一个鼻咽癌相关EST的鉴定及其全长cDNA序列分析

鼻咽癌是我国南方及东南亚地区常见的恶性肿瘤之一.通过对鼻咽癌染色体高频率杂合性丢失区域3p21的表达序列标签(expressedsequencetag,EST)进行同源性比较分析,运用逆转录聚合酶链式反应的方法,筛选到一个在41.18%(14/34)的鼻咽癌活检组织及20.0%(1/5)的鼻咽癌细胞系中表达下调的ESTBG772301;并用Northern杂交方法,检测了该EST在多种正常成人组织中的表达状况及其所代表基因的转录本大小.在此基础上,对该EST来源的cDNA克隆(IMAGE:4839190)进行直接测序,获得了一个全长为2377bp的新cDNA序列;经生物信息学分析,发现它与已知基因序列无明显同源性,属于一个新基因,定位于染色体3p21.3,被命名为鼻咽癌表达下调基因(NPCEDRG,GenBank登录号:AF538150).其编码的蛋白质含169个氨基酸,与一个已报道的在进化上相对保守、功能未知的人类蛋白Nicolin1(简称NICN1)N端170个氨基酸残基的序列同源性为97%,但缺少NICN1蛋白C端43个氨基酸残基,可能是nicolin1基因不同剪接本的编码产物.     

Molecular cloning, expression, localization, and gene organization of PTX1, a human nuclear protein that is downregulated in prostate cancer.

A cDNA, designated PTX1, has been isolated by subtractive hybridization on the basis that it is expressed in normal prostate but not in prostate carcinoma. The full-length cDNA was subsequently established by 5' and 3' RACE. Nucleotide sequence analysis of the 5'- and 3'-RACE clones yielded a composite cDNA of 1327 bp, which predicted a protein of 377 amino acid residues with a putative nuclear import signal (RRLNRKK) at its N terminus. The PTX1 gene was localized to human chromosome 12 and was found to be ubiquitously expressed. A segment of the cDNA was expressed in E. coli to produce a fragment of the PTX1 protein for the generation of specific antibodies. The resulting antibodies detected a 73-kDa protein in both nuclear and cytoplasmic extracts of prostate, although the level in the cytoplasmic extract was much lower. Using immunohistochemical analysis, the PTX1 protein was localized mainly in the nuclei of glandular epithelia of normal prostate. The nuclear staining was greatly reduced in prostate carcinoma. The gene organization of PTX1 was established by comparing the cDNA sequence with the published human genomic sequence.     

Primary structure of pregnancy zone protein. Molecular cloning of a full-length PZP cDNA clone by the polymerase chain reaction

A full-length cDNA clone of the human pregnancy zone protein (PZP) was cloned from the hepatocellular carcinoma cell line Hep3B. Based on the exon sequences of the PZP gene (Devriendt et al. (1989) Gene 81, 325-334; Marynen et al., unpublished data), primer pairs were designed to amplify six overlapping fragments of the PZP cDNA. The obtained cDNA is 4609 bp long and contains an open reading frame coding for 1482 amino acids, including a signal peptide of 25 amino acid residues. Comparison with the published partial PZP amino acid sequence (Sottrup-Jensen et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7353-7357) and the PZP genomic sequences confirmed the identity as a PZP cDNA. 71% of the corresponding amino acid residues in PZP and human alpha 2-macroglobulin (alpha 2M) are identical and all cysteine residues are conserved. A typical internal thiol ester site and a bait domain were identified. A Pro/Thr polymorphism was identified at amino acid position 1180, and an A/G nucleotide polymorphism at bp 4097.     

A novel human insulinoma-associated cDNA, IA-1, encodes a protein with "zinc-finger" DNA-binding motifs.

A subtraction library was constructed from human insulinoma (beta cell tumor) and glucagonoma (alpha cell tumor) cDNA phagemid libraries. Differential screening of 153 clones with end-labeled mRNAs from insulinoma, glucagonoma, and HeLa cells resulted in the isolation of a novel cDNA clone designated IA-1. This cDNA clone has a 2838-base pair sequence consisting of an open reading frame of 1530 nucleotides, which translates into a protein of 510 amino acids with a pI value of 9.1 and a molecular mass of 52,923 daltons. At the 3'-untranslated region there are seven ATTTA sequences between two polyadenylation signals (AATAAA). The IA-1 protein can be divided into two domains based upon the features of its amino acid sequence. The NH2-terminal domain of the deduced protein sequence (amino acids 1-250) has four classical pro-hormone dibasic conversion sites and an amidation signal sequence, Pro-Gly-Lys-Arg. The COOH-terminal domain (amino acids 251-510) contains five putative "zinc-finger" DNA-binding motifs of the form X3-Cys-X2-4-Cys-X12-His-X3-4-His-X4 which has been described as a consensus sequence for members of the Cys2-His2 DNA-binding protein class. Northern blot analysis revealed IA-1 mRNA in five of five human insulinoma and three of three murine insulinoma cell lines. Expression of this gene was undetectable in normal tissues. Additional tissue studies revealed that the message is expressed in several tumor cell lines of neuroendocrine origin including pheochromocytoma, medullary thyroid carcinoma, insulinoma, pituitary tumor, and small cell lung carcinoma. The restricted tissue distribution and unique sequence motifs suggest that this novel cDNA clone may encode a protein associated with the transformation of neuroendocrine cells.     

Cloning and characterization of a novel gene（C17orf25）from the deletion region on chromosome 17p13.3 in hepatocelular carcinoma

INTRODUCTIONLoss of heterozygosity (LOH) at chromosoma1loci associated with tumor suppressor genes has beenimplicated in the genesis of many types of humanmalignancies. On the basis of frequent LOH in tu-mors, coupled with linkage analysis in some heredi-tary cancer syndromes, a number of tumor suppres-sor genes, such as RB[l], DCC[2], NF2[3], VHLI4],MTh1[5], DML/OM1[6], and PrsN/rmC1[7l have been successively isolated.It has beell reported that LOH occurred at l7p invarious ty…     

A novel gene (PLU-1) containing highly conserved putative DNA/chromatin binding motifs is specifically up-regulated in breast cancer.

A novel human gene (PLU-1) has been identified which shows a highly restricted expression in normal adult tissues but which is consistently expressed in breast cancers. A fragment of the PLU-1 cDNA was identified by differentially screening a fetal brain library with cDNAs prepared from ce-1 cells (a human mammary epithelial cell line overexpressing c-ErbB2) treated or untreated with the antibody 4D5, which inhibits c-ErbB2 phosphorylation. Clones covering the full cDNA sequence of 6.4 kilobases were isolated from a breast cancer cDNA library. Although expression of PLU-1 in ce-1 cells is regulated by signaling from c-ErbB2, the gene is expressed in all the breast cancer cell lines examined, in cells cultured from primary breast cancers, and in the invasive and in situ components of primary breast cancers. Translation of the open reading frame predicts a protein of 1544 amino acids, which contains three PHD/LAP motifs, a specific DNA-binding domain found in a Drosophila protein (dri) and novel domains showing extensive homology with other human and non human gene products. Transient transfection of cell lines with MYC-tagged PLU-1 showed the protein to be localized in the nucleus and associated with discrete foci. The presence of the dri motif and PHD/LAP fingers together with the clear nuclear localization and consistent expression in breast cancers, suggest a role for PLU-1 in regulating gene expression in breast cancers.     

编码新型转录因子样蛋白的小鼠及人类同源cDNA的克隆(英文)

 通过检索GenBank的表达序列标签 (EST)数据库并结合cDNA末端快速扩增法 (RACE) ,从小鼠胸腺克隆到一个新的cDNA序列 ,并从人类肝癌组织中克隆出了其同源cDNA .根据读码框架分析 ,这两个cDNA分别编码 541和 555个氨基酸的蛋白质 两个蛋白质之间氨基酸序列一致率为77% ,和已知蛋白无显著同源性 .分子生物学软件和网上分析表明 ,两个蛋白质所含功能序列与STAT家族成员极为相似 ,均含有包括酪氨酸蛋白激酶在内的多种蛋白激酶的磷酸化位点和核定位信号 (NLS) ,可能是一种新型转录因子 .RT PCR分析显示 ,两个基因在正常组织中选择性表达 ,其分布相似 ,而且都具有一定程度的与分化或增殖相关的趋势 .     

Primary structure of human hepatocellular carcinoma-associated aldehyde dehydrogenase

Tumor-associated aldehyde dehydrogenase (T-ALDH) is strongly expressed in hepatocellular carcinoma (HCC) but undetectable in normal liver. In the present study, this enzyme from human HCC, HCC T-ALDH, was purified and the partial amino acid sequences (384 residues) determined by direct protein sequencing matched the amino acid sequence (453 residues) deduced from cloned HCC T-ALDH cDNAs with an open reading frame. The coding sequences of HCC T-ALDH cDNA, human stomach ALDH3A1 cDNA [Hsu et al., J. Biol. Chem. 267 (1992) 3030-3037] and human squamous cell carcinoma (SCC) T-ALDH cDNA (Schuuring et al., GenBank I.D. M74542) matched one another except for discrepancies at four positions, with consequent P12R, I27F and S134A substitutions. R and A were found in HCC and SCC T-ALDHs, whereas P and S were present in stomach ALDH3A1. To confirm that these discrepancies would have general occurrence, coding sequences of HCC T-ALDH cDNAs from six patients and stomach ALDH3A1 cDNAs from two individuals were examined and all were found to encode ALDH3A1 having R, I and A at protein positions 12, 27 and 134, respectively, indicating HCC T-ALDH to be variant ALDH3A1 which is common in human stomach tissues.     

HCRP1, a novel gene that is downregulated in hepatocellular carcinoma, encodes a growth-inhibitory protein

One of the most frequent allelic deletions in hepatocellular carcinoma (HCC) has been found at chromosome 8p21-23. We reported here the identification and characterization of a novel gene for a hepatocellular carcinoma related protein 1 (HCRP1) localized at 8p22, which was isolated by positional candidate cloning. The expression of the gene for HCRP1 was most abundant in normal human liver tissue and significantly reduced or undetected in HCC tissues. The analysis of subcellular distribution showed that HCRP1 diffused in the cytoplasm with a significant fraction accumulated in the nuclei. After introduction of the sense and antisense cDNA of HCRP1 into HCC cell line SMMC-7721, we observed that the overexpression of HCRP1 significantly inhibited both anchorage-dependent and anchorage-independent cell growth in vitro. Using the transgenic short hairpin RNA (shRNA) to knock down the expression of HCRP1 gene in the other HCC cell line BEL-7404 resulted in the cell growth greatly enhanced. Moreover, reduction of the HCRP1 gene expression could also elevate the invasive ability of BEL-7404 cells. Our results strongly suggest that HCRP1 might be a growth inhibitory protein and associated with decreasing the invasion of HCC cells.     

Molecular Cloning and Characterization of a Novel Retinoblastoma-Binding Protein

We describe the isolation and characterization of a novel cDNA encoding a polypeptide that interacts in a yeast two-hybrid system as well as in mammalian cells with the retinoblastoma (RB) protein. This new protein, which we call Rim, consists of 897 amino acids, has two leucine zipper motifs, and has a LECEE sequence previously identified as an RB-binding domain. Rim also has an E1A/CtBP-binding motif and four putative nuclear localization signals.RimmRNA is expressed ubiquitously at low levels in all human adult tissues tested and at much higher levels in several tumor cell lines. TheRimgene (HGMW-approved symbol RBBP8) is localized on human chromosome 18q11.2.     

Structural analysis of the Drosophila rpA1 gene, a member of the eucaryotic ''A'' type ribosomal protein family.

The expression of ribosomal protein (r-protein) genes is uniquely regulated at the translational level during early development of Drosophila. Here we report results of a detailed analysis of the r-protein rpA1 gene. A cloned DNA sequence coding for rpA1 has been identified by hybrid-selected translation and amino acid composition analysis. The rpA1 gene was localized to polytene chromosome band 53CD. The nucleotide sequence of the rpA1 gene and its cDNA have been determined. rpA1 is a single copy gene and sequence comparison between the gene and its cDNA indicates that this r-protein gene is intronless. Allelic restriction site polymorphisms outside of the gene were observed, while the coding sequence is well conserved between two Drosophila strains. The protein has unusual domains rich in Ala and charged residues. The rpA1 is homologous to the "A" family of eucaryotic acidic r-proteins which are known to play a key role in the initiation and elongation steps of protein synthesis.