Characterization of the human p53 gene.

Cosmid and lambda clones containing the human p53 gene were isolated and characterized in detail. The gene is 20 kilobases (kb) long and has 11 exons, the first and second exons being separated by an intron of 10 kb. Restriction fragments upstream of sequences known to be within the first identified exon were tested for promoter activity by cloning them in front of the chloramphenicol acetyltransferase gene and transfecting the resulting constructs into HeLa cells. A 0.35-kb DNA fragment was identified that had promoter activity. Results of primer extension experiments indicated that the mRNA cap site falls within this fragment, as expected. Analysis of the sequence upstream of the presumptive cap site indicated that the human p53 promoter may be of an unusual type.     

杜氏盐藻rbcS启动子的克隆和功能分析

为提高转基因盐藻的表达效率,利用基因组步行方法和巢式PCR,从盐藻中克隆了1,5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）的小亚基基因rbcS 的5'上游调控序列,并对其进行序列分析和转基因功能分析。采用Dra I、EcoR V、Pvu II和Stu I四种平端限制内切酶分别酶切盐藻基因组DNA,并与接头连接,构建基因组步行文库GWL 1、GWL 2、GWL 3和GWL 4;设计特异引物从这四种文库中扩增rbcS基因的5'上游调控序列。在GWL 1、GWL 4中分别扩增出约1.2 kb的片段。对该序列的分析表明,它的3'端与已知盐藻rbcS cDNA 的5'端序列完全一致,说明是该基因的5'端上游区,并且包含多个与转录调控有关的保守序列（如TATA-box、CAAT-box）,富含GT的重复序列。此序列EcoR I下游的片段与除草剂抗性基因bar相融合,构建表达载体,电击法转化盐藻。通过对转化藻株的抗性筛选以及PCR和Southern blot检测,表明该区域能驱动外源基因bar在转基因盐藻中的表达,推断是盐藻rbcS基因的启动子调控区。     

Analysis of Cre1 binding sites in the Trichoderma reesei cbh1 upstream region

Abstract A 1.5-kb XbaI-SacII fragment containing the upstream region of the Trichoderma reesei cellobiohydrolase I gene ( cbh1 ) has been sequenced. The 1.5-kb fragment contains eight 6-bp sites having an identical or similar sequence to the consensus sequence for binding a catabolite repressor, Aspergillus nidulans CreA. Results of binding assays with the maltose-binding protein: :Cre1(10–131) fusion protein (Cre1 is a catabolite repressor of T. reesei ) and the cbhI upstream region revealed that a 504-bp XbaI-NspV fragment (nucleotide position − 1496 to − 993) bearing three 6-bp sites, Al, A2, and A3, and a 356-bp NspV-MunI fragment (nucleotide position −994 to −639) bearing three 6-bp sites, B1, B2, and B3, were shifted in the electrophoretic mobility shift assay. DNase I footprinting experiments showed that the 6-bp sites A2, B1, B2, and B3 were protected from DNase I digestion.     

Restriction endonuclease mapping of the hepatitis B viral genome isolated from Taiwan

The Eco RI fragment of hepatitis B virus (HBV) DNA isolated from human blood plasma Dane particles were inserted into plasmid pUC8 Eco RI site and transformed into E. coli JM103 host. Two recombinants pTWL1 and pTWL2 were found to carry 3.2 kbp fragment and proved to have HBV genome by Southern hybridization method. The 1.4 kbp Bam HI fragment which carried the hepatitis B viral surface antigen (HBsAg) gene, obtained via Bam HI digestion of Dane particles DNA which was made fully double stranded by endogenous DNA polymerase reaction, was also inserted into plasmid pUC8 Bam HI site. Four recombinant clones, pTWS1, pTWS2, pTWS3, and pTWS4 were found. Only one of the clones pTWS1 carried the HBsAg gene in a correct orientation with respect to the lac promoter sequence. The physical mapping of HBV DNA was performed with several restriction endonucleases. Our results indicated that the HBV DNA insert contains unique XbaI and HpaI cleavage sites and lacks the cleavage sites for the HindIII, SmaI, KpnI, SalI, and SstI endonucleases. The locations of Bam HI, BglII, and HincII endonucleases cleavage sites within the cloned HBV DNA of the pTWL1 plasmid were similar to that HBV DNA of adw and adw2 subtypes.     

New type of linker useful for cloning experiments

A new class of linker oligodeoxynucleotide sequences is described which allows the original sequence of a foreign DNA to be restored after cloning. In the standard linker approach the terminal base pairs of the linker-sequences are irreversibly attached to the cloned DNA, thus altering the genetic information. The use of the new type of linker is demonstrated by the transformation of the unique Pvu II site in pBR322 into Bam HI site using the ISO ('in-site-out') linker d(GATCCGGATC). Possible further applications of these linker sequences are described.     

人SATB1基因5′上游序列的转录激活功能分析

在对人SATB1基因进行生物信息学分析的基础上 ,采用PCR技术 ,扩增人基因组DNA中SATB1基因 5′上游序列的 - 2 95 5～ - 9片段 ,构建了 3个分别由SATB1基因 5′上游 - 2 95 5～ - 9,- 172 7～ - 9和 - 76 0～ - 9序列片段驱动的报告载体 -pGL3 SP2 94 6 luc ,pGL3 SP1718 luc和pGL3 SP75 1 luc ,分别瞬时转染JurkatT ,K5 6 2 ,U937和HeLa细胞 ,通过测定荧光素酶的表达活性 ,观察SATB1基因 5′上游序列片段 3个删除突变体在不同细胞内活性的差异 .结果显示 ,SATB1上游序列- 2 95 5 - 9在 4种细胞中的转录激活能力为U937>JurkatT >K5 6 2 ,在HeLa细胞中基本无激活 ,提示SATB1的转录激活可能具有一定的细胞类型特异性 .3种 5′删除突变体转录激活性由大至小顺序为 - 76 0 - 9>- 2 95 5 - 9>- 172 7 - 9,提示SATB1的核心启动子可能存在于其 5′上游序列的- 76 0至 - 9bp区域中 .     

VIP receptor 1 (VPAC1) promoter targets the expression of a reporter gene to cerebellum and adrenal medulla in transgenic mice

Vasoactive intestinal peptide (VIP) is a neurotransmitter with neurotropic effects. VIP functions through two distinct G-protein-coupled receptor subtypes (VPAC1 and VPAC2). We have demonstrated expression of VPAC1 in pediatric nervous system tumors, including medulloblastoma arising in the cerebellum and neuroblastoma arising in the adrenal medulla. More recently, we have reported the differentiation of neuroblastoma cells by upregulation of VIP type 1 receptor suggesting a role for VPAC1 in neuronal development.To understand the molecular mechanisms regulating VPAC1 expression in both cerebellum and adrenal medulla, we have cloned the human VPAC1 gene and sequenced 2.6-kb of the 5'-flanking sequence. Expression of the luciferase reporter gene under the control of this 2.6-kb human VPAC1 promoter was induced 35-fold in a human medulloblastoma cell line (DAOY) and 36-fold in a human neuroblastoma cell line (SKNSH). Analysis of 5'-unidirectional deletion derivatives of the 2.6-kb fragment demonstrated that a 241-bp sequence immediately upstream of the VPAC1 coding region retains high activity, suggesting that it contains the core promoter region. Quantitative RT-PCR analysis demonstrated that VPAC1 is expressed in mouse cerebellar and adrenal tissues. The VPAC1 promoter also directed expression of a reporter gene in cerebellum and adrenal medulla in transgenic mice. Along with our previous findings, these results suggest that VPAC1 may play a functional role in development of both cerebellum and adrenal medulla.     

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。