Metalloregulatory DNA-binding protein encoded by the merR gene: isolation and characterization

The MerR protein mediates the induction of the mercury resistance phenotype in bacteria; it has been isolated in order to study the effects of metal-ion induced changes in the metabolism of prokaryotic cells at the molecular level. After DNA sequences responsible for negative autoregulation were removed, the 16-kilodalton protein was overproduced and purified to more than 90 percent homogeneity by a salt extraction procedure that yields about 5 milligrams of protein per gram of cells. Complementation data, amino terminal analysis, gel filtration, and deoxyribonuclease I protection studies demonstrate that the purified merR gene product is a dimer under nondenaturing conditions and that it binds specifically to DNA, in the presence and absence of mercury, at a palindromic site which is directly between the -10 and -35 regions of the structural genes and adjacent to its own promoter. These initial results indicate that MerR is a DNA-binding metalloregulatory protein that plays a central role in this heavy metal responsive system and they delineate an operator site in the mer operon.     

Identification of the protein encoded by the E6 transforming gene of bovine papillomavirus

Papillomaviruses (PV) contain several conserved genes that may encode nonstructural proteins; however, none of these predicted gene products have been identified. Papillomavirus E6 genes are retained and expressed as RNA in PV-associated human and animal carcinomas and cell lines. This suggests that the E6 gene product may be important in the maintenance of the malignant phenotype. The E6 open reading frame of the bovine papillomavirus (BPV) genome has been identified as one of two BPV genes that can independently transform mouse cells in vitro. A polypeptide encoded by this region of BPV was produced in a bacterial expression vector and used to raise antisera. The antisera specifically immunoprecipitated the predicted 15.5-kilodalton BPV E6 protein from cells transformed by the E6 gene. The E6 protein was identified in both the nuclear and membrane fractions of these transformed cells.     

Nuclear localization and DNA binding properties of a protein expressed by human c-myc oncogene

Antisera to the human cellular myc oncogene product were used to identify a human c-myc specific protein with a molecular weight of 65,000. Subcellular fractionation showed that the human c-myc protein is predominantly found in the cell nucleus. The p65Kc-myc protein binds to double- and single-stranded DNA as measured by a DNA affinity chromatography assay.     

G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb

Exposure of peripheral blood mononuclear cells (PBMC) to an 18-base c-myb antisense oligomer before mitogen or antigen stimulation resulted in almost complete inhibition of c-myb messenger RNA and protein synthesis and blockade of T lymphocyte proliferation. Expression of early and late activation markers, interleukin-2 receptor and transferrin receptor, respectively, by PBMC was unaffected by antisense oligomer exposure as was the expression of c-myc messenger RNA. In contrast, histone H3 messenger RNA levels and DNA content were selectively decreased. These results suggest that c-myb protein deprivation does not perturb T lymphocyte activation or early molecular events that may prepare the cell for subsequent proliferation. Rather, it appears to specifically block cells in late G1 or early S phase of the cell cycle.     

猪轮状病毒VP7蛋白模拟表位的噬菌体展示技术筛选与鉴定

猪轮状病毒(Po RV)是引起仔猪腹泻的重要病原之一,其主要结构蛋白衣壳蛋白VP7可诱导机体产生中和抗体。文章以制备的抗Po RV VP7单克隆抗体(MAb)3B6作为靶分子,利用噬菌体十二肽库展示技术对其模拟表位进行筛选。四轮筛选后随机挑取阳性克隆扩增后进行ELISA鉴定,同时提取其基因组DNA测序,10个阳性噬菌体亲和肽拥有共同的外源肽序列"VPLGTDNGDIWV",而且与靶分子有很高亲和力。阳性噬菌体亲和肽与VP7蛋白进行序列比对,结果表明,十二肽中有4个氨基酸(第167位P、第178位T、第179位D和第184位W)与VP7蛋白167~184位氨基酸有一定的相似性。竞争抑制ELISA证明亲和多肽降低Po RV与抗VP7单克隆抗体之间的作用,病毒竞争抑制试验表明亲和多肽可以竞争性地抑制Po RV感染细胞。因此由这4个氨基酸构成的基序很有可能作为猪轮状病毒VP7蛋白的一个模拟表位。     

Activated expression of the N-myc gene in human neuroblastomas and related tumors

In neuroblastoma lines in which the N-myc gene is present as a single copy, the expression of N-myc as messenger RNA is increased relative to that in nonneuroblastoma cell lines and tumors. The increase of expression in neuroblastomas with amplified N-myc genes is the result of (i) an increase in the absolute amount of expression of each N-myc gene and (ii) an increase in the copy number of the N-myc gene. A second gene--which is amplified in many of the same lines as N-myc--is expressed to about the same degree in most human cell lines and primary tumors regardless of origin (when normalized to gene copy number). Thus, a change in the regulation of N-myc expression in neuroblastomas and certain other tumors results in greatly increased expression of each N-myc gene copy.     

A potassium channel protein encoded by chlorella virus PBCV-1

The large chlorella virus PBCV-1, which contains double-stranded DNA (dsDNA), encodes a 94-codon open reading frame (ORF) that contains a motif resembling the signature sequence of the pore domain of potassium channel proteins. Phylogenetic analyses of the encoded protein, Kcv, indicate a previously unidentified type of potassium channel. The messenger RNA encoded by the ORF leads to functional expression of a potassium-selective conductance in Xenopus laevis oocytes. The channel blockers amantadine and barium, but not cesium, inhibit this conductance, in addition to virus plaque formation. Thus, PBCV-1 encodes the first known viral protein that functions as a potassium-selective channel and is essential in the virus life cycle.     

Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21

The crystal structure at 2.7 A resolution of the normal human c-H-ras oncogene protein lacking a flexible carboxyl-terminal 18 residue reveals that the protein consists of a six-stranded beta sheet, four alpha helices, and nine connecting loops. Four loops are involved in interactions with bound guanosine diphosphate: one with the phosphates, another with the ribose, and two with the guanine base. Most of the transforming proteins (in vivo and in vitro) have single amino acid substitutions at one of a few key positions in three of these four loops plus one additional loop. The biological functions of the remaining five loops and other exposed regions are at present unknown. However, one loop corresponds to the binding site for a neutralizing monoclonal antibody and another to a putative "effector region"; mutations in the latter region do not alter guanine nucleotide binding or guanosine triphosphatase activity but they do reduce the transforming activity of activated proteins. The data provide a structural basis for understanding the known biochemical properties of normal as well as activated ras oncogene proteins and indicate additional regions in the molecule that may possibly participate in other cellular functions.     

Transformation of human bronchial epithelial cells transfected by Harvey ras oncogene

Transfection of normal human bronchial epithelial (NHBE) cells with a plasmid carrying the ras oncogene of Harvey murine sarcoma virus (v-Ha ras) changed the growth requirements, terminal differentiation, and tumorigenicity of the recipient cells. One of the cell lines isolated after transfection (TBE-1) was studied extensively and shown to contain v-Ha ras DNA. Total cellular RNA from TBE-1 cells hybridized to v-Ha ras structural gene fragment probes five to eight times more than RNA from parental NHBE cells. The TBE-1 cells expressed phosphorylated v-Ha ras polypeptide p21, showed a reduced requirement for growth-factor supplements, and became aneuploid as an early cellular response to v-Ha ras expression. As the transfectants acquire an indefinite life-span and anchorage independence they became transplantable tumor cells and showed many phenotypic changes suggesting a pleiotropic mechanism for the role of Ha ras in human carcinogenesis.     

A second nuclear protein is encoded by Epstein-Barr virus in latent infection

A region of the Epstein-Barr virus (EBV) genome that is important in inducing cell proliferation includes a single long open reading frame. Part of this open reading frame has been fused to the lacZ gene and expressed in Escherichia coli. Antisera to the fusion protein identify a protein in the nuclei of latently infected growth-transformed lymphocytes and in Burkitt tumor cells grown in vitro. This nuclear protein is encoded by a different virus-gene than that which encodes the previously described EBV nuclear antigen, EBNA.     

Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage

A domain of DNA designated N-myc is amplified 20- to 140-fold in human neuroblastoma cell lines but not in cell lines from other tumor types. N-myc has now been found to be amplified in neuroblastoma tissue from 24 of 63 untreated patients (38 percent). The extent of amplification appears to be bimodal, with amplification of 100- to 300-fold in 12 cases and 3- to 10-fold in 10 others. Amplification was found in 0 of 15 patients with stage 1 or 2 disease, whereas 24 of 48 cases (50 percent) with stage 3 or 4 had evidence of N-myc amplification. These data indicate that N-myc amplification is a common event in untreated human neuroblastomas. Furthermore, N-myc amplification is highly correlated with advanced stages of disease (P less than 0.001) and with the ability to grow in vitro as an established cell line, both of which are associated with a poor prognosis.     

Antibodies to human c-myc oncogene product: evidence of an evolutionarily conserved protein induced during cell proliferation

Antisera to a synthetic c-myc peptide and to c-myc antigens synthesized from various portions of the human gene expressed in Escherichia coli were used in order to characterize the protein product of the human c-myc oncogene. Although the deduced molecular weight of the human c-myc protein is 49,000, these antisera precipitate a protein from human cells that migrates in sodium dodecyl sulfate-polyacrylamide gel as if its molecular weight were 65,000. In addition, the mouse c-myc protein, whether synthesized in cells or in a cell-free system directed by pure, synthetic messenger RNA, has analogous properties and is immunoprecipitated by the antiserum to the human c-myc protein. Similar proteins are immunoprecipitated from monkey, rat, hamster, and frog cells, suggesting evolutionary conservation of antigenic structure of the c-myc protein among vertebrates. In addition, and in a manner consistent with the behavior of its messenger RNA, the immunoprecipitable c-myc protein is sharply induced by the action of mitogens on resting human T cells.     

Translocation and rearrangements of the c-myc oncogene locus in human undifferentiated B-cell lymphomas

The locus for the cellular myc (c-myc) oncogene in humans is located on the region of chromosome 8 that is translocated to chromosome 14 in cells from most undifferentiated B-cell lymphomas. It is shown in this study that the c-myc locus is rearranged in 5 out of 15 cell lines from patients with undifferentiated B-cell lymphomas, and that the rearrangement involves a region at the 5' side of an apparently intact c-myc gene. In at least three patients, this rearranged region appears to contain immunoglobulin heavy chain mu sequences that are located on chromosome 14. The data indicate that this region contains the crossover point between chromosomes 8 and 14. The break point can occur at different positions on both chromosomes among individual cell lines.     

猪链球菌9型噬菌体裂解酶Ply5218最小功能域及关键氨基酸位点的鉴定

为了鉴定猪链球菌9型噬菌体裂解酶Ply5218的最小活性功能域及关键氨基酸位点,利用PCR技术对全长裂解酶Ply5218进行截短并对可能与活性相关的关键氨基酸位点进行定点突变,研究截短与突变后各个蛋白的活性并与全长蛋白活性对比。结果显示,截短片段Ply5218_(1-147)可在平板上形成裂菌圈,仍保持裂菌活性,而比该片段更短的截短片段则失去裂菌活性,推测Ply5218_(1-147)为Ply5218的最小活性相关功能域;第8、58、136和142位点的氨基酸突变后,裂菌活性部分降低,第34和144位点的氨基酸突变后,则彻底失去裂菌活性。研究结果为深入揭示Ply5218的裂菌机制和进一步改造裂解酶提供了基础数据。     

A new HTLV-III/LAV protein encoded by a gene found in cytopathic retroviruses

The DNA of the HTLV-III/LAV group of retroviruses contains certain additional open reading frames that are not found in typical avian or mammalian retroviruses. The role of these sequences in encoding for gene products that may be related to pathogenesis remains to be resolved. An open reading frame whose 5' end overlaps with the pol gene, but is unrelated to the env gene, has been observed in HTLV-III/LAV and visna virus, both cytopathic mammalian retroviruses. Evidence presented here shows that this open reading frame is a bona fide coding sequence of HTLV-III/LAV and that its product, a protein with a molecular weight of 23,000, induces antibody production in the natural course of infection.     

Transfer of a protein encoded by a single nucleus to nearby nuclei in multinucleated myotubes

Specialized regions of muscle fibers may result from differential gene expression within a single fiber. In order to investigate the range of action of individual nuclei in multinucleated myotubes, C2 myoblasts were transfected to obtain stable cell lines that express a reporter protein that is targeted to the nucleus. Hybrid myotubes were then formed containing one or a few transfected nuclei as well as a large number of nuclei from the parental strain. In order to determine how far the products of a single nucleus extend, transfected nuclei were labeled with [3H]thymidine before fusion and the myotubes were stained to identify the reporter protein. In such myotubes the fusion protein was not confined to its nucleus of origin, but was restricted to nearby nuclei.     

辣椒疫霉中一个具有转录因子序列特征的外泌蛋白的分析鉴定

植物病原细菌分泌核调控蛋白进入寄主细胞核调控其基因表达以利于自身的侵染,但目前对卵菌中该类蛋白知之甚少.前期生物信息学分析从辣椒疫霉中获得一个具有DNA结合域的外泌蛋白PcSEP9,利用qRT-PCR检测其在辣椒疫霉侵染前期(菌丝、游动孢子囊、游动孢子、休止孢萌发)和侵染阶段[灌根接种本氏烟(Nicotiana ben...