A novel tenascin type III repeat is part of a complex of tenascin mRNA alternative splices.

Sequence analysis of two human tenascin encoding cDNA clones from a cDNA library of the U251 glioblastoma cell line revealed the presence of a novel 276 bp tenascin type III fibronectin like repeat. This alternatively spliced type III repeat designated AD1 is located between the previously identified repeats 10 and 11 and has sequence homology with human, chicken and mouse tenascin type III repeats. These results show that tenascin has at least 16 consecutive fibronectin like type III repeats. PCR amplification of random primed mRNA with specific type III repeat primers revealed a pattern of multiple alternative splices of AD1 and flanking type III repeats. The alternative splice variants were confirmed by direct sequencing. Differences were observed in the expression of the various alternative splices of tenascin mRNA between tumor and normal cells and may thus indicate differences in tenascin isoform expression and function in normal and tumor cells. PCR and Southern analysis of genomic DNA indicate that AD1 is coded by a single exon present in both human and mouse genome.     

鼻咽癌相关基因NAP1的克隆及鉴定

从UniGene库中选取编号为BG231197,来自人鼻咽组织的EST序列.利用Blast检索GenBank的nr数据库和EST数据库,构建EST重叠群.利用人类基因组草图搜索法从成人正常鼻咽组织中PCR扩增获得该基因全长cDNA,命名为NAP1,GenBank登录号为AY190326.NAP1基因cDNA序列全长为573 bp,编码由85个氨基酸组成,相对分子质量为9 700的多肽.用α-³²P-dCTP标记NAP1基因片段,与含15种正常成人组织的多组织RNA印迹膜杂交,结果表明NAP1基因在淋巴结和气管中高表达,转录本大小约为0.6 kb,在其他组织中不表达.NAP1蛋白质与滤泡树突状细胞（follicular dendritic cell, FDC）的一种分泌肽前体（FDC -SP）（AF435080）同源,与其他已知蛋白质无明显同源性.NAP1基因定位在染色体4q13,基因组跨越9 179 bp,含5个外显子和4个内含子.采用差异RT-PCR检测了40例经病理诊断为低分化鳞状上皮癌的鼻咽活检组织及其对侧相应部位的正常鼻咽组织中该基因的表达差异.在40例鼻咽癌中,NAP1基因表达下调的有17例（42.5%）,表达上调的有6例（15%）,无明显表达差异的有17例（42.5%）.原位杂交和免疫组化结果显示该基因在正常鼻咽和鼻咽癌组织间质中的树突状细胞中表达,在其他间质细胞和鼻咽上皮中均不表达.以上结果表明,NAP1为树突状细胞的一种新的多肽,该基因在鼻咽癌组织中表达下调的原因,及其与鼻咽癌发生、发展的关系值得进一步探讨.     

Cell surface-expressed Thomsen-Friedenreich antigen in colon cancer is predominantly carried on high molecular weight splice variants of CD44.

Increased mucosal expression of TF, the Thomsen-Friedenreich oncofetal blood group antigen (galactose beta1-3 N-acetylgalactosamine alpha-) occurs in colon cancer and colitis. This allows binding of TF-specific lectins, such as peanut agglutinin (PNA), which is mitogenic to the colorectal epithelium. To identify the cell surface TF-expressing glycoprotein(s), HT29 and Caco2 colon cancer cells were surface-labeled with Na[(125)I] and subjected to PNA-agarose affinity purification and electrophoresis. Proteins, approximately 110-180 kDa, present in HT29 but not Caco2 were identified by Western blotting as high molecular weight splice variants of CD44 (CD44v). Selective removal of TF antigen by Streptococcus pneumoniae endo-alpha-N-acetylgalactosaminidase substantially reduced PNA binding to CD44v. Immunoprecipitated CD44v from HT29 cell extracts also expressed sialyl-Tn (sialyl 2-6 N-acetylgalactosaminealpha-). Incubation of PNA 15 microg/ml with HT29 cells caused no additional proliferative effect in the presence of anti-CD44v6 mAb. In colon cancer tissue extracts (N = 3) PNA bound to CD44v but not to standard CD44. These data show that CD44v is a major PNA-binding glycoprotein in colon cancer cells. Because CD44 high molecular weight splice variants are present in colon cancer and inflammatory bowel disease tissue but are absent from normal mucosa, these results may also explain the increased PNA reactivity in colon cancer and inflammatory bowel disease. The coexpression of oncofetal carbohydrate antigens TF and sialyl-Tn on CD44 splice variants provides a link between cancer-associated changes in glycosylation and CD44 splicing, both of which correlate with increased metastatic potential.     

Genomic organization, physical mapping, and expression analysis of the human protein arginine methyltransferase 1 gene

Protein arginine methyltransferases (PRMTs) regulate mRNA processing and maturation by modulating the activity of RNA-binding proteins through methylation. The cDNA for human PRMT1 (HRMT1L2) was recently identified. In this paper, we describe the complete genomic organization of the human PRMT1 gene (GenBank Accession No. AF222689), together with its precise chromosomal localization in relation to other neighboring genes. We have also examined its expression in a total RNA panel of 26 human tissues, the BT-474 breast carcinoma cell line, and 16 breast tumors. PRMT1, which spans 11.2 kb of genomic sequence on chromosome 19q13.3, is located in close proximity to the IRF3 and RRAS genes and is transcribed in the opposite direction. It is formed of 12 coding exons and 11 intervening introns, and shows structural similarity to other PRMT genes. Three PRMT1 isoforms exist as a result of alternative mRNA splicing. Amino acid sequence comparison of the splicing variants indicates that they are all enzymatically active methyl transferases, but with different N-terminal hydrophobic regions. PRMT1 expression was detected in a variety of tissues. We have shown that the relative prevalence of alternatively spliced forms of PRMT1 is different between normal and cancerous breast tissues. Although PRMT1 was not found to be hormonally regulated by steroid hormones in breast cancer cells, our results suggest that two variants of PRMT1 are down regulated in breast cancer.     

Gene cloning of immunogenic antigens overexpressed in pancreatic cancer

The serological analysis of recombinant cDNA expression libraries (SEREX) by utilizing a library derived from a human pancreatic adenocarcinoma cell line and IgG antibodies from an allogeneic patient serum led to the identification of 18 genes: 13 of these were known genes, and 5 were unknown genes. In Northern and RT-PCR analyses, we found that the expression of mRNA of 14 genes was elevated in pancreatic cancer cell lines compared with the levels in normal pancreatic tissues. In addition, the expression of mRNA of hsp105 in colon cancer was greater than that in normal colon tissue. Immunohistochemical analysis using anti-hsp105 antibody revealed that an increased expression of hsp105 is a characteristic feature of pancreatic ductal and colon adenocarcinoma. Furthermore, hsp105 immunoreactivity in some cases of gastric, esophageal, and hepatocellular carcinoma was much stronger than that in normal corresponding tissues. These molecules identified may provide good diagnostic markers for cancer cells.     

Different expression patterns for ubiquitous calpains and Capn3 splice variants in monkey ocular tissues

The purpose of the present investigation was to compare the expression of ubiquitous and tissue-specific calpains in ocular tissues from the Macaca fascicularis monkey. Calpain isoforms in retina and corneal epithelium from adult M. fascicularis monkeys were characterized by RT-PCR, cDNA cloning and sequencing. Calpain isoform activities in ocular tissues were investigated by fractionation on DEAE-HPLC, immunoblotting, and casein zymography. Capn3 splice variants in the ocular tissues from rat, rabbit and monkey were compared after RT-PCR. RT-PCR analysis revealed that numerous splice variants of Capn3 were expressed in the epithelium from monkey cornea. The variants contained deletions or insertions in or around the IS1, IS2, and NS regions. The cDNAs for Capn3 variants were highly conserved, yet the expression patterns of the Capn3 isoforms were widely different among the mammalian species. In contrast, the expression patterns of ubiquitous calpains in ocular tissues were conserved among the mammalian species, and similarities between monkey and human cDNAs for Capn1 (mu-calpain) and Capn2 (m-calpain) were 98 and 99%, respectively. These results suggested that differences in expression patterns of Capn3 variants might be related to the function of each variant in a particular tissue or species.     

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.     

Expression of splice variants of 1alpha-hydroxylase in mcf-7 breast cancer cells

1,25-Dihydroxyvitamin D(3) (calcitriol) is the most active natural metabolite of Vitamin D(3). It has strong antiproliferative and differentiating effects on various cell types including breast cancer cells. 25-Hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase, CYP27B1) is one of the key enzymes in the formation of calcitriol. It has been found in breast cancer cells suggesting an autocrine regulation of formation of calcitriol in these cells. Alternative splicing of the encoding genes for this enzyme can possibly play a role in regulating the enzyme level and can explain tissue specific variations of 1alpha-hydroxylase activity. Splice variants containing intron 1 may encode for truncated proteins with deletion of protein domains which are essential for its enzymatic activity. In order to obtain more information on the abundance of 1alpha-hydroxylase splice variants, we performed a highly specific nested touchdown PCR in MCF-7 cells. The full-length sequence of 1alpha-hydroxylase and two different splice variants of this enzyme containing intron 1 were isolated. By Western blot technique we then confirmed the protein products of the full-length enzyme and its splice variants. We hypothesize that that the expression of splice variants can lead to a quantitatively lower expression of the mRNA of the full-length enzyme. The abundance of less active 1alpha-hydroxylase protein variants can alter the local synthesis of calcitriol in the cells and may explain variations of enzymatic activity in different cells and tissues.