Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1

The product of the gene RCC1 (regulator of chromosome condensation) in a BHK cell line is involved in the control of mitotic events. Homologous genes have been found in Xenopus, Drosophila and yeast. A human genomic DNA fragment and complementary DNA that complement a temperature-sensitive mutation of RCC1 in BHK21 cells encode a protein of relative molecular mass 45,000 (Mr 45K) which is located in the nucleus and binds to chromatin. We have recently isolated a protein from HeLa cells that strongly binds an anti-RCC1 antibody and has the same molecular mass, DNA-binding properties, and amino-acid sequence as the 205 residues already identified. HeLa cell RCC1 is complexed to a protein of Mr 25K. We have shown that this 25K protein has a sequence homologous to the translated reading frame of TC4, a cDNA found by screening a human teratocarcinoma cDNA library with oligonucleotides coding for a ras consensus sequence, and that the protein binds GDP and GTP. We have referred to this protein as the Ran protein (ras-related nuclear protein). In addition to the fraction of Ran protein complexed to RCC1, a 25-fold molar excess of the protein over RCC1 was found in the nucleoplasm of HeLa cells. Here we show that RCC1 specifically catalyses the exchange of guanine nucleotides on the Ran protein but not on the protein c-Ha-ras p21 (p21ras).     

Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity

Alzheimer's disease is characterized by cerebral deposits of amyloid beta-protein (AP) as senile plaque core and vascular amyloid, and a complementary DNA encoding a precursor of this protein (APP) has been cloned from human brain. From a cDNA library of a human glioblastoma cell line, we have isolated a cDNA identical to that previously reported, together with a new cDNA which contains a 225-nucleotide insert. The sequence of the 56 amino acids at the N-terminal of the protein deduced from this insert is highly homologous to the basic trypsin inhibitor family, and the lysate from COS-1 cells transfected with the longer APP cDNA showed an increased inhibition of trypsin activity. Partial sequencing of the genomic DNA encoding APP showed that the 225 nucleotides are located in two exons. At least three messenger RNA species, apparently transcribed from a single APP gene by alternative splicing, were found in human brain. We suggest that protease inhibition by the longer APP(s) could be related to aberrant APP catabolism.     

人核蛋白P68 cDNA基因克隆及全序列测定

靠人合成４个混合聚核苷酸探针，从一个人盘λｇｔ１１ ｃＤＮＡ库筛选到含有ｐ６８ ｃＤＮＡ基因的阳性噬菌斑。经次克隆得到含ｐ６８ ｃＤＮＡ基因的ｐＢＲＢｔ７－ｐ６８质粒。用末端终止法对此ｃＤＮＡ基因进行了全序列测定。分析结构证明得到的ｐ６８ｃＤＮＡ基因含有２２８７３个苷酸，具有一个可阅读框架，共编码６１４个氨基酸。其５＇端非编码区含有１３０个碱基。３＇端非编码区含有３１５个碱基，比已发表的３＇端非     

Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2

A human homologue of the cdc2 gene has been cloned by expressing a human cDNA library in fission yeast and selecting for clones that can complement a mutant of cdc2. The predicted protein sequence of the human homologue is very similar to that of the yeast cdc2 gene. These data indicate that elements of the mechanism by which the cell cycle is controlled are likely to be conserved between yeast and humans.     

人SVOP基因的cDNA克隆及表达

SV2和SVOP都是大鼠中包含有12个跨膜结构域的突触小泡蛋白.笔者从人类大脑的cDNA文库中分离得到了一个类似于大鼠突触小泡蛋白SV2和SVOP的人类新基因的全长cDNA序列,它包含一个含有1 646个核苷酸的开放阅读框,编码一个含548个氨基酸的蛋白质.生物信息学分析表明:该基因定位在第12号染色体的12q24.12区域.Northern杂交结果表明:该基因只在人类大脑组织中表达,且与无脊椎动物线虫、疟蚊和果蝇的同源基因有50%的氨基酸同源性,与脊椎动物大鼠和小鼠的同源基因有90%以上的氨基酸同源性.     

Identification of reciprocal translocation sites within the c-myc oncogene and immunoglobulin mu locus in a Burkitt lymphoma

The association between certain human tumours and characteristic chromosomal abnormalities has led to the hypothesis that specific cellular oncogenes may be involved and consequently 'activated' in these genetic recombinations. This hypothesis has found strong support in the recent findings that some cellular homologues of retroviral onc genes are located in chromosomal segments which are affected by specific tumour-related abnormalities (see ref. 4 for review). In the case of human undifferentiated B-cell lymphoma (UBL) and mouse plasmacytomas, cytogenetic and chromosomal mapping data have identified characteristic chromosomal recombinations directly involving different immunoglobulin genes and the c-myc oncogene (for review see refs 5, 6). In UBLs carrying the t(8:14) translocation it has been shown that the human c-myc gene is located on the region of chromosome 8 (8q24) which is translocated to the immunoglobulin heavy-chain locus (IHC) on chromosome 14. Although it is known that the chromosomal breakpoints can be variably located within or outside the c-myc locus and within the IHC mu (refs 9, 11) or IHC gamma locus, the recombination sites have not been exactly identified and mapped in relation to the functional domains of these loci. We report here the identification and characterization of two reciprocal recombination sites between c-myc and IHC mu in a Burkitt lymphoma. Nucleotide sequencing of the cross-over point joining chromosomes 8 and 14 on chromosome 14q--shows that the onc gene is interrupted within its first intron and joined to the heavy-chain mu switch region. This recombination predicts that the translocated onc gene would code for a rearranged mRNA but a normal c-myc polypeptide.     

Identification and nucleotide sequence of a human locus homologous to the v-myc oncogene of avian myelocytomatosis virus MC29

Avian myelocytomatosis virus MC29 is a replication-defective acute leukaemia virus which induces a variety of tumours in chickens including sarcomas, renal and hepatic carcinomas, and myelocytomatosis. The oncogenic potential of the virus is mediated by the gene v-myc, acquired from sequences (c-myc) present in normal uninfected chicken DNA. Sequences closely related to chicken c-myc have been highly conserved throughout evolution, from Drosophila to vertebrates. The hypothesis that c-myc may be involved in neoplastic transformation has been strengthened by the finding that B-cell lymphomas induced in chickens by avian leukosis virus (ALV) are often associated with increased expression of c-myc resulting from integration of the ALV provirus adjacent to the c-myc gene. More recently, it has been demonstrated that the malignant human cell line HL-60, derived from the peripheral blood leukocytes of a patient with acute promyelocytic leukaemia, expresses elevated levels of myc-related mRNA associated with an amplification of the c-myc gene. To explore the relationship of the human cellular myc gene with the corresponding viral oncogene from MC29, and to provide a framework for the analysis of the mechanism and significance of c-myc amplification in human tumours, we have isolated and determined the nucleotide sequence of a genomic clone prepared from a normal human library which contains all domains sharing homology with v-myc.     

人类磷酸泛酰巯基乙胺结合蛋白新基因的分离和鉴定

在对AD293和HEK293进行差减杂交以探索两者在吸附和凋亡特性上的差异时,从AD293的高表达文库中分离得到一段新的cDNA片段.从人类胎脑文库克隆得到该基因,全长2 745 bp,编码的蛋白含518个氨基酸,被预测为磷酸泛酰巯基乙胺结合蛋白.该基因在染色体上定位于2p22.3,包含8个外显子.该cDNA编码的蛋白序列含有一个凋亡抑制蛋白5结构域,外皮蛋白重复片段和铜结合辛肽重复片段.RT-PCR分析显示该基因在人类正常组织和癌组织中广泛表达,但在癌组织中表达量相对较低,提示其可能对细胞凋亡有抑制作用.该基因在进化过程中高度保守.     

Molecular cloning and expression analysis of a novel human cDNA fragment encoding a putative Ser/Thr protein kinase

A novel full-length cDNA encoding a putative serine/threonine kinase has been isolated from a human testis cDNA library. A nucleotide sequence of 1225 bp length has been determined containing an open reading frame of 1 044 nucleotides (encoding 348 amino acids). In view of its degree of homology to members of the Ser/Thr protein kinase family and the closest relationship toMus musculus STK-1, the predicted product was designated by the name of HUMSTK-1. Its mRNA is present in large amounts in thymus, and small amounts in testis, small intestine and colon.     

Insertional mutagenesis of the myc locus by a LINE-1 sequence in a human breast carcinoma

The proto-oncogene c-myc is the cellular homologue of the transforming sequence carried by the avian myelocytomastosis virus MC29. A growing body of evidence implicates structural and functional alterations in and around proto-oncogenes such as c-myc in tumorogenesis. Here we report that comparison of the structure of myc from a ductal adenocarcinoma of the breast and from normal breast tissue of the same patient (Sc) revealed a tumour-specific rearrangement of one myc locus and amplification of the other myc locus. (For myc reviews see refs 1-4; for myc involvement in breast neoplasia see refs 5-7.) Within the second intron of the rearranged locus was a non-myc sequence with nearly complete homology to a long interspersed repetitive element (a LINE-1 sequence or L1). In this case, the L1 sequence has functioned as a mobile genetic element to produce a somatic mutation.     

Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently, Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.     

Reciprocal chromosome translocation between c-myc and immunoglobulin gamma 2b genes

Specific chromosome translocations have been observed in transformed cell lines of both man and mouse and may be implicated in the origin or maintenance of malignancy. In mouse plasmacytomas, translocations have been identified that bring the immunoglobulin alpha heavy-chain gene (C alpha, normally located on chromosome 12) into proximity with c-myc (normally located on chromosome 15), c-myc being the mouse cellular homologue of the avian myelocytomatosis virus transforming gene (v-myc). Here we identify a DNA rearrangement in a mouse hybridoma that has brought c-myc close to C gamma 2b and show that this rearrangement occurred by reciprocal chromosome translocation, as recombinant clones were isolated from the same cell line in which a rearranged variable-region (VH) gene has been brought close to 5' c-myc sequences. The translocation has resulted in the net loss of 7 base pairs (bp) of chromosome 15 sequence as well as in the presence of an additional base of unknown provenance. This reciprocal translocation was analysed in DNA from a mouse hybridoma cell line but is shown to be characteristic of the X63Ag8 myeloma parent.     

马铃薯卷叶病毒中国分离株外壳蛋白基因及其上游先导序列的cDNA克隆和序列分析

按照马铃薯卷叶病毒（ＰＬＲＶ）核苷酸序列，针对ＣＰ基因及其上游基因间隔区全长约０．８ｋｂ的区段设计合成两个特异性引物，以马铃薯卷叶病毒中国分离株（ＰＬＲＶ－Ｃｈ）的ＲＮＡ为模板，反转录合成ＣＤＮＡ第一条链，再经ＰＣＲ扩增合成ｃＤＮＡ，将ＣＤＮＡ克隆于ｐＵＣ１９质粒．限制性酶切分析和核苷酸序列测定表明克隆的ＰＬＲＶ－Ｃｈ外壳蛋白（ＣＰ）基因及其上游基因间隔区的全长ＣＤＮＡ共８２４个核苷酸，与国外报道的４个ＰＬＲＶ分离株的核苷梳序列相比具有高度同源性．ＰＬＲＶ的外壳蛋白基因序列与其上游基因间隔区相比保守性更强．     

Cloning and identification of a novel human glioma differentiation related gene GDR1

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed gene GDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame of GDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows that GDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns of GDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.     

一个新的水稻GST类似蛋白基因XIG的克隆与分析

根据编码一种玉米GST类似蛋白的mRNA In2-1序列设计引物，以水稻cDNA文库为模板，PCR扩增得到一条270bp的DNA片段，以此片段为探针分别筛选水稻根cDNA文库及水稻基因文库，获得一条全长cDNA及其相应的全长基因，并通过测序得到了两者的全序列，该基因编码的蛋白具有GST的典型特征，在水稻中尚未见报道。