IS1237, a Repetitive Chromosomal Element from Clavibacter xyli subsp. cynodontis, Is Related to Insertion Sequences from Gram-Negative and Gram-Positive Bacteria

We describe here a repetitive chromosomal element, which appears to be an insertion sequence, isolated from Clavibacter xyli subsp. cynodontis, a gram-positive plant-associated bacterium. The element, IS1237, is 905 bp in size, is bounded by 19-bp perfect inverted repeats and 3-bp direct repeats, and appears at least 16 times in the genome. It contains three open reading frames which show similarity to open reading frames from various other insertion sequences. We have found that there are two groups of related mobile elements: one in which two open reading frames are read separately and the other in which these two open reading frames are fuse together to give one predicted protein product. Using one of these open reading frames to search amino acid sequence databases, we found two instances in which similar reading frames flank genes carried on plasmids. We believe therefore that these plasmid-borne genes may be parts of previously unidentified mobile elements. For IS1237, a frameshift in two of the open reading frames and a stop codon in the third may indicate that this particular copy of the element is no longer active in transposition. The similarity of IS1237 to other elements from both gram-negative and gram-positive bacteria provides further evidence that mobile elements have been transferred between these two bacterial groups.     

备孕二胎女性人群中MTHFR基因多态性及相关环境因素的调查分析

目的:调查评估备孕二胎女性人群中MTHFR基因多态性及其生活习惯、一胎孕期情况等相关环境因素,以发挥孕前检查在子代先心病一级预防中的作用。方法:选取江苏部分地区参加备孕二胎优生健康检查妇女共402名进行问卷调查,并检测外周血中MTHFR基因rs1801131、rs1801133两个位点的突变情况。结果:收回有效问卷389份,其中备孕二胎女性平均年龄为27.04±2.68,最大43岁,最小19岁。MTHFR rs1801131纯合突变(CC型)为3.5%、杂合突变(AC型)为29.1%;rs1801133纯合突变(TT型)22.9%、杂合突变(CT型)48.5%;rs1801131、rs1801133双杂合突变为16.7%。结论:备孕二胎女性人群中存在一定的先心病环境危险因素暴露和MTHFR相关位点基因多态性。加强女性孕前保健,为高危人群制定个性化叶酸增补方案并降低先心病危险因素的暴露,从而做好出生缺陷一级预防,提高出生人口质量。     

Isolation of a Second S-Locus-Related Cdna from Brassica Oleracea: Genetic Relationships between the S Locus and Two Related Loci

Self-incompatibility in Brassica oleracea is controlled by the highly polymorphic S locus. Isolation and subsequent characterization of the S-locus-glycoprotein (SLG) gene, which encodes the S-locus-specific glycoprotein (SLSG), has revealed the presence of a self-incompatibility multigene family. One of these S-locus-related genes, SLR1, has been shown to be expressed. In this study we present the isolation and preliminary characterization of a second expressed S-locus-related sequence, SLR2. Through restriction fragment length polymorphism (RFLP) linkage analysis we demonstrate that the SLR1 and SLR2 loci reside approximately 18.5 map units apart in one linkage group that segregates independently of the S-locus. The identification of a second SLR gene expressed in stigmas suggests that loci unlinked to the S-locus may play a role in the self-incompatibility response, or in pollination in general.     

GPC6, a Novel Member of the Glypican Gene Family, Encodes a Product Structurally Related to GPC4 and Is Colocalized withGPC5on Human Chromosome 13

Glypicans are a family of cell surface heparan sulfate proteoglycans that appear to play an important role in cellular growth control and differentiation, as is supported by the observation that mutations in GPC3 are responsible for Simpson-Golabi-Behmel syndrome (SGBS) in humans. Recently it has been shown that the GPC4 gene is tightly clustered with GPC3 on the X chromosome and that some patients with SGBS apparently have deletions affecting both genes. We report here the identification of a human cDNA encoding a novel glypican family member, glypican-6. This cDNA encodes a predicted protein of 554 amino acids and is structurally analogous to other members of the glypican gene family, but most highly related to glypican-4. A single GPC6 mRNA of 6.2 kb is detected most abundantly in the ovary, liver, and kidney, with lower levels of mRNA expression also detected in a wide range of other adult tissues. Radiation hybrid analysis mapped the GPC6 gene to human chromosome 13 very near the GPC5 gene, a member of the glypican family bearing strong similarity to GPC3.     

The karyopherin Msn5/Kap142 requires Nup82 for nuclear export and performs a function distinct from translocation in RPA protein import

Protein transport between the nucleus and cytoplasm requires interactions between nuclear pore complex proteins (nucleoporins) and soluble nuclear transport factors (karyopherins, importins, and exportins). Exactly how these interactions contribute to the nucleocytoplasmic transport of substrates remains unclear. Using a synthetic lethal screen with the nucleoporin NUP1, we have identified a conditional allele of NUP82, encoding an essential nuclear pore complex protein in Saccharomyces cerevisiae. This nup82-3 allele also exhibits synthetic genetic interactions with mutants of the karyopherin MSN5. nup82-3 mutants accumulate the Msn5 export substrate Pho4 within the nucleus at non-permissive temperatures. The nuclear import of the RPA complex subunit Rfa2 is impaired in nup82-3 and in mutants of the karyopherin KAP95, but is not affected by the loss of MSN5. Interestingly, deletion of MSN5 results in retention of Rfa2-GFP within the nucleus under conditions in which it normally diffuses out. These data provide evidence that Nup82 is important for Msn5-mediated nuclear protein export and Kap95-mediated protein import. In addition, Msn5 may play a role independent of import in the localization of Rfa2.     

Obesity Resistance and Enhanced Insulin Sensitivity in Ahnak-/- Mice Fed a High Fat Diet Are Related to Impaired Adipogenesis and Increased Energy Expenditure

ObjectiveRecent evidence has suggested that AHNAK expression is altered in obesity, although its role in adipose tissue development remains unclear. The objective of this study was to determine the molecular mechanism by which Ahnak influences adipogenesis and glucose homeostasis.DesignWe investigated the in vitro role of AHNAK in adipogenesis using adipose-derived mesenchymal stem cells (ADSCs) and C3H10T1/2 cells. AHNAK-KO male mice were fed a high-fat diet (HFD; 60% calories from fat) and examined for glucose and insulin tolerances, for body fat compositions, and by hyperinsulinemic-euglycemic clamping. Energy expenditures were assessed using metabolic cages and by measuring the expression levels of genes involved in thermogenesis in white or brown adipose tissues.ResultsAdipogenesis in ADSCs was impaired in AHNAK-KO mice. The loss of AHNAK led to decreased BMP4/SMAD1 signaling, resulting in the downregulation of key regulators of adipocyte differentiation (P<0.05). AHNAK directly interacted with SMAD1 on the Pparγ2 promoter. Concomitantly, HFD-fed AHNAK-KO mice displayed reduced hepatosteatosis and improved metabolic profiles, including improved glucose tolerance (P<0.001), enhanced insulin sensitivity (P<0.001), and increased energy expenditure (P<0.05), without undergoing alterations in food intake and physical activity.ConclusionAHNAK plays a crucial role in body fat accumulation by regulating adipose tissue development via interaction with the SMAD1 protein and can be involved in metabolic homeostasis.     

Overexpression of lncRNA Gm15621 alleviates apoptosis and inflammation response resulting from sevoflurane treatment through inhibiting miR-133a/Sox4

Sevoflurane is the most widely used anesthetic administered by inhalation. Exposure to sevoflurane can elicit learning deficits and abnormal cognitive disorder. In this study, we investigated the function of long noncoding RNA (lncRNA) Gm15621. Primary hippocampal neuron cells were used to analyze the function of lncRNA Gm15621 in vitro. The tunel, inflammation markers, and cell survival rates were detected to evaluate the function of lncRNA Gm15621. Dual-luciferase reporter assay was used to identify the interaction between microRNA 133a and Gm15621. We found that lncRNA Gm15621 located in the cytoplasm. The expression of lncRNA Gm15621 was decreased with the development of sevoflurane exposure. Overexpression of lncRNA Gm15621 significantly reduced the apoptosis and cell survival rates. The inflammation response was also attenuated in lncRNA Gm15621 overexpressed group. The dual-luciferase assay revealed that miR-133a was the direct target of lncRNA Gm15621. In addition, we also found that Sox4 was a downstream target of miR-133a and lncRNA Gm15621 exerted its biological functions by regulating the expression of Sox4. In summary, our findings revealed that lncRNA Gm15621 ameliorated the sevoflurane-induced neurotoxicity and the important role of Gm15621/miR-133a/Sox4 axis in cognitive disorder.     

Apolipophorin II/I, Apolipoprotein B, Vitellogenin, and Microsomal Triglyceride Transfer Protein Genes Are Derived from a Common Ancestor

Large lipid transfer proteins (LLTP) are nonexchangeable apolipoproteins and intracellular lipid-exchange proteins involved in the assembly, secretion, and metabolism of lipoproteins. We have identified contiguous conserved sequence motifs in alignments of insect apolipophorin II/I precursor (apoLp-II/I), human apolipoprotein B (apoB), invertebrate and vertebrate vitellogenins (VTG), and the large subunit of mammalian microsomal triglyceride transfer protein (MTP). Conserved motifs present in the N-terminal part of nonexchangeable apolipoproteins encompass almost completely the large subunit of MTP, suggesting a derivation from a common ancestral functional unit, termed large lipid transfer (LLT) module. Divergence of LLTP from a common ancestor is supported by (1) the statistical significance of the combined match scores obtained after motif-based database searches, (2) the presence of several identical amino acid residues in all LLTP sequences currently available, (3) the conservation of hydrophobic clusters in an α-helical domain, (4) the phylogenetic analysis of the conserved sequences related to the von Willebrand factor D (VWD) module identified in nonexchangeable apolipoproteins, and (5) the presence of four and one ancestral exon boundaries in the LLT and VWD modules, respectively. Our data indicate that the genes coding for apoLp-II/I, apoB, VTG, and the MTP large subunit are members of the same multigene superfamily. LLTP have emerged from an ancestral molecule designed to ensure a pivotal event in the intracellular and extracellular transfer of lipids and liposoluble substances. Received: 8 June 1998 / Accepted: 15 February 1999     

Cloning and Sequence Analysis of a Novel Insertion Element from Plasmids Harbored by the Carbofuran-Degrading Bacterium,Sphingomonassp. CFO6

Sphingomonassp. CFO6 (a member of the alpha group ofProteobacteria) was isolated from a Washington soil by enrichment on the insecticide carbofuran as a sole source of carbon and energy. This strain has been shown to harbor five plasmids, at least some of which are required for catabolism of carbofuran. Rearrangements, deletions, and loss of individual plasmids resulting in the loss of the carbofuran-degrading phenotype were observed following treatment with heat or introduction of Tn5.Several putative insertion sequence elements of different sizes were cloned from these plasmids by trapping in pUCD800, a positive selection vector for isolation of transposable elements. Three of the most common putative IS elements (designated IS1412,IS1487,and IS1488) in the clone library were of different sizes and cross-hybridize with each other. An element hybridizing with IS1412,IS1487,and IS1488was mobilized during growth of CFO6 at 42°C and inserted into one of CFO6's plasmids (pCFO4), corresponding to a deletion in the plasmid and a loss of catabolic function. IS1412was completely sequenced and its sequence analyzed. IS1412is 1656 bp in length and possesses terminal partially matched inverted repeats of unequal length (17 and 18 bp). In addition, IS1412contains an open reading frame which encodes a putative transposase with significant homology to the putative transposases of IS1380fromAcetobacter pasteurianus,HRS1 fromBradyrhizobium japonicum,and IS1247fromXanthobacter autotrophicus.These related IS elements form part of a family of common IS elements distributed among members of the alpha group of theProteobacteria.     

1株来自海洋的芽胞杆菌dhs-330转座突变库的构建及表面活性相关基因的克隆

为研究产生物表面活性剂的海洋芽胞杆菌dhs-330合成活性产物的分子机制,应用质粒pIC333介导的mini-Tn10转座子随机突变技术,构建了芽胞杆菌dhs-330的突变体库。通过表面活性测定和反向PCR克隆,从300个突变株中筛选出产表面活性剂水平提高的突变株2株,分别在ycsG和yvkC基因发生插入突变;表面活性降低的突变株4株,分别在fenC、yrkF、kinE和sigD基因发生插入突变。这些基因可能与芽胞杆菌dhs-330中表面活性剂的合成代谢和调控有关。     

A Novel Electrochemically Active and Fe(III)-reducing Bacterium Phylogenetically Related to Clostridium butyricum Isolated from a Microbial Fuel Cell

An obligatory anaerobic bacterium was isolated from a mediator-less microbial fuel cell using starch processing wastewater as the fuel and designated as EG3. The isolate was Gram-positive, motile and rod (2.8–3.0 μm long, 0.5–0.6 μm wide). The partial 16S rRNA gene sequence and analysis of the cellular fatty acids profile suggested that EG3 clusters with Clostridium sub-phylum and exhibited the highest similarity (98%) with Clostridium butyricum. The temperature and pH optimum for growth were 37°C and 7.0, respectively. The major products of glucose and glucose/Fe(O)OH metabolism were lactate, formate, butyrate, acetate, CO₂and H₂. Growth was faster at the initial phase and the cell yield was higher when the medium was supplemented with Fe(O)OH than without Fe(O)OH. These results suggest that Fe(III) ion is utilised as an electron sink. Cyclic voltammetry showed that Clostridium butyricum EG3 cells were electrochemically active. It is a novel characteristic of strict anaerobic Gram-positive bacteria.     

CHK2 1100delC,IVS2 + 1G>A and I157T mutations are not present in hepatocellular cancer cases from a Turkish population

Aim

The cell cycle checkpoint kinase 2 (CHK2) protein participates in the DNA damage response in many cell types. Germline mutations in CHK2 (1100delC, IVS2 + 1G>A and I157T) have been associated with a range of cancer types. This study aimed to investigate whether CHK2 1100delC, IVS2 + 1G>A and I157T mutations play an important role in the development of hepatocellular carcinoma (HCC) in a Turkish population.

Methods

A total of 165 hepatocellular cancer cases and 446 cancer-free controls were genotyped for CHK2 mutations by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele specific-polymerase chain reaction (AS-PCR) methods.

Results

We did not find CHK2 1100delC, IVS2 + 1G>A and I157T mutations in any of 611 Turkish subjects.

Conclusion

Our results demonstrate for the first time that CHK2 1100delC, IVS2 + 1G>A and I157T mutations have not been a genetic susceptibility factor for HCC in the Turkish population. Overall, our data suggests that genotyping of CHK2 mutations in clinical settings in the Turkish population should not be recommended. Independent studies are needed to validate our findings in a larger series, as well as in patients of different ethnic origins.