Evidence for membrane-bound oligomerization of bacteriophage phi X174 lysis protein-E

The expression of cloned bacteriophage phi X174 lysis gene E was analyzed in minicells of Escherichia coli using two-dimensional gel electrophoresis. Beside the 10-11-kDa protein-E, at least two additional protein bands were detected, associated with the inner membrane, which showed the same isoelectric point as E. To clarify whether these proteins were E-specific, two different antibodies directed against a beta-galactosidase-E' hybrid protein and a synthetic oligopeptide corresponding to the C-terminal end of protein-E were raised. Immunoadsorption studies with anti-peptide-specific antibodies resulted in the detection of protein-E as well as in the detection of proteins of higher molecular weight. Two of these protein bands were positively recognized by anti beta-galactosidase-E' antibodies. The latter protein bands had the same molecular weight as the putative protein-E bands detected by two-dimensional gel electrophoresis indicating that these bands represent protein-E-specific oligomers. These data support the idea that an E-specific oligomeric structure penetrating the inner and outer membrane of E. coli is formed during the lytic action of protein-E.     

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5''-disrupted myb loci.

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.     

Cloning,expression and immunogenicity of ferric enterobactin binding protein Fep B from <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7

The gene coding for ferric enterobactin binding protein from E. coli O157:H7 was amplifi ed. This gene was cloned and expressed as C-terminal His (6)-tagged protein. The SDS-PAGE analysis of the total protein revealed only two distinct bands, with molecular masses of 31kDa and 34kDa. The Ni-NTA chromatography purifi ed FepB and the osmotically shocked periplasmic fraction of IPTG induced cells showed only a single band of 31 kDa. Polyclonal mouse antibody was raised against the recombinant protein during 4 weeks after immunization. Western blot analysis of the recombinant FepB with mouse antiserum revealeda single band of 31 kDa. Identification and purification of FepB helped reveal its appropriate molecular mass. Polyclonal antibody raised against the recombinant protein reacted with bacterial FepB. The recombinant protein FepB could have a protective effect against E. coli O157:H7 and might be useful as an effective vaccine.     

Multisite and bidirectional exonic splicing enhancer in CD44 alternative exon v3

The human CD44 gene encodes multiple isoforms of a transmembrane protein that differ in their extracellular domains as a result of alternative splicing of its variable exons. Expression of CD44 is tightly regulated according to the type and physiological status of a cell, with expression of high molecular weight isoforms by inclusion of variable exons and low molecular weight isoforms containing few or no variable exons. Human CD44 variable exon 3 (v3) can follow a specific alternative splicing route different from that affecting other variable exons. Here we map and functionally describe the splicing enhancer element within CD44 exon v3 which regulates its inclusion in the final mRNA. The v3 splicing enhancer is a multisite bipartite element consisting of a tandem nonamer, the XX motif, and an heptamer, the Y motif, located centrally in the exon. Each of the three sites of this multisite enhancer partially retains its splicing enhancing capacity independently from each other in CD44 and shows full enhancing function in gene contexts different from CD44. We further demonstrate that these motifs act cooperatively as at least two motifs are needed to maintain exon inclusion. Their action is differential with respect to the splice-site target abutting v3. The first X motif acts on the 3' splice site, the second X motif acts on both splice sites (as a bidirectional exonic splicing enhancer), and the Y motif acts on the 5' splice site. We also show that the multisite v3 splicing enhancer is functional irrespective of flanking intron length and spatial organization within v3.     

Primary structure of the gene for the murine Ia antigen-associated invariant chains (Ii). An alternatively spliced exon encodes a cysteine-rich domain highly homologous to a repetitive sequence of thyroglobulin.

The gene for murine Ia-associated invariant (Ii) chains (Ii31 and Ii41) was characterized by sequence analysis. The gene extends over approximately 9 kb and is organized in nine exons. Exon 1 encodes the 5' untranslated region and the cytoplasmic segment, exon 2 the membrane spanning segment and adjacent amino acids and exons 3-8 the extracytoplasmic portion of Ii31. Putative promoter sequences were found upstream of the start of the coding sequence. Between exons 6 and 7 an additional, alternatively spliced exon 6b has been identified. This exon is spliced into the mRNA coding for the Ii-related Ii41 protein. Exon 6b encodes a cysteine-rich domain of 64 amino acids. It shows a remarkably high homology to the repetitive elements in thyroglobulin, a precursor for thyroid hormone. Based on this homology, it is suggested that this domain (TgR) in Tg and in Ii41 may play a role either in hormone formation or as a carrier in the transport of molecules (thyroid hormone or processed antigen respectively) between intracellular compartments.     

Expression and characterization of Ov-47, a dominant antigen of Onchocerca volvulus

The expression and characterization of a recombinant antigen termed Ov-47 are described. Ov-47 was identified and isolated from a lambda gt-11 cDNA expression library derived from adult female Onchocerca volvulus mRNA using rabbit antiserum raised against the surface proteins of O. volvulus female worms. The antiserum was earlier found to mediate, in vitro, cytoadherence and cytotoxicity reactions to microfilariae in the presence of heat-labile serum factors. The deduced amino acid sequence of the gene was assigned the EMBL GenBank Accession No. Y15993. The open reading frame (1077 bp) of the gene was then subcloned into pQE-60 and expressed in Escherichia coli JM109 cells. The gene encodes a protein with an apparent molecular weight of 47,000 Da as revealed by SDS-PAGE. Up to 100 micrograms/ml pure Ov-47 recombinant protein could be isolated from E. coli cultures by Ni-agarose affinity chromatography. The 47-kDa protein was recognized by sera from both infected and endemic normal subjects. The parent protein was found to have a molecular weight of 60 kDa. IgG3 subclass responses to Ov-47 were significantly higher in endemic normals than in infected subjects (P < 0.05). In contrast, IgG4 responses were higher in infected subjects than in endemic normals (P < 0.05). IgG2 response exhibited marked age dependency with lower responses in younger patients, which rose to higher levels in elderly patients. IgG1, IgG3, and IgG4 responses did not show any age dependency. This study clearly shows that Ov-47 is a dominant antigen of O. volvulus adult worms with an important role in the host-parasite-interplay.     

Two mechanisms involving the autophagic and proteasomal pathways process the metastasis suppressor protein,N-myc downstream regulated gene 1

N-myc downstream regulated gene 1 (NDRG1) is an intriguing metastasis suppressor protein, which plays an important role in suppressing multiple oncogenic signaling pathways. Interestingly, multiple isoforms of NDRG1 have been identified, although the molecular mechanisms involved in their generation remains elusive. Herein, we demonstrate the role of two mechanisms involving autophagic and proteasomal machinery as part of an intricate system to generate different NDRG1 isoforms. Examining multiple pancreatic cancer cell-types using immunoblotting demonstrated three major isoforms of NDRG1 at approximately 41-, 46- and 47-kDa. The top NDRG1 band at 47-kDa was shown to be processed by the proteasome, followed by autophagic metabolism of the middle NDRG1 band at 46-kDa. The role of the proteasomal and autophagic pathways in NDRG1 processing was further confirmed by co-localization analysis of confocal images using PSMD9 and LC3 as classical markers of these respective pathways. All NDRG1 isoforms were demonstrated to be, at least in part, phosphorylated forms of the protein. Inhibition of two well-characterized upstream kinases of NDRG1, namely GSK3β and SGK1, resulted in decreased levels of the top NDRG1 band. Studies demonstrated that inhibition of GSK3β decreased levels of the top 47-kDa NDRG1 band, independent of its kinase activity, and this effect was not mediated via the proteasomal pathway. In contrast, the decrease in the top NDRG1 band at 47-kDa after SGK1 inhibition, was due to suppression of its kinase activity. Overall, these studies elucidated the complex and intricate regulatory pathways involving both proteasomal and autophagic processing of the metastasis suppressor protein, NDRG1.     

Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.     

Multiple Trp isoforms implicated in capacitative calcium entry are expressed in human pregnant myometrium and myometrial cells

Capacitative Ca2+ entry plays a role in thapsigargin- and oxytocin-mediated increases in intracellular free Ca2+ in human myometrium. Members of the Trp protein family have been implicated in capacitative Ca2+ entry in a number of tissues. Pregnant human myometrium and the human myometrial cell line PHM1-41 expressed mRNA for hTrp1, hTrp3, hTrp4, hTrp6, and hTrp7. A number of known splice variants of hTrp1 and hTrp4 were expressed in these cells. In addition, novel splice variants for hTrp1 and hTrp3 were discovered. hTrp1gamma1 and hTrp1gamma2 contain insertions between previously described exons 9 and 10 that would alter reading frame and produce Trp proteins truncated in the membrane spanning region if expressed. The hTrp3 variant introduces sequence between exons 8 and 9 that would insert 16 amino acids in the C-terminal region of the protein upstream of the calmodulin and inositol 1,4,5-triphosphate receptor interaction domain. hTrp1, hTrp3, and hTrp4 proteins were detected in both pregnant human myometrial and PHM1-41 membranes; a weak band consistent with hTrp6 expression was detected in pregnant human myometrium. These data are consistent with the presence of proteins that could form putative capacitative Ca2+ channels in human myometrium. Control of the activity of these channels may be important for the control of uterine contractile activity.     

Alternative splicing in intracellular loop connecting domains II and III of the alpha 1 subunit of Cav1.2 Ca2+ channels predicts two-domain polypeptides with unique C-terminal tails

Novel splice variants of the alpha(1) subunit of the Ca(v)1.2 voltage-gated Ca(2+) channel were identified that predicted two truncated forms of the alpha(1) subunit comprising domains I and II generated by alternative splicing in the intracellular loop region linking domains II and III. In rabbit heart splice variant 1 (RH-1), exon 19 was deleted, which resulted in a reading frameshift of exon 20 with a premature termination codon and a novel 19-amino acid carboxyl-terminal tail. In the RH-2 variant, exons 17 and 18 were deleted, leading to a reading frameshift of exons 19 and 20 with a premature stop codon and a novel 62-amino acid carboxyl-terminal tail. RNase protection assays with RH-1 and RH-2 cRNA probes confirmed the expression in cardiac and neuronal tissue but not skeletal muscle. The deduced amino acid sequence from full-length cDNAs encoding the two variants predicted polypeptides of 99.0 and 99.2 kDa, which constituted domains I and II of the alpha(1) subunit of the Ca(v)1.2 channel. Antipeptide antibodies directed to sequences in the second intracellular loop between domains II and III identified the 240-kDa Ca(v)1.2 subunit in sarcolemmal and heavy sarcoplasmic reticulum (HSR) membranes and a 99-kDa polypeptide in the HSR. An antipeptide antibody raised against unique sequences in the RH-2 variant also identified a 99-kDa polypeptide in the HSR. These data reveal the expression of additional Ca(2+) channel structural units generated by alternative splicing of the Ca(v)1.2 gene.     

猪丹毒丝菌C43311株spaA基因N端免疫保护区的克隆和表达

采用PCR从猪丹毒丝菌C43311株基因组中扩增出编码表面保护性抗原A的spaA基因片段,将其克隆到pMD18-T载体并对插入片段进行测序.用spaA基因N端免疫保护区(spaA-N)的特异引物从重组质粒pMD18-spaA中扩增得到spaA-N片段,将其定向插入原核表达载体pGEX-6p-2中,构建重组表达质粒pGEX-spaA-N,转化大肠杆菌BL21(DE3),在IPTG诱导下表达融合蛋白GST-SpaA-N,用SDS-PAGE和Western印迹检测表达产物.测序结果表明spaA基因片段大小为1881bp,与已报道的不同血清型菌株spaA基因的核苷酸序列比较,核苷酸序列同源性在93%～99%之间.SDS-PAGE结果显示表达蛋白约为64kDa,与预期的大小相近.Western印迹检测结果表明,表达的融合蛋白GST-SpaA-N能与C43311株SpaA蛋白的抗血清发生特异性反应,证明原核融合表达蛋白具有免疫反应性.     

Genomic organization and tissue-specific expression of splice variants of mouse organic anion transporting polypeptide 2

cDNAs that code for mouse organic anion transporting polypeptide 2 (oatp2) have been cloned. At least three forms of mouse oatp2 cDNAs containing the same coding sequence were isolated. The common coding sequence is for a protein of 670 amino acids with 12 putative transmembrane domains. The deduced amino acid sequence of the mouse oatp2 shares 89% identity with the reported rat oatp2. Cloning and analysis of mouse oatp2 gene indicates that these isoforms are alternatively spliced products from the same gene. Heterogeneity was observed in the 5'-untranslated region of the cDNAs. Two of the three isoforms lacked the noncoding exon 3 sequence. Northern-blot hybridization analysis using the exon 3-specific probes demonstrated that mouse oatp2 mRNA containing exon 3 sequence is expressed in heart and lung, whereas exon 1-, 2-, and 17-specific probes detected mRNA only in brain and liver. The mouse oatp2 gene consists of 17 exons, including three noncoding exons, and 16 introns. All of the introns are flanked by GT-AG splice sequences except for intron 10 that is flanked by GC-AG splice sequence.     

Lens epithelium-derived growth factor (LEDGF/p75) and p52 are derived from a single gene by alternative splicing

A human gene that encodes lens epithelium-derived growth factor (LEDGF) was isolated, and the DNA sequence and the exon/intron organization was determined. The gene contains at least 15 exons and 14 introns and encodes LEDGF mRNA and p52 mRNA. Exons 1-15 encode LEDGF mRNA, and exons 1-9, and a part of the ninth intron encode a splice variant (p52). Sequences of the exon/intron junctions of the gene have the highly conserved GT/AG rule. Most intron/exon junctions correspond to junctions of individual protein motifs. Almost equal amounts of LEDGF and p52 are expressed in lens epithelial cells in culture. The LEDGF gene is assigned to chromosome 9p22.2, which is adjacent to the major cell malignancy locus.