Identification of a unique Marek's disease virus gene which encodes a 38-kilodalton phosphoprotein and is expressed in both lytically infected cells and latently infected lymphoblastoid tumor cells.

The identification of unique Marek's disease (MD) virus (MDV) antigens expressed not only in lytically infected cells but also in latently infected MD lymphoblastoid tumor cell lines is important in understanding the molecular mechanisms of latency and transformation by MDV, an oncogenic lymphotropic herpesvirus of chickens. Through cDNA and nucleotide sequence analysis, an open reading frame (designated the pp38 ORF) which encodes a predicted polypeptide of 290 amino acids was identified in BamHI-H. Demonstration that the pp38 ORF spans the junction of the MDV long unique and long internal repeat regions (MDV has an alphaherpesvirus genome structure) precludes the presence of the gene encoding the B-antigen complex (gp100, gp60, and gp49) in the same region of BamHI-H, where it was originally thought to exist. Duplication of the complete pp38 ORF was not observed in BamHI-D, but part of it (encoding 45 amino acids) was found in the long terminal repeat region of the fragment. By use of trpE-pp38 fusion proteins, antisera against pp38 were prepared. By immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a predominant virus-specific 38,000-dalton polypeptide (designated pp38) and a minor 24,000-dalton polypeptide (designated p24) were found. No precursor-product relationship was found between pp38 and p24 by pulse-chase analysis, and only pp38 was detected by Western blot (immunoblot) analysis with antiserum to pp38. pp38 was found to be phosphorylated and present in oncogenic serotype 1-but not nononcogenic serotype 3-infected cells. Expression of the gene encoding pp38 was relatively insensitive to phosphonoacetic acid inhibition, suggesting that pp38 may belong to one of the early classes of herpesvirus proteins. pp38 was also detected in the latently infected MSB-1 lymphoblastoid tumor cell line. The detection of antibody against pp38 in immune chicken sera indicates that pp38 is an immunogen in birds with MD. Most of the properties described here for a protein detected by methods based on finding the ORF first are identical to those of a 38-kDa phosphoprotein reported by others, suggesting that they are the same. Collectively, the data reported here provide (i) more definitive information on the complete ORF of another MDV gene and the protein that it encodes, (ii) clarification of the gene content within a specific region of the MDV genome, and (iii) the molecular means to conduct further studies to determine whether pp38 plays a role in MDV latency and transformation.     

马立克氏病病毒pp38基因上游的一个双向启动子研究

马立克氏病病毒（MDV）pp38基因上游是病毒基因组DNA复制原点。在其两侧均含有启动子TATAbox、CAATbox等特征性的保守基元,推测是一个天然的双向启动子。为了在体外验证其双向启动活性,本研究以MDVpp38为报告基因,并将其ORF插入到pUC18中,构建了pUCpp38质粒。将包含该启动子完整区域的789bp序列分别以正反两个方向克隆进pUCpp38质粒中pp38报告基因的上游,获得的重组质粒pProfpp38和pProrpp38。将所获得的重组质粒分别转染鸡胚成纤维细胞（CEF）,通过间接免疫荧光试验检测pp38基因的表达以验证该启动子的双向启动活性。结果表明,马立克氏病病毒复制原点区的启动子无论以何种方向插入pUCpp38质粒中,在转染细胞24h内能检测到pp38基因的表达,48h后能获得高效和持续的表达。逐渐缩小该启动子的范围,最终在320bp时,仍能检测到两个方向较强的启动活性。     

Characterization of a gene involved in histidine biosynthesis in Halobacterium (Haloferax) volcanii: isolation and rapid mapping by transformation of an auxotroph with cosmid DNA.

Techniques for the transformation of halophilic archaebacteria have been developed recently and hold much promise for the characterization of these organisms at the molecular level. In order to understand genome organization and gene regulation in halobacteria, we have begun the characterization of genes involved in amino acid biosynthesis in Halobacterium (Haloferax) volcanii. These studies are facilitated by the many auxotrophic mutants of H. volcanii that have been isolated. In this project we demonstrate that cosmid DNA prepared from Escherichia coli can be used to transform an H. volcanii histidine auxotroph to prototrophy. A set of cosmid clones covering most of the genome of H. volcanii was used to isolate the gene which is defective in H. volcanii WR256. Subcloning identified a 1.6-kilobase region responsible for transformation. DNA sequence analysis of this region revealed an open reading frame encoding a putative protein 361 amino acids in length. A search of the DNA and protein data bases revealed that this open reading frame encodes histidinol-phosphate aminotransferase (EC 2.6.1.9), the sequence of which is also known for E. coli, Bacillus subtilis, and Saccharomyces cerevisiae.     

马立克氏病病毒pp38基因和1.8kb转录子之间双向启动子的特性研究

从马立克氏病病毒(MDV)基因组DNA复制原点区某一点,将介于MDVpp38基因和1 8kb转录子之间的双向启动子分割成两个单方向的启动子。以pp38为报告基因,pUC18质粒为载体,构建了含不同方向完整启动子序列的pProfpp38和pProrpp38质粒,以及含分割后单方向启动子序列的pdProfpp38和pdProrpp38质粒。4种质粒分别转染鸡胚成纤维细胞(Chickenembryofibroblast,CEF)后,均能检测到pp38基因的表达。进一步以氯霉素乙酰转移酶(Chloramphenicolacetyltransferase,CAT)为报告基因,构建了含不同方向完整双向启动子的pProfCAT和pProrCAT质粒,以及含分割后单方向启动子序列的pdProfCAT和pdProrCAT质粒。通过转染试验,定量分析了完整启动子和分割后启动子在两个方向上的启动活性。实验结果表明,分割后的启动子在两个方向上的启动活性均比相应方向上完整启动子的活性低,其中1 8kb转录子方向上的活性下降了4 1倍     

Molecular analysis of the Escherichia coli recO gene.

The plasmid pLC7-47, which contains lep, rnc, and era, was found to complement the UV-sensitive and recombination-deficient phenotypes caused by the recO1504::Tn5 mutation. Southern blotting analysis demonstrated that pLC7-47 contained a segment of Escherichia coli DNA that covered the region of the E. coli chromosome containing the recO1504::Tn5 mutation. A combination of deletion mapping and insertional mutagenesis localized the recO-complementing region to an approximately 1-kilobase region of a 1.6-kilobase BamHI fragment. The DNA sequence of the 1.6-kilobase BamHI fragment was determined and contained part of era and a 726-base-pair recO open reading frame. The recO open reading frame contained three possible translation start codons and could potentially encode a polypeptide of Mr 26,000. Computer analysis indicated that the putative RecO protein had suboptimal codon usage and did not show significant homology with previously identified proteins whose sequences were present in protein data bases. A combination of primary sequence analysis and secondary structure predictions suggested that recO contains a mononucleotide-binding fold.     

马立克氏病病毒强毒GA株基因组BamH Ⅰ-L片段的序列分析

马立克氏病病毒（Ｍａｒｅｋ’ｓｄｉｓｅａｓｅｖｉｒｕｓ,ＭＤＶ）是一种能诱导鸡淋巴组织增生或淋巴肿瘤的细胞结合性疱疹病毒,由此而引起的马立克氏病（Ｍａｒｅｋ’ｓｄｉｓｅａｓｅ,ＭＤ）也是迄今为止唯一可以利用疫苗进行有效控制的肿瘤性疾病。鉴于此,作为研...     

Isolation and sequencing of the replication region of Mycobacterium avium plasmid pLR7.

The Mycobacterium avium plasmid pLR7 is representative of a group of small plasmids that are common in isolates from AIDS patients with disseminated M. avium infections. Determination of the functions of these and other plasmids has been hampered by the lack of methods for genetic manipulation of M. avium. In this study, the region of pLR7 capable of replication was identified and sequenced. Fragments of pLR7 were cloned into a pUC18 derivative carrying a kanamycin resistance marker and introduced into a plasmid-free M. avium strain by electroporation. The origin of replication was located on a 1.8-kb PvuII-to-SmaI fragment. An open reading frame encoding a putative Rep protein was identified. Two other open reading frames were identified in this region. A shuttle vector, pMB351, was constructed with the pLR7 origin of replication, pUC18, and the kanamycin resistance gene from Tn5. This vector was successfully transformed into M. avium, Mycobacterium tuberculosis, and Mycobacterium bovis.