Expression of histocompatibility antigens H-2K, -D, and -L is reduced in adenovirus-12-transformed mouse cells and is restored by interferon gamma.

Primary mouse cells transformed by adenovirus type 12 (Ad12) expressed negligible amounts of class I antigens H-2K, -D, and -L on the cell surface and were capable of forming tumors in syngeneic animals, whereas cells transformed by Ad5 continued to express class I antigens and were nontumorigenic. Cells from a tumor, generated by injection of Ad12-transformed mouse cells into a syngeneic mouse, also expressed low levels of H-2 antigens, indicating that this phenotype is maintained in vivo. In all Ad12-transformed cells, synthesis of the H-2 heavy chain was not detected whereas the beta 2-microglobulin light chain was synthesized. Furthermore, the level of cytoplasmic H-2 mRNA in the Ad12 lines was greatly reduced. Reduction of H-2 expression is instructed solely by the transforming region of the viral genome, since this repression occurred in cells transformed by a DNA fragment containing only Ad12 E1A and E1B genes. Addition of recombinant murine interferon gamma strongly stimulated expression of class I antigens in the Ad12 transformants as well as in cells from the Ad12 tumor. This result indicates that Ad12 does not preferentially transform cells that are deficient for class I genes and that Ad12 does not mutate the class I genes in cells it transforms. The correlation between tumorigenicity and loss of H-2 expression in Ad12-transformed cells is discussed.     

Adenovirus type 12 early region 1A proteins repress class I HLA expression in transformed human cells

The adenovirus type 12 (Ad12) early region 1A (E1A) gene is thought to play a major role in repressing class I major histocompatibility complex expression in transformed rodent cells. However, since transformation by adenovirus requires both E1A and E1B genes, it has not been demonstrated whether the Ad12 E1A gene acts alone or synergistically with the E1B gene to accomplish this effect. Moreover, it is not known whether the repression of class I antigen synthesis by Ad12-transforming gene products occurs only in rodent cells. We show that the Ad12 E1A gene, in the absence of the E1B gene, is capable of greatly reducing the levels of class I HLA antigens and mRNAs in primary human cells transformed by the E1A gene of Ad12 and the large tumor antigen (T-antigen) gene of BK virus; control cells transformed by BK virus T-antigen gene alone or the highly related simian virus 40 T-antigen gene showed no apparent alteration in class I HLA expression. Human recombinant interferon gamma was able to restore synthesis of class I HLA antigens in transformed cells that produced Ad12 E1A proteins, indicating that these cells were not deficient for class I genes. These results strongly indicate that the Ad12 E1A proteins modulate class I gene expression by similar mechanisms in both transformed rodent and human cells.     

Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression.

The adenovirus type 12 (Ad12) DNA sequences integrated into the DNA of four lines of Ad12-transformed hamster cells are extensively methylated. Methylation in mammalian cell DNA is believed to occur predominantly at 5'-C-G-3' sequences. The majority, although not all, of the 5'-C-C-G-G-3' sequences present in integrated Ad12 DNA are methylated. Ad12 DNA isolated from purified virions, on the other hand, is not methylated to any significant extent. The segments of the integrated viral DNA comprising early genes, which are expressed as mRNA in two lines of Ad2-transformed hamster cells, are undermethylated in comparison to late viral segments, which are not expressed and are extensively methylated. In contrast, in two lines of Ad12-induced rat brain tumor cells, some of the late viral genes have been shown to be expressed as mRNA. The segment of the integrated Ad12 DNA that comprises these late genes, the EcoRI B fragment, is undermethylated in comparison to the extensive methylation of the same fragment in Ad12-transformed hamster cells. Thus, there appears to exist a striking inverse correlation between the levels of methylation of specific DNA segments and the extent to which these segments are expressed as mRNA. The functional significance of this correlation remains to be determined. It may provide a clue to understanding the regulation of gene expression in transformed cells and perhaps in eukaryotic cells in general.     

丙型肝炎病毒非结构蛋白NS4B反式激活基因的克隆化研究

目的应用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建丙型肝炎病毒(HCV)非结构蛋白4B(NS4B)转染细胞差异表达cDNA消减文库,克隆HCV NS4B蛋白反式激活相关基因.方法以HCV NS4B表达质粒pcDNA3.1(-)-NS4B转染HepG2细胞,以空载体pcDNA3.1(-)为对照,制备转染后的细胞裂解液,提取mRNA并逆转录为cDNA,进行抑制性消减杂交分析.将富集的二次PCR产物与T/A载体连接,并转染大肠杆菌进行文库扩增,随机挑取克隆聚合酶链反应(PCR)扩增后进行测序及同源性分析.结果文库扩增后得到33个阳性克隆,经菌落PCR分析显示其中28个克隆含有大小不等的200～1000 bp插入片段.测序及同源性分析显示,12种已知基因编码蛋白,包括一些与细胞周期、信号传导及肿瘤发生等细胞生长调节密切相关的蛋白编码基因,可能是NS4B反式激活靶基因.结论成功构建了HCV NS4B反式激活基因差异表达的cDNA消减文库,为进一步阐明HCV NS4B反式调节的靶基因在肝炎、肝纤维化和肝细胞癌发生的分子生物学机制提供理论依据.     

马尔尼菲青霉酵母相消减cDNA文库的构建及初步筛选

目的构建马尔尼菲青零酵母相抑制性消减cDNA文库，寻找其在酵母相中的差异表达基因。方法分别提取马尔尼菲青零菌丝相和酵母相的总RNA并合成cDNA，然后应用抑制性消减杂交技术（SSH）。以酵母相为tester（检测子），菌丝相为driver（驱赶子），连接不同的接头，通过两轮杂交和两次抑制性PCR后，将产物与T载体连接并转染大肠杆菌。经PCR鉴定，共得到480条插入片段。序列分析和同源性比较表明一些基因与细胞壁抗原、转运蛋白、氧化还原酶等具有同源性。结果成功构建了一个以酵母相为tester（检测子）的抑制性消减cDNA文库。结论所构建的cDNA消减文库为进一步筛选马尔尼菲膏零致病相关基因奠定了基础。     

Evidence that a second tumor antigen coded by adenovirus early gene region E1a is required for efficient cell transformation.

The expression of the adenovirus (Ad) early coding region 1a (E1a) is required for virus-induced cell transformation and for the activation of other viral early genes and some cellular genes. Two overlapping early mRNAs of 13S and 12S that are transcribed from this region code for a 289-amino acid protein and a 243-amino acid protein, respectively. Earlier studies have shown that the 289-amino acid protein is essential for cell transformation. We have constructed an Ad type 2 (Ad2) deletion mutant (dl231) in which the intervening sequence for the 13S mRNA is precisely removed. Mutant dl231 is completely viable in human KB cells and produces normal amounts of 13S mRNA but much reduced amounts of a defective 12S mRNA. Mutant dl231 induces focal transformation of established rat embryo fibroblasts at a frequency one-fifth to one-half that of wild-type virus. However, the transformed cells are defective in their ability to form anchorage-independent colonies on semisolid medium. Therefore, our results demonstrate that the 243-amino acid protein is required for full transformation of rat embryo cells.     

应用抑制性消减杂交技术筛选丙型肝炎病毒E1蛋白反式激活基因

目的　应用抑制性消减杂交 (SSH)技术构建丙型肝炎病毒 (HCV)E1蛋白反式激活基因差异表达的cDNA消减文库 ,克隆HCVE1蛋白反式激活相关基因。方法　以HCVE1表达质粒pcDNA3 .1( -) E1转染肝母细胞瘤细胞系HepG2细胞 ,以空载体pcDNA3 .1( -)为对照 ;制备转染后的细胞裂解液 ,从中提取mRNA并逆转录为cDNA ,经RsaI酶切后将实验组cDNA分成 2组 ,分别与 2种不同的接头衔接 ,再与对照组cDNA进行 2次消减杂交及 2次抑制性PCR ,将产物与T/A载体连接 ,构建cDNA消减文库 ,并转染大肠杆菌进行文库扩增 ,随机挑选克隆PCR扩增后进行测序及同源性分析。结果　成功构建人HCVE1蛋白反式激活基因差异表达的cDNA消减文库。文库扩增后得到 89个阳性克隆 ,进行菌落PCR分析 ,均得到 10 0 10 0 0bp插入片段。挑取 46个含有插入片段的阳性克隆测序分析 ,获得 44个已知基因序列和 2个未知基因。通过生物信息学分析获得其全长序列 ,已被GenBank收录。结论　应用SSH技术成功构建了HCVE1反式激活基因差异表达的cDNA消减文库。该文库的建立为进一步阐明HCVE1反式调节的靶基因及致肝脏疾病发生的分子生物学机制提供理论依据     

Selective inhibition of Escherichia coli protein synthesis and growth by nonionic oligonucleotides complementary to the 3' end of 16S rRNA

A series of nonionic oligonucleotide analogues, the deoxyribooligonucleoside methylphosphonates, were synthesized. The base sequences of these compounds, d(ApGpGp), d(ApGpGp)(2), and d[(ApGpGp)(2)T], are complementary to the Shine-Dalgarno sequence (-A-C-C-U-C-C-U-) found at the 3' end of bacterial 16S rRNA. These nonionic oligonucleotide analogues were tested for their ability to inhibit the in vitro translation of mRNAs in cell-free systems of Escherichia coli and rabbit reticulocyte. In the E. coli system, both d(ApGpGp)(2) and d[(ApGpGp)(2)T] effectively inhibited MS-2 RNA-directed protein synthesis but they had much less effect on either poly(U)- or poly(A)-directed polypeptide synthesis. In the reticulocyte system, these compounds had no significant effect on the translation of globin mRNA. The observation that d[(ApGpGp)(2)[(3)H]T)] binds to 70S ribosomes (association constant, 2.0 x 10(4) M(-1), 37 degrees C) together with the specificity of the inhibitory action of these compounds on protein synthesis strongly suggests that inhibition of translation is a consequence of analogue binding to Shine-Dalgarno sequence of 16S rRNA. The oligonucleoside methylphosphonates inhibited both protein synthesis (without concurrent inhibition of RNA synthesis) and colony formation by E. coli ML 308-225 (a permeable mutant) whose cell wall contains negligible quantities of lipopolysaccharide but had no effect on wild-type E. coli B. Our preliminary results on the uptake of oligodeoxyribonucleoside methylphosphonates by E. coli B show that these cells are not permeable to oligomers longer than 4 nucleotidyl units. Although oligodeoxyribonucleoside methylphosphonates are taken up by mammalian cells in culture, this series of analogues had negligible inhibitory effects on colony formation by transformed human cells. This study indicates that this class of nonionic oligonucleotide analogues can be used to probe and regulate the function and structure of nucleic acids of defined sequence within living cells.     

Initiation of protein synthesis from a termination codon.

We show that the amber termination codon UAG can initiate protein synthesis in Escherichia coli. We mutated the initiation codon AUG of the chloramphenicol acetyltransferase (CAT) gene to UAG (CATam1) and translated mRNA derived from the mutant CAT gene in E. coli S-30 extracts. A full-length CAT polypeptide was synthesized in the presence of tRNA(fMetCUA), a mutant E. coli initiator tRNA which has a change in the anticodon sequence from CAU to CUA. Addition of purified E. coli glutaminyl-tRNA synthetase substantially stimulated synthesis of the CAT polypeptide. Thus, initiation of protein synthesis with UAG and tRNA(fMetCUA) most likely occurs with glutamine and not methionine. The UAG codon also initiates protein synthesis in vivo. To eliminate a weak secondary site of initiation from AUC, the fifth codon, we further mutagenized the CATam1 gene at codons 2 (GAG----GAC) and 5 (AUC----ACC). Transformation of E. coli with the resultant CATam1.2.5 gene yielded transformants that synthesized CAT polypeptide and were resistant to chloramphenicol only when they were also transformed with the mutant tRNA(fMetCUA) gene. Immunoblot analyses and assays for CAT enzyme activity in extracts from transformed cells indicate that initiation from UAG is efficient, 60-70% of that obtained from AUG. Initiation of protein synthesis from UAG using a mutant initiator tRNA allows tightly regulated expression of specific genes. This may be generally useful for overproduction in E. coli and other eubacteria of proteins which are toxic to these cells.     

应用抑制性消减杂交技术克隆乙型肝炎病毒全S蛋白反式激活基因

目的应用抑制性消减杂交(SSH)技术构建乙型肝炎病毒(HBV)全S蛋白反式激活基因差异表达的cDNA消减文库,克隆HBV全S蛋白反式激活相关基因.方法以HBV全S表达质粒pcDNA3.1(-)-全S转染HepG2细胞,以空载体pcDNA3.1(-)为对照;制备转染后的细胞裂解液,从中提取mRNA并逆转录为cDNA,经RsaI酶切后将实验组cDNA分成两组,分别与两种不同的接头衔接,再与对照组cDNA进行两次消减杂交及两次抑制性PCR,将产物与T/A载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增,随机挑选克隆PCR扩增后进行测序及同源性分析.结果成功构建人HBV全S蛋白反式激活基因差异表达的cD-NA消减文库.文库扩增后得到86个白色克隆,进行菌落PCR分析,均得到100-1000 bp插入片段.挑取35个含有插入片段的阳性克隆测序分析,获得33个已知基因序列,和2个未知基因,通过生物信息学分析获得其全长序列,其中之一命名为全S蛋白反式激活基因1(CSTP1),已在GenBank中注册,注册号:AY553877.未知基因的功能还正在研究中.结论应用SSH技术成功构建了HBV全S反式激活基因差异表达的cDNA消减文库.该文库的建立为进一步阐明HBV全S反式调节的靶基因及致肝病发生的分子生物学机制提供理论依据.     

家蝇抗菌肽MAF-1A的串联表达与抗白念珠菌体外活性检测

目的 构建家蝇抗菌肽MAF-1A基因原核串联表达体系,在大肠杆菌中表达具有抗菌活性的MAF-1A。方法 根据大肠杆菌密码子的偏嗜性进行密码子优化,设计并合成含有5个拷贝的MAF-1A串联基因序列;将合成的串联基因序列克隆到表达载体pET28a并转化大肠杆菌Roseeta(DE3),应用IPTG诱导表达5×MAF-1A串联重组蛋白;通过SDS-PAGE电泳、Western Blot对表达产物进行分析;用肠激酶专一性切割经Ni-NTA纯化后的5×MAF-1A重组串联蛋白,得到MAF-1A单体;采用微量稀释法检测MAF-1A单体对白念珠菌的体外抗菌活性。结果 5×MAF-1A蛋白在大肠杆菌中呈可溶性表达,28 ℃、0.8 mmol/L IPTG诱导12 h可达最大表达量;酶切后的抗菌肽MAF-1A单体对白念珠菌的MIC和MBC分别为0.5 mg/mL、1.0 mg/mL。结论 成功构建MAF-1A原核串联表达系统,重组表达的MAF-1A对白念珠菌具有较高的抑杀活性。     

Isolation, sequence, and bacterial expression of a cDNA for (S)-tetrahydroberberine oxidase from cultured berberine-producing Coptis japonica cells.

cDNA clones for the (S)-tetrahydroberberine (H4Ber) oxidase of cultured berberine-producing Coptis japonica cells were isolated by screening a C. japonica cDNA library with synthetic nucleotides that can encode the NH2-terminal sequence of this enzyme. Analyses of the nucleotide sequences of the cloned cDNA inserts revealed a 759-base-pair open reading frame that encoded a 253-amino acid polypeptide with a Mr of 27,089 and NH2-terminal and internal sequences identical with those of the (S)-H4Ber oxidase, as determined by microsequencing methods. Escherichia coli were transformed with an expression vector carrying (S)-H4Ber oxidase cDNA. The transformed bacteria were induced to overproduce a 28-kDa protein that reacted with Coptis (S)-H4Ber oxidase-specific antibody. A comparison of the derived amino acid sequence of (S)-H4Ber oxidase with sequences in the protein data base of the Protein Research Foundation showed a marked similarity between (S)-H4Ber oxidase and the NH2-terminal portion of mouse P1-450, which is encoded by a single exon of the mouse P1-450 gene. The availability of cloned cDNA for (S)-H4Ber oxidase allows use of the methods of molecular biology to study the regulation of (S)-H4Ber oxidase gene expression in cultured C. japonica cells in relation to berberine biosynthesis.     

HLA-DR gene expression in a proliferating human thyroid cell clone (12S)

We have used a retroviral vector carrying the adenovirus E1A oncogene and the neomycin phosphotransferase gene to establish a human thyroid-derived cell line that exhibits TSH-mediated cAMP generation as well as the differential expression of HLA class II antigens in response to recombinant gamma-interferon. Twenty-two-week gestation, histologically confirmed, human fetal thyroid was collagenase digested, cultured as a monolayer, and infected directly with 12S or 13S E1A-containing retrovirus constructs. Infected clones (n = 30) were selected in a hormone-supplemented medium containing bovine TSH (bTSH; 1 mU/ml), 10% fetal bovine serum, and 0.5 mg/ml G418 antibiotic. A rapidly growing clone (designated 12S) was chosen for detailed analysis over 18 months of continuous culture. The 12S clone was sensitive to less than 10 microU/ml bTSH when assessed by extracellular accumulation of cAMP, but TSH had no influence on 72-h incorporation of [3H]thymidine. Clone 12S responded to recombinant human gamma-interferon (1-10(4) U/ml) by induction of HLA DR alpha-chain-specific mRNA and the surface expression of HLA-DR antigen detected by fluorescein isothiocyanate-labeled monoclonal antibody to nonpolymorphic HLA-DR regions using flow cytometry. These studies indicate the potential for immortalizing human thyroid cells for use as targets of anti-TSH receptor immune responses and for long term studies of human throcyte HLA gene regulation.     

Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus.

Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [ with an oligo(dA)10 primer] functioned as template only with E. coli polymerase. (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1. The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.     

类泛素FAT10高表达肝癌细胞株Hep3B酵母双杂交cDNA文库的构建

目的:构建类泛素FAT10高表达肝癌细胞株Hep3B的酵母双杂交用cDNA文库.方法:从肝癌细胞Hep3B中提取总RNA,分离mRNA.利用反转录酶M-MLV与Oligo(dT)AnchorPrimer合成1stStrandcDNA,用E.coliDNAPolymerase与E.coliDNALigase将RNA链置换成DNA链,合成2ndStrandcDNA.将双链cDNA与EcoRⅠAdaptor连接,然后用EcoRⅠ/XhoⅠ进行酶切.使用SpinColumn除去短链cDNA与pGADT7载体连接,转化入E.coliDH10B,建成原始文库.然后对其进行扩增并随机挑取单菌落,酶切鉴定重组子插入片段大小.结果:提取的总RNA降解少且分子完整;RNA纯度高,相对分子质量为400-5000bp;成功合成双链cDNA,均符合建库要求;库容量达到1.03×106克隆,原始文库滴度为2.50×109cfu/L,扩增后的文库滴度为3.60×1012cfu/L.插入片段大小分布为0.5-3.5kb,平均长度约为2.0kb.结论:所构建文库的各项指标均达到要求,为筛选FAT10作用蛋白奠定了重要基础.     

The bicistronic nature of lens alpha-crystallin 14S mRNA.

The A2 and B2 polypeptide chains of calf lens alpha-crystallin are synthesized on a 14S, 1500-nucleotide mRNA and a 10S, 735-nucleotide mRNA, respectively. The 10S mRNA is theoretically compatible with the size of the B2 chain, but the 14S mRNA contains approximately twice the required number of nucleotides necessary for A2 chain synthesis. This fact raises the question of the function of the additional nucleotide sequence in the 14S mRNA. The following observations on 14S mRNA suggest that it may contain an additional cistron. (i) Under a number of denaturing conditions, 14S mRNA continues to retain its initial size characteristics. (ii) In addition to synthesis of the A2 chain, 14S mRNA directs the synthesis of another polypeptide with the same electrophoretic mobility as that of the B2 chain. (iii) Molecular hybridization of the 14S mRNA with the cDNA produced from the 10S mRNA suggests that 2 mol of the cDNA bind to 1 mol of the 14S mRNA. (iv) Examination of the nucleotide sequences of the 10S and 14S mRNAs by two-dimensional maps of RNase A and T1 digests indicates marked similarity. The overall data suggest that the additional cistronic component may carry coding information for an alpha-crystallin polypeptide or a closely related polypeptide species.     

人胰腺癌抑制消减cDNA文库的构建

目的应用抑制消减杂交技术(SSH)构建胰腺癌和正常胰腺组织间差异表达的抑制消减cDNA文库。方法分别提取胰腺癌(tester)和癌旁正常胰腺组织(driver)中的总RNA和mRNA．合成双链cDNA，经RsaI酶切后，将胰腺癌双链cDNA分为两组，分别加上不同的接头，再与正常胰腺组织cDNA进行两次消减杂交及两次抑制性PCR，分离出胰腺癌差异表达基因的cDNA片段。将该差异表达片段克隆至T／A载体，并转化大肠杆菌TOP10F’，经蓝白斑筛选后，再用PcR方法筛选阳性克隆，从而构建胰腺癌抑制消减cDNA文库。结果文库扩增后得到257个白色克隆，随机挑取50个阳性克隆进行PCR扩增分析，其中47个克隆有插入片段．克隆阳性率为94％，片段大小主要集中在300～600bp之间。结论成功构建了人胰腺癌抑制消减cDNA文库，为进一步筛选、克隆胰腺癌特异性表达基因奠定了基础。