Spliced RNA from the IR1-U2 region of Epstein-Barr virus: presence of an open reading frame for a repetitive polypeptide.

We have constructed a cDNA library from the cytoplasmic RNAs of Raji cells, a Burkitt's lymphoma cell line latently infected with Epstein-Barr virus. We report here the characterization of a cDNA representing a spliced RNA transcribed from the IR1-U2 region of the viral genome. The cDNA is 1007 bp long. The 5' region contains three tandem repeats of two exons, 66 and 132 bp, which are transcribed from the IR1 repeats. The 3' region is formed from four exons transcribed from U2. An open reading frame extends from the 5' end to position 784, and includes the repeats. This reading frame presumably corresponds to the carboxy-terminal 261 amino acids of a polypeptide containing several repeats of a 66 amino acid sequence. Since it would be encoded by the IR1-U2 region of the viral genome, the putative polypeptide might be involved in the process of growth-transformation of B-lymphocytes.     

Structure of the human TNF receptor 1 (p60) gene (TNFR1) and localization to chromosome 12p13 [corrected]

Clones encoding the entire coding and 3' untranslated region of the human type I tumor necrosis factor receptor (p60) gene (TNFR1) were isolated by hybridization using probes derived from TNFR-1 cDNA. The gene was characterized by restriction mapping. DNA blot analysis and sequence analysis. The coding region and the 3' untranslated region are distributed over 10 exons. Each of the four repeats, comprising the extracellular ligand binding domain and characterizing a receptor superfamily, is interrupted by an intron. However, the intron-exon structure is not conserved in the nerve growth factor receptor gene, another member of this superfamily. By PCR analysis of human-mouse somatic cell hybrids and in situ hybridization using biotinylated genomic TNFR1 DNA, we localized the gene to human chromosomal band 12p13. This corresponds to the homologous murine gene localized at the distal region of mouse chromosome 6.     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

Comparison of the whey acidic protein genes of the rat and mouse.

Whey acidic protein (WAP), a hormonally-regulated 14,000 dalton cysteine-rich protein, is the principal whey protein found in rodent milk. Genomic clones encompassing both the 2.8 Kb rat and 3.3 Kb mouse WAP genes have been characterized. The genes consist of four exons and three introns. The middle two exons encode the two cysteine-rich regions which probably form separate protein domains. Homology in the 5' flanking DNA of the mouse and rat extends at least 325 bp upstream of the putative CAP site, including a precisely conserved stretch of 50 bp around the unusual TATA and CAAT sites. The homology previously observed between the 3' noncoding sequences of the rat and mouse mRNAs extends at least 20 bp into the 3' flanking region. Several potential glucocorticoid receptor binding sites have been found in the 5' flanking region of the WAP gene. The conservation of the 5' flanking region of the WAP genes may be related to regulation of expression of WAP by peptide and/or steroid hormones.     

Genomic organization of the channel catfish CD45 functional gene and CD45 pseudogenes

CD45 is a transmembrane protein tyrosine phosphatase, which in mammals plays an important role in T and B cell receptor and cytokine signaling. Recently, a catfish cDNA was shown to contain all characteristic CD45 features: an alternatively spliced amino-terminus, a cysteine-rich region, three fibronectin domains, a transmembrane region, and two phosphotyrosine phosphatase domains. However, analyses of CD45 cDNAs from various catfish lymphoid cell lines demonstrated that catfish CD45 is unique in that it contains a large number of alternatively spliced exons. Sequence analyses of cDNAs derived from the catfish clonal B cell line 3B11 indicated that this cell line expresses up to 13 alternatively spliced exons. Furthermore, sequence similarity among the alternatively spliced exons suggested duplication events. To establish the exact number and organization of alternatively spliced exons, a bacterial artificial chromosome library was screened, and the catfish functional CD45 gene plus six CD45 pseudogenes were sequenced. The catfish functional CD45 gene spans 37 kb and contains 49 exons. In comparison, the human and pufferfish CD45 genes consist of 34 and 30 exons, respectively. This difference in the otherwise structurally conserved catfish gene is due to the presence of 18 alternatively spliced exons that were likely derived through several duplication events. In addition, duplication events were also likely involved in generating the six pseudogenes, truncated at the 3 ends. A similarly 3 truncated CD45 pseudogene is also present in the pufferfish genome, suggesting that this specific CD45 gene duplication occurred before catfish and pufferfish diverged (400 million years ago).