation. In contrast, chromosome synapsis cannot attenuate DSB-dependent and Red1-independent Zip1 phosphorylation. These results reveal how DNA replication, DSB repair, and chromosome synapsis are differentially monitored by the meiotic checkpoint network. Meiosis generates four haploid daughter cells from a diploid parental cell. The central steps of meiosis are the pairing and recombination of homologous chromosomes, followed by their segregation in two rounds of cell division. A key step of meiosis occurs in the pachytene stage, in which the homologous chromosomes (i.e., the parental chromosomes, each containing two sister chromatids) align (synapsis) (1, 2). Meiotic recombination is initiated by DNA double-strand breaks (DSBs) induced by Spo11, and chromosome synapsis is mediated by a tripartite structure named the synaptonemal complex (SC). The SC is a zipper-like protein complex that consists of a central element and two dense lateral/axial elements. The tripartite structure of the SC is strikingly conserved from budding yeast to humans, underscoring its prominent function during meiosis (2-4). In the budding yeast Saccharomyces cerevisiae, the central element of the SC includes a major component (Zip1) and the SC initiating proteins (Zip2-4, Mer3, Msh4-5, Spo16, and Ecm11-Gcm2) (5-15). The axial elements include the sister chromatid cohesin complex (Rec8/Scc3/ Smc1/Smc3) and three meiosis-specific components, Hop1, Red1, and Mek1 (1, 16, 17). Red1 and Hop1, like Rec8, assemble along the rod-like cores of meiotic chromosomes (16). The SC proteins have different impacts on meiotic DNA recombination and cell cycle progression. In cell cycle progression, checkpoint m...
Nine isolates of Klebsiella pneumoniae, obtained from one colonized and eight bacteraemic patients on a paediatric ward, were shown to be identical by PFGE, indicating an outbreak. Screening for extended-spectrum beta-lactamase (ESBL) production using the double-disc synergy test, Etest for ESBLs and agar diffusion tests indicated ESBL production. The isolates showed reduced susceptibility to cefotaxime but not to other third-generation cephalosporins. Molecular studies revealed production of TEM-1 and SHV-1 but no ESBLs were identified. Deficiency in expression of an outer membrane protein (OmpK35) was also observed. These observations led us to postulate that the extremely low level of OmpK35 expression and the co-existence of TEM-1 and SHV-1 resulted in an increased MIC of cefotaxime and the false designation of the isolates as ESBL producers. All the infected infants were treated with either third-generation cephalosporins alone or multiple antibiotics including a third-generation cephalosporin, and recovered and were discharged without sequelae.
Thirteen patients who had 16 episodes of bacteremia were observed between 1993 and 1997 in a pediatric oncology ward with a high background isolation rate of cefotaxime- or aztreonam-resistant gram-negative bacteria. Four blood isolates were Escherichia coli and 12 were Klebsiella pneumoniae, and these isolates harbored extended-spectrum β-lactamases (ESBLs). All episodes of bacteremia were nosocomial, all except one of the episodes occurred in neutropenic patients, and all patients were treated with piperacillin or ceftazidime with amikacin and cefazolin prior to the onset of bacteremia. Nine of 13 patients were receiving extended-spectrum β-lactam treatment when the bacteremias caused by ESBL producers occurred. Molecular studies revealed that four K. pneumoniae SHV-2-producing isolates from 1994 were of the same clone. Other ESBL producers, including six that carried both TEM-1 and SHV-5, five that carried SHV-5, and one that carried SHV-2 alone, were unrelated. In conclusion, SHV-5 was present in 11 of the 16 isolates and coexisted with TEM-1 in 6 isolates. Acquisition of resistance genes probably occurred under antibiotic selection pressure. This study highlights the importance of routine checks for and detection of ESBL producers. Effective therapy against ESBL producers should be considered early for children who have malignancies and neutropenia and who are septic, despite treatment with a regimen that includes an extended-spectrum β-lactam, in a clinical setting of an increased incidence of ESBL-producing bacteria.
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