EXPRESSION OF N-ras GENE IN HUMAN PRIMARY HEPATOCARCINOMA

Poly(A)~+ RNA was isolated from 9 specimens of human primary hepatic carcinoma, 1 non-tumorous liver tissue adjacent to cancer and 1 normal liver tissue samples. The Oligo-dT cellulose-purified poly(A)~+ RNAs were subjected to formaldehyde agarose gel electrophoresis, Northern transfer and hybridization with various ancogene probes. Two RNA species, 5.6 kb and 2.2 kb wore identified by N-ras gone hybridization in 6 out of 9 mRNA samples from primary hepatic carcinoma specimen. N-ras specific mRNA was not detectable in mRNA samples from normal human liver and tumor surrounding cirrhotic tissue. No detectable hybridization of mRNA from hepatoma and normal liver with Ki-ras or Ha-ras was observed.As human N-ras gene has been identified in DNA of mouse transfeetants transformed with PHC DNA, it strongly suggests that N-ras gene might be responsible for the transforming activity of part of cases of human liver cancer.     

IDENTIFICATION OF HUMAN N-ras AS THE COMMON ONCOGENE IN NIH 3T3 CELLS TRANSFORMED WITH DNAs FROM HUMAN PRIMARY HEPATIC CANCER AND HEPATOMA 7402 LINE

DNA was extracted from NIH 3T3 cells transformed with DNAs from human primary hepatic cancer (PHC) and Hepatoma 7402 cell line. The transformant DNA was analyzed by Southern transfer and hybridization with ~(32)P-labeled probes of various oncogenes.The EcoRI 7.2 and 9.0 kb bands characteristic of human N-ras gene were identified in transformed NIH 3T3 cells derived both from PHC and 7402 DNA. The BamHI 6.6kb band characteristic of human c-Ha-ras I was present only in 7402 transformants, but not in PHC transformants.Using ~(35)S-methionine incorporation, immunoprecipitation with anti-p21 monoclonal antibodies, SDS-PAGE and autoradiography, it was demonstrated that p21 synthesis was remarkably enhanced in 1402 cells as well as in transformed cells derived from both 7402 and PHC DNA.Taking the data together, it. strongly implies that N-ras is one of the transforming genes for human liver cancer.     

Nucleotide Sequence of a Cloned Human HBV Mutant (pDKHBV) in Duck Hepatoma of Qidong County

This is the first report to describe the presence of an HBV-like DNA sequence in two hepatoma and their tumor surrounding liver tissues, one precancerous and one non-malignant liver tissue of ducks collected from Qidong County of China. The HBV-like sequences were either in an episomal form of 3.2 kb or in an integrated form of various sizes, while the DHBV DNA sequences (3.0 kb) were either present or absent in these tissues and in different size pattern. Furthermore, there was no evidence of cross-hybridization between HBV-like DNA sequences in duck and DHBV DNA. A 3.2 kb HBV-like DNA sequence has been cloned from one duck hepatoma (40 K), designated as pDKHBV. The 3218 bp full-length nucleotide sequence of this clone has been determined, which had no apparent homology with Duck Hepatitis B Virus (DHBV) genome, but was highly homologous to human HBV adw_2 subtype (99.0%). The sequence was composed of four open reading frames for HBV gene Pre-S/S, X, C and P respectively. In addition to multiple sites     

RESTRICTION ENDONUCLEASE CLEAVAGE MAP OF MITOCHONDRIAL DNA FROM PEKING DUCK LIVER

Mitochondrial DNA of the liver from Peking duck was cleaved by restricition endonu-cleases EcoRI, BamHI, PstⅠ and BglⅠ into 1, 2, 4 and 5 fragments, respectively, while the BglⅡ was without any cleavage. The restriction map of this mtDNA was constructed by measuring the length of restriction fragments using both electrophoresis analysis and electron microscopy. The position of D-loop and the direction of replication of the mtDNA were also determined.     

TRANSTHYRETIN (PREALBUMIN) GENE IN HUMAN PRIMARY HEPATIC CANCER

From a subtracting cDNA library constructed from normal liver versus human primary hepatic cancer (PHC), a cDNA clone pG8 was isolated. Using it as a probe, RNA extracted from one human liver and 9 PHC samples were analyzed by Northern hybridization. As expected, its mRNA was highly expressed in liver; however, the expression was strikingly suppressed in PHC. Only weak signal was observed in 2 out of 9 PHC, while no signal was detectable in the other 7 samples. Utilizing pG8 as a probe, DNA from the same PHC specimens was analyzed after MspI digestion and Southern hybridization. Deletion of DNA fragment was observed in 4 out of 9 samples. In further study of cancer and non-cancerous liver from other 7 PHC patients, similar deletion of DNA fragments in cancer was observed in 4 out of 7 samples. After sequencing of the clone of 572 bp, it was unexpectedly found that pG8 was completely homologous to the coding sequence of transthyretin, TTR gene, as TTR (or prealbumin) gene has been known to be linked to a     

STUDY ON BclI/St14 RFLPs IN CHINESE FOR DNA DIAGNOSIS FOR HEMOPHILIA A

The BclI polymorphism within DXS52 (St14) was reported. It was composed of 4 allelic fragments 4.0 kb, 3.3 kb, 3.0 kb and 2.3 kb, The frequency of these fragments were 0.09, 0.12, 0.44 and 0.35 respectively in the Chinese. The polymorphism provided the PIC of 0.66. DNA analysis of families, with hemophilia A showed that the confidence of the RFLPs was the same as the TaqI/St14 RFLPs and for carrier detection the former is much better than that of the TaqI/St14 RFLPs.     

MOLECULAR CLONING AND NUCLEOTIDE SEQUENCE ANALYSIS OF A TRANSFORMING GENE FROM HUMAN GASTROCARCINOMA CELL LINE

Mouse and rat fibroblasts were transfected with total DNA from human gastrocarcinoma cell line BGC-823. It was shown by hybridization assay that the genome of one of the rat secondary foci contains transforming genes from the human gastrocarcinoma cell line, which are homologous to the protooncogene c-Ha-ras in the normal cells. The genomic library of the rat secondary foci was constructed, using λ phage EMBL3 as the vector. The transforming gene Ha-ras of the human gastrocarcinoma cell was thus cloned by screening the library with the probes of human Alu repeat sequence and c-Ha-ras. The nucleotide sequences of the first and second exons were analysed by M13-dideoxy method. The result shows that the nucleotide sequence of the transforming gene is the same as that of the normal protooncogene except one nucleotide difference in the first exon.     

INHIBITED NEOPLASTIC PHENOTYPE BY THE c-Ha-ras ANTISENSE RNA

A 2.0 kb fragment DNA plasmid which expresses antisense to the upstream first exon of c-Ha-ras oncogene was transfected into Ha-ras transformed cell lines, GCM-3T3 and REF-4.3. The transfection leads to the inhibition of malignant behaviour, shown by decreasing of growth speed, colony forming ability on soft agar, tumorigenicity in nude mice and increasing ot differentiation degree. In GCM-3T3 cells the lung metastasis frequency became much less (from 60% to 12.5%) after the transfection and the expression of ras oncogene product, and p21 protein was obviously decreased in the transfected cells. This work has first shown the inhibitory effect of antisense RNA on neoplastic behaviour in China.     

STUDIES ON THE MECHANISM OF CARCINOGENESIS(IX)——DIFFERENTIAL EXPRESSION OF PROLIFERATING AND TISSUE-SPECIFIC ENZYME GENES-DURING HEPATOCARCINOGENESIS

With cDNA fragments of CPSI, OCT and ACT as probes, dot and Northern blot analyses of poly(A)+-RNA from rat liver with different pathological lesions during carcinogenesis induced by di-ethylnitrosamine were conducted. It was shown that the level of mRNA of tissue-specific enzymes, CPSI and OCT decreased while that of the proliferating enzyme ACT mRNA increased, and the alteration was correlated with the degree of pathological changes in each case. The relative changes in the mRNA level of these enzymes during hepatocarcinogenesis were correlated with that of enzyme activities. Implication of these findings in the mechanism of carcinogenesis in terms of cell proliferation and differentiation was discussed.     

STUDIES ON RNA LIGASE ACTIVITY IN THE BRAIN AND LIVER CELLS OF MOUSE

RNA ligase in eukaryotic mammalian cells was studied by using mouse brain and livercell extracts as enzyme sources and Oligo A as substrates. RNA ligase activity was deter-mined by measuring the formation of alkaline phosphate-resistant product from 5'-~32P-termi-hated Oligoribonucleotides. Under appropriate conditions, the activity of this enzyme inbrain and liver cells may vary between 16-49 mU/ml. The joining way between donorand acceptor is 5'-P→3'-OH. Further studies wore carried out by using synthetic UpCpUand ~32pNp as substrates and crude enzyme preparations from extracts of cell unclei of brainand liver as enzyme sources. RNA ligase activity was examined by homochromatographyand autoradiography. A clear joining product was demonstrated and then isolated from thereaction mixture by DEAE-Sephadex A25 column chromatography. The eluted fractionswere identified by DEAE-cellulose thin layer chromatography. The joining product washydrolyzed either with KOH or with alkaline phosphatase, the autoradiog     

THE POLYHEDRIN GENE OF Bombyx mori NUCLEAR POLYHEDROSIS VIRUS

Cloned SalI fragments of Bombyx mori nuclear polyhedrosis virus (BmSNPV) DNA were screened with the polyhedrin gene of Autographa californica nuclear polyhedrosis virus as a probe. One positive clone, pBN61, with an insert of 1.65 Kb, was obtained. The Hind-Ⅲ, HpaⅡ and AluⅠ maps of the insert were constructed. Part of its nucleotide sequence has been determined. The 46 amino acid sequence, as determined from the nucleotide sequence, was compared with the reported sequence of BmSNPV polyhedrin. Only ono amino acid difference has been found. It is likely that clone pBN61 contains the whole BmSNPV polyhedrin gene.     

STUDY ON THE GENOME OF CAULIFLOWER MOSAIC VIRUS (XINJIANG ISOLATE)——RESTRICTION MAPPING OF VIRION AND CLONED VIRAL DNA

The genomie DNA of CaMV (Xinjiang isolate) was mapped on the virion DNA with anumber of restriction endonueleases by double digestions and partial digestions of one-end~(32)P-labelled fragments. It contained the unique sites of BglI, SalI and XhoI. BamHI andHhaI each cut it at two sites, EeoRI at five sites (three of which were located), Hpa Ⅱ atseven sites, BglⅡ at eight sites, and HaeⅢ at ten sites. The sites of all the enzymes usedabove, as well as HindⅢ which cut at ton sites were also mapped on the cloned CaMVDNA. Virion DNA and cloned viral DNA gave the same results in the number and locationof cleavage sites of the restriction enzymes tested on both DNAs. Like most of the otherisolates, CaMV-Xinjiang isolate has three single-stranded discontinuities. From the diges-tion patterns of SalI, XhoI, EcoRI and HhaI, it is concluded that the restriction map ofCaMV-XJ genome is closely related to that of BKMV isolate from East Germany.     

New process of low-temperature methanol synthesis from CO/CO_2/H_2 based on dual-catalysis

A new process of low-temperature methanol synthesis from CO/CO2/H2 based on dual-catalysis has been developed. Some alcohols, especially 2-alcohol, were found to have high catalytic promoting effect on the synthesis of methanol from CO hydrogenation. At 443 K and 5 MPa, the synthesis of methanol could process high effectively, resulting from the synergic catalysis of Cu/ZnO solid catalyst and 2-alcohol solvent catalyst. The primary results showed that when 2-butanol was used as reaction solvent, the one-pass average yield and the selectivity of methanol, in 40 h continuous reaction at temperature as low as 443 K and 5 MPa, were high up to 46.51% and 98.94% respectively. The catalytic activity was stable and the reaction temperature was 80 K or so lower than that in current industry synthesis process. This new process hopefully will become a practical method for methanol synthesis at low temperature.     

AN EFFICIENT SITE-DIRECTED MUTAGENESIS USING POLYMERASE CHAIN REACTION

We report here a simple and efficient method for site-directed mutagenesis using polymer-ase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restrictiongene, we made two point mutations. While one created a new Sall site prior to the SDsequence, the other replaced Glu144 with Lys. A 1.5 kb Sall-PstI fragment isolated frompER101 was used as the template. Two 25 mer oligonucleotide primers containing the de-sired mutations were synthesized and used to direct PCR amplification with Taq DNA poly-merase. About 0.5μg of the 0.49 kb fragment was obtained from 0.05 μ of the 1.5 kb frag-ment by carrying out polymerase chain reaction for 30 cycles. As calculated theoretically,99% of the product contained the desired mutations. The product was cloned into pUC19using Sall and PstI, two of the transformed colonies were randomly chosen for sequence anal-ysis, and both of them were shown to contain the desired mutations. Finally, the amplifiedfragment was cloned into pER304 to place the     

ANALYSIS OF FRAGMENT INTERACTION AND ASSIGNMENT OF ELECTRONIC SPECTRUM FOR COMPLEX ION[S2MoS2FeCl2]^2—

<正> The electronic structures of complex ion [S2MoS2FeCl2]2- (1) and its fragments MoS42- (2) and FcCl2(3) have been calculated base on the LCAO-HFS method with restricted open shell or closed shell. The interaction between the fragments 3 and 2 and the formation of complex ion 1 have been discussed. It was found that the Fe(Ⅱ) donated electrons to the Mo(Ⅵ)and accepted electrons from the sulphur ligand and that the stability of complex ion 1 is contributed from both direct and indirect interactions through the bridging sulphur atoms. In addtion, the electron transition energies of complex ion 1 were calculated and its electronic absorptions were assigned. It was shown that the calculated wavelengths of the absorption bands are in agreement with the observed ones.     

MOLECULAR CLONING OF A NEW VARIANT OF HUMAN PAPILLOMAVIRUS TYPE 6 ISOLATED FROM A CHINESE WOMAN AND EXPRESSION OF ITS L1 GENE IN E. coli——MOLECULAR CLONING & EXPRESSION OF HPV6 GENE

A human papillomavirus genome DNA of 7.9 kb from a Chinese woman with genital condyloma acuminata was cloned in Bam HI site of pAT153. According to the results obtained from Southern blotting, restriction mapping as well as partial DNA sequencing, the isolated genome (HPV6BV) had obvious variance and was referred to as a new variant of HPV6 found in China the first time. HPV6BV L1 gene was successfully expressed in E. coli as a fusion protein with pUR288. The β-galactosidase/L1 fusion protein reacted with both β-galactosidase antiserum and HPV antibody using Western blot technique. The E. coli-produced fusion protein, possessing HPV antigenicity, may provide a reagent for clinical diagnosis and epidemiological survey.     

THE PREPARATION OF FRUCTOSE-MODIFIED CHITOSAN MICROCARRIERS AND CULTURE OF PRIMARY RAT HEPATOCYTES

1. INTRODUCTION The development of cell based organs and tissue-engineering devices such as hybrid artificial liver requires a large number of cells be cultured for the replacement of damaged tissue [1,2]. Since its introduction in 1967, microcarriers culture has been applied successfully in growing primary cells and cell lines with the advantage of attaining high cell density [3,4]. Spheroid culture is another promising hepatocytes culture method to enhance the cell density and metaboli…     

Studies on the Antiviral Activities in vitro of Polysaccharide from Eucheuma striatum

To assay the antiviral activities on HSV-1 and CVB3 in vitro of the polysaccharide from Eucheuma striatum, its antiviral mechanism was explored. Vero cells were infected by HSV-1 and CVB3, and they were cultured with serial dilutions of polysaccharide. The cells cytotoxicity of Polysaccharide was evaluated by the MTT method. The inhibitory effects were evaluated by the cytopathic effect (CPE). Its antiviral mechanism was researched by the method of giving samples in different time. The polysaccharide could inhibit the CPE of cells infected by HSV-1 and CVB3. It showed low cytotoxicity on vero cells. Its antiviral activities were better than those of acyclovir and ribavirin which were run in parallel as the positive control samples. The polysaccharide from Eucheuma striatum has potent antiviral activities. Its antiviral mechanism is that it can prevent the virus from absorbing to the cell surface.     

COLIPHAGE M13 CLONING SYSTEM AND ddXTP CHAIN-TERMINATION METHOD FOR DNA SEQUENCING

Two DNA fragments of cytochrome b gene in yeast mitochondral DNA were sequenced by Messing's M13 cloning system and Sanger's ddXTP chain-termination method. M13mp8 and M13mp9 serve as vectors for insert fragment. The recipient strain is E. coli JM103 for transfection. When the ratio of insert/vector was 3:1, high frequencies of recombination and positive recombination were obtained. The two fragments, which have 575 bp and 709 bp, were sequenced. To read more bases, the ratio of ddXTP/dXTP must be reduced. On one X-ray film of 80×17cm 394 bp were read.     

MEIOTIC CHROMOSOME BEHAVIOR IN FEMALE INTERSEXES OF ARTIFICIAL TRIPLOID TRANSPARENT-COLORED CRUCIAN CARP

Chromosome behavior in meiosis was studied by ail-drying, C-banding and surface-spreading methods in female intersexes of artificial triploid transparent-colored crucian carp (Corassius auratus). Chromosome pairing and contraction were obviously asynchronous. The preferential pairing of two homologous chromosomes was the major pattern of chromosome pairing, and a few triple pairing, repeated pairing, telomere or centromere associating and multiple pairing were also observed in the pachytene cells. The metaphase I cells were main-ly composed of univalents, bivalents and trivalents, as well as a few of other multivalents, such as tetravalents, pentavalents, hexavalents and heptavalents, were also found in some metaphase I cells. The chromosome elements including uni-, bi-, tri- and other multivalents varied considerably among the metaphase I cells, and the associating patterns of multivalents were also diverse. Some 6 n and 12 n cells, in which premeiotic endomitosis occurred once or twice, were found at