Vol 7 No.1 1997

Replication of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion by Escherichia coil cell extracts

WANG GE,PAUL M DUNMAN,M ZAFRI HUMAYUN
Depertment of Microbiology and Molecular Genetics UMD New Jersey Medical School 185, South ORange Avenue,MSB F607 Newwark, HJ 07103-2714,USA

ABSTRACT
Previous investigation on the mutagenic effects of 3, N4-Ethenocytosine (εC) , a nonpairing DNA lesion, revealed the existence of a novel SOS-independent inducible mutagenic mechanism in E.coli termed UVM for UV modulation of mutagenesis. To investigate whether UVM is mediated by an alteration of DNA replication, we have set up an in vitro replication system in which phage M13 viral single-stranded DNA bearing a single site-specific.
(εC) residue is replicated by soluble protein extracts from E.coil cells, Replication products were analyzed by agarose gel electrophoresis and the frequency of transleasion analyses, Our data indicate that DNA replication is strongly inhibited by εC,but that translesion DNA synthesis does occur in about 14% of the replicated DNA molecules. These results are very similar to those observed previously in vivo, and suggest that this experimental system may be suitable for evaluating a lterations in DNA replication in UVM-induced cells.

Key words: Ethenocytosine,M13, in vitro replication, cell extract

Human chromosome pellicle antibody recognizing centromere pretein-C(CENP-C), the main component of the kinetochore

XIE YONG,ZU MEI NI,JIAN RENGU,PHIL WONG,WEN QING WU,GUO WEI XU
Hongkong University of Science and Technology, Department of Biology , Hongkong
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai
Shanghai Cancer Institute ,National Labortory for Incogenes and Related Genes, Shanghai

ABSTRACT
Recently the antichromosome antisera form several scleroderma patients have been found to recognize the pellicle of metaphase and anaphase chromosomes . Tn order to indetify the pellicle component s, we used these antichromoseme antisera to serene a human e,bryonic cDNAlibrary. The sequences of the positive clones are identical to the cDNA gene sequence of CENP-C(centromere protein C),a human centromere aitoantigen. This result suggests that CENP-C is a component of the pellicle of human metaphase and anaphase chromosomes.
Key words: Human antibody, scleroderma,CENP-C(cnetromere protein C), metaphase,chromosome pellicle, indirect imminofluorescent staining.

Increment of hFIX expression with endogenous intron1 in vitro

ZHENGBING, XIAO YUN QIN,MIN TAN ,YONG NA XING,DARU LU, JING LUN XUE,XIN FANG QIU
Institute of Genetics, Fudan University , Shanghai 200433

ABSTRACT
This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous intro 1 sequence . hFIX minigene was obtained with middle sequence truncated intron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i’ix,retroviral vector G1NaCiIX were constructed . These vectors were transduced into target cells of PA317,C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/Pkg5Iix,151 ng/106 cells/24h; PA317/G1NaCiIX,308 ng/106 cells/24h; C2c12/G1 NaCi’IX, 186 ng/106 cells/24 h; RSF/G1NaCiIX, 1929ng/10/106 cells/24 h;;HSF/G1NaCi’IX,1646 ng/106 cells/24 h. These results indicated that hFIX minigene with intron 1 is able to increase the expression level to about 3 times of that of hFIX minigene in the retroviral-medicated gene transger system and regrain from intron splicing during viral production, a retroviral vector G1NaCi’IXR with reversely inserted hFIX minigene expression cassette was constructed/ The expression level if reverse constructor in PA317 cells was 390 ng/106 cells/24 h.with 79% of bioactivity. PCR detection of HT/G1NaCi’IXR cells infected with PA 317/G1NaCi’IXR supernatant confirmed the existence of intron 1 sequence. These results suggested that expression vector with forward-inserted intron1-carrying hFIX expression cassette can be used in directed gene Human factor IX expression with itron transfer, but when using the retroviral-mediated gene transfer system, resersely-inserted intron1-carrying hFIX expression cassette should be considered.

Ket words: Intron 1 , human clotting factor IX (hIFX), gene transfer, gene expression, reverse inserted sequence.

Cytological indentification of an isotetrasomic in rice and its application to centromere mapping

CHENGZHUKUAN,HENG XIUYU,CHANG JIE YAN, LI HUANG ZHU,MING HONG GU
Depertment of Agronomy, Agriculure College, Yangzhou University,Yangzhou 255009
Institute of Genetics, Chinese Academy of Sciences, Beijing 100101

ABSTRACT
The aneuploid with isochromosome or telochromosome is ideal material for exploring the position of centromere in lingkage map. For obtaining these aneuploids in rice, the primary trisomics from triplo-1 to triplo-12 and the aneuploids derived from a troploid of indica rice barety Zhongxian 3037 were carefully investageted. From the offsprings of troplo-10, a primary trisomic of chromosome 10 of the variety, an isotetrasomic “triplo-10-2”, in which the trasomic, a secondary trisomic “triplo-10-2”,in which the extra-chromosome was an isochromosome derived from the short arm of chromosome 10, was identified. With the isotetrasomic, secondary trisomic, primary trisomic and diploid of variety Zhongxian 3037, different molecular markers were ised for exploring the position of the centromere of chromosome 10. Based on the DNA dosage effect, it was verified that the molecular markers G1125, G333 and L169 were located on the short arm, G1084 and other 16 available molecular markers were on the long arm of chromosome 10. So the centromere of chromosome 10 was located somewhere between G1125 and G1084 according to the RFLP linkage map given by Kurata et al. The distance from G1125 to G1084 was about 3.2cM.
Key words: Rice, isotetrasomic, centromere mapping

High efficiency DNA delivery into swine swine oocytes and embryos by electronic pulse delibery (EPD)

YANG YANG,SHAO HUA HAUNG,XI ZHAO
Incell, Inc 2310 Walsh Avenue, santa Clara, California 95051,USA

ABSTRACT
The production of transgenic swine for xennotransplantation has been proposed as an option to overcome the chronic shortage of human organ donors. Generation of genetically engineered swine has been elusibe due to the difficulties in gene transfer. In order to achieve effective gene delivery, a key step for the genetic modification we applied electronic pulse delivery (EPD) technology to introduce H2Kb-DC DNA construct into swine eggs. Using the developed EPD ProtocolsTM, ,we have achieved good viability of the EPD treated oocytes, satisfactory embryonic development of the EPD treated embryos with high efficiency. Thus, application of the EPD technology promises to effectively facilitate the generation of large transgenic mammals.
Key words: Xentransplantation, transgenic pig, electronic pulse delivery (EPD), swine embryos, DNA transfer.

Identification and expression of epidermal growth factor gene in mouse testis

ZHANG MEI LAN, YUAN CHANG YAN,YA PING SUN, S S KOIDE
Shanghai Institute of Cell Biology, Chinese Academy of Sciences 320 Yo-yang Road Shanghai 200031,China
Center for biomedical Research, The population council, 1230 York Avenue, New York,NY 10021,USA

ABSTRACT
Epidermal growth factor(EGF) is produced primarily by Leydig cells of human testis. Expression of the EGF gene was assessed in mouse testis during the course of sexual maturation by the application of the RT-PCR method and the use of specific oligonucleotide primers. Testis EGF mRNA content increased with the developmental age of the mice,i.e., day 15<day<30<day 45 postnatal. The expression of the EGF gene appears to correlate with maturation of the testis and proliferation of Leydig cells
Key words: Epidermal growth factor, testis, gene expression, spermatogenesis, sexual maturation.

Characterization of 5’-proximal sequence of mouse GABA transporter gene(GAT-1)

FEI JIAN,FANG HUANG,YIN HUA MA,LI HE GUO
Shanghai Institute of Cell Biology, Chinese Academy of Sciences ,Shanghai 200031

ABSTRACT
The cDNA molecule encoding the mouse GABA transporter gene(GAT-1) was used as probe for selecting GAT-1 gene from mouse genomic library . A positive clone, harboring the whole open reading frame of the GAT-1 protein and designated as MGABAT-G, was fished out from the library , the 5’ proximal region and intron 1 were found in the above region between GAT-1 genes grom mouse and human except some short conserved sequences, The DNA protein interaction between DNA fragments containing the conserved sequences in the 5’s proximal region and nuclear proteins from different tissues of mouse were studied by means of gel-shirt assay, and Southern-Western blot. The results indicate a possible positive-negative regulation mode controlling the expression of the mouse GAT-1 gene.
Key words: Mouse GABA transporter gene, expression regulation, cloning.

Molecular characterization and functional expression of opioid receptor-like receptor

WU YA LAN, LU PU ,KUN LING,JIAN ZHAO,ZHI JIE CHENG,LAN MA, CANG PEI
Shanghai Institute of Cell Biology, Chinese Academy of Sciences ,Shanghai 200031
Shanghai Medical University, Shanghai 200032, China

ABSTRACT
The opioid receptor-like receptor-like receptor(ORL1), an orphan receptor whose human and murine complementary DNAs, has been characterized recently. OLR1 transcripts are particularly abundant in the central nervous system. We demonstrated that ORL1 expressed in human neuroblastoma SK-N-SH and SH-SY5Y cell lines by radioligigand binding saasy, reverse transcription polymerase chain reaction (RT-PCR) and Northern analysis in the present study. Stimulation with ORL1 specific agonist, nociceptin/orphanin FQ, increased GTPγS binding to SK-N-SH cell membranes (EC50=140.45 nM),and attenuated forskolin-stimulated accumulation of cellular cAMP (EC50=0.80±0.45nM), indicative that actovation of ORL1 activates G proteins and inhibits adenylyl cyclase. Activation of ORL1 receptor was also accessed using CHO:hORL1 cell line by microphysiometer. Treatment of nociceptin/orphanin FQ increased extracellular acidification rate sighificantly.

Key words: Opioid receptor-like receptor (ORL1), nociceptin/orphanin FQ(N/OFQ), SK-N-SH cells, extracellular acidification, functional expression.

Differential expression if a cNDA clone in human liver veruss hepatic cancer-highly homologous to aryl-dialkylphosphase

WANG KAN KAN, DA FANG WAN,XIAO KUN QIU, PEI XIN LU, JIAN REN GU
National Laboratory for Oncogenes and Related Genes Shanghai Cancer Institute, Shanghai 200032
Qidong Cancer Institute, Jiangsu 226200

ABSTRACT
We applied the technique of mRNA differential display to normal liver tissue and hepatoma ell line Hep3B. One of the isolated cDNA clones was expressed in human normal liver tissue but of mRNA was expressed in human normal liver tissue from hepatoma patients. Low level or no expression was observed in human hepatoma tissue . One of these transcripts was about 1.8 kb in length. Southern Blot analysis showed that ir was a single copy gene. We obtained a full length cDNA clone of 2,395 bp by screening human liver 5'-stregth plus cDNA library. Nucleotide sequence indicated that this clone was highly homologous to aryldialkyl-phosphatase which has a prominent role in the metabolism of several toxic, synthetic compounds , may be potentially related to human hepatocarcinoma suspectibility. The biological significance of tis differential expression in normal versus malignant tissue is discussed.

Key words: Hepatocarrinoma, mRNA DD, aryl-dialkyl- phosphatase.

The apaptosis of HEL cells induced by hydroxyurea

GUI CHANG YUN, CHU JIANG, HENG YUE XIE,RUO LAN QIAN
Shanghai Institute of Cell Biology, Chinese Academ of Sciences, Shanghai 200031

ABSTRACT
Hydroxyurea has been used to synchronize cultured cells to S-phase and used to treat patients with sichlecell anemia . Recently, we found that hydroxyurea can induce the apotosis of HEL (human ceythroleukemia) cells. The induced HEL cells shoued ultrastructurally chromatin condensation with regular crescents at the nuclear edges and apoptotic bodies. However , the cells of K562, another humanerythreleukemia cell line did not show such morphological changes. Under fluoroscope , the HEL cells after induction often displayed a clear reduction in nuclear diameter and nuclear ring and apoptotic bodies. Analysis with flow cytometry showed that the percentage of apoptotic cells is about 30%-40%after HEL cells were induced by hydroxyurea for 3 days. DNA ladder can be observed by electrophoretic analysis.

Key words: HEL cells ,hydroxyurea, apoptosis

RAPD analysis of natural populations of ACanthopanax brachypus

YAN JUN, SI LAN DAI, NAI HU WU
Institute of Developmental Biology,Chinese Academy of Sciences , Beijing 100080

ABSTRACT
Random Amplified Polymorphic DNA(RAPD)
analyisi is a new technology of molecular marking which has proved very powerful in detecting genetic diversity at the level of population. The genomic DNAs used in our experiment were extracted from fresh leaves taken from 59 individuals sampled from three natural populations in Yan an , Shanxi Province . Through more than 2,000 PCRs, deep-going RAPD analysis was carried out on DNA sanples from 49 individuals . The percentage of polymorphic RAPD loci found in these three populations were respectively 27.2%,18.6%,and 5.4%; the average genetic distances within population , 0.055,0.036 and 0.060. The genetic diversity of A. brachypus within and between populations was found , for the first time, to be rather poor, thus revealing status. In addition, our work also provieds basic materials for elucidating the underlying cause of its endangerment and for its protein biology.

Key words: Ananthopanax brachypus; genetic diversity; radom amplified polymorphic DNA (RAPD) analysis.

Associatation of DNA with nuclear nuclear matrix in in vitro assembled nucleiinduced by rDNA from Terahymena shanghaiensis in Xenopus egg extracts

CHEN YING , BO ZHANG,XIU FEN LI ,ZHONGHE ZHAI
Department of Cell Biology and Genetics , College of Life Sciences, Peiking University, Beijing 100871

ABSTRACT
The nuclei assembled from exogenous DNA or chromatin in egg extracts resemble their in vivo counterparts in many aspects. However, the distribution pattern of DNA in these nuclei remains unknow. We introduced rDNA from the macronuclei of Tetrahymena into Xenopus cell-free extracts to examine the association of specific DNA sequences with nuclei aeeembled in votro. Our previous works showed the 5'NTS (non-transcription sequences) of the rDNA specifically bind to the NM system in the macronuclei . We show now the rDNA could inducd chromatin assembly and nuclear formation in Xenopus cell-free system . When we extracted the NM system and compared the binding affinity of different regions of rDNA with the NM system ,we found that the 5' NTS still hod their bing affintiy with insoluble structure of the assembled nuclei in the extracts in the extracts of Xenopus eggs.

Key words: Nuclear assembly, nuclear matrix, Xenopus egg extracts, Tetraphymena rDNA.

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