Integration and expression of the human dihydrofolate reductase gene in the Bacillus subtilis chromosome

Integration of functionally active human dihydrofolate reductase (hDHFR) gene into the Bacillus subtilis chromosome was performed. The clones obtained contained 1 to 7 copies of hDHFR gene per chromosome equivalent and were resistant to trimethoprim. In cell lysates of such clones a protein with the molecular mass of hDHFR was detected. The hDHFR gene was stably maintained in all clones having this gene integrated into the bacterial chromosome, when grown under non-selective conditions.     

Free and integrated forms of hepatitis B virus DNA in human hepatocellular carcinoma cells (PLC/342) propagated in nude mice.

Primary hepatocellular carcinoma cells (PLC/342) propagated in nude mice produce hepatitis B surface antigen of subtype adr, as well as core particles containing viral DNA and DNA polymerase. Free and integrated forms of hepatitis B virus (HBV) DNA in the tumor were isolated by molecular cloning, and their nucleotide sequences were determined. Both of the two representative clones of free HBV DNA had the same genomic length (3,158 base pairs) and had two stop codons as well as two deletions in the envelope gene. None of the seven distinct clones of integrated HBV DNA possessed the entire viral genome. The integrated clone sequences had deletions and rearrangements, and only two clones possessed the envelope gene including the promoter and enhancer sequences. The C gene, which codes for core protein, was preserved in the two free clones and one of the integrated clones. The P gene, which codes for DNA polymerase, had deletions at two positions of 21 and 36 base pairs in both free clones, but was carried in toto by one of the integrated clones. The nucleotide sequences of the S genes of two free and four integrated clones, as well as their two inverted repeats, were compared. All of the eight sequences of the S gene possessed two nucleotide substitutions in common that were not displayed by any of the reported HBV genomes. The sequences differed from one another by only 1.2%. They differed, however, from 11 reported HBV genomes of subtype adr by 2.4%, from an ayr genome by 1.9%, from 2 adw genomes by 6.9%, and from 2 ayw genomes by 5.9%. These results indicate that all free and integrated HBV DNA species in the PLC/342 tumor cell evolved from a common progenitor. The free HBV DNA underwent nucleotide substitutions during several integration events, resulting in integrated HBV DNA copies that were similar in sequence but distinct from the reported HBV genomes.     

Carrier DNA affects the integration of HSV-1 TK gene in the genome of L929TK- cells.

L929TK- cells were cotransfected with DNA mixtures containing tk gene of HSV-1, plasmids carrying LTR of MoMLV or RSV and carrier DNA of salmon sperm or chromosomal DNA of recipient cells. Selection of TK+ transformants was conducted in DMEM supplemented with HAT. Plasmids carrying LTR sequences of MoMLV or RSV retroviruses showed enhancing effect on the frequency of TK+ transformation. Southern blot analysis of chromosomal DNA of TK+ transformants demonstrated in clones deriving from cotransfections of tk gene and carrier DNA of L929TK- cells multiple copies of tk gene integrated into several genomic sites of host. Single copies of tk gene integrated into different sites of host genome occurred in chromosomal DNA of TK+ clones deriving from cotransfections of tk gene and carrier DNA of salmon sperm. Cells cotransfected with tk gene and plasmids carrying LTR sequences of MoMLV or RSV formed three dimensional colonies in semisolid agar medium. No effect of carrier DNA on the morphology of TK+ transformant clones was noticed.     

Avian sarcoma virus-transformed quail clones defective in the production of focus-forming virus.

Quail embryo fibroblasts were infected at low multiplicity with avian sarcoma virus, and transformed cells were selected by their ability to form colonies in agar. Five clones that failed to produce focus-forming virus were examined for (i) intactness of the integrated proviral DNA, (ii) intracellular viral RNA production, (iii) intracellular viral antigen production, (iv) production of virus particles, and (v) rescue of a functional src gene and of parental host range determinants by superinfection with Rous-associated virus-60, an avian leukosis virus of subgroup E. Deletions in the integrated viral DNA were apparent in three of the five nonproducer clones. In one clone producing focus-forming virus, analysis of the integrated viral DNA revealed an insertion in the region of the genome that codes for src.     

Integrated structures of duck hepatitis B virus DNA in hepatocellular carcinoma.

The structure of integrated viral DNA in a hepatocellular carcinoma of a duck from Chi-tung county in China was analyzed. Three different clones of integrated viral DNA, lambda DHS 6-1, lambda DHS 6-2, and lambda DHE 6-2, were obtained from the neoplastic portion of the liver by molecular cloning. One of the three clones, lambda DHS 6-1, showed inverted repetition of integrated viral DNA with chromosomal flanking sequences. Another clone, lambda DHS 6-2, showed a head-to-head configuration of the core and surface gene regions of duck hepatitis B virus (DHBV) DNA. The virus-chromosome junctions were often located near direct repeat 1 or 2 of DHBV DNA in three independent clones. Nucleotide sequences at the virus-virus junctions in two clones, lambda DHS 6-1 and 6-2, indicated the possible rearrangement of chromosomal DNA and recombination of viral DNA. DHBV DNA appears to be integrated into the genome of hepatocytes in a manner similar to that of human and woodchuck hepatitis viruses. Thus, the duck system may serve as a useful animal model for the study of human hepatocarcinogenesis.     

与甘蓝型油菜重要经济性状有关的DNA克隆在拟南芥遗传图谱中的整合

拟南芥与油菜同属十字花科植物芸寡族,亲缘关系很近,基因组间的同源性很高,在用拟南芥EST克隆和油菜DNA克隆作探针定位了甘蓝型油菜一系列重要性状的基础上,对25个与油菜雄性不育恢复基因,硼高效利用基因,抗菌核病QTL及油菜种间杂种营养优势相关联的克隆进行了测序,在拟南芥基因组数据库中寻找到与这25个克隆高度同源的序列,根据这些高度同源序列在拟南芥染色体上的相位位置,将油菜DNA克隆整合到了拟南芥遗传图谱上,其中油菜硼高效基因BE1两侧的标记克隆整合在拟南芥第一染色体长臂一个较小的区段内,以该目标区段内的拟南芥EST克隆PA24为探针对甘蓝型油菜基因组比较作图,将该克隆定位在油菜连锁图BE1两侧标记之间,表明了利用基因组间的相互比较作图来精细定位芸薹属作物重要基因的可能性。     

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.     

Unintegrated viral sequences in rat cells transformed by simian virus 40 and a temperature sensitive A gene mutant

The status of viral sequences in rat cells transformed by simian virus 40 (SV40) and its temperature sensitive A gene mutant was investigated. Agarose gel electrophoresis of cell DNA prepared from clones picked from soft-agar and blot hybridization showed that sequences specific to SV40 genome were present both as integrated and unintegrated structures in rat clones. Digestion of rat cell DNA with various endonucleases with or without recognition sites in SV40 DNA and analysis indicated that the unintegrated viral genomes existed as full-length, covalently closed circular molecules. No differences in the free viral genomes were apparent between the clones transformed by the wild type and the mutant virus. The importance of the existence of free viral genomes in nonpermissive cells is discussed.     

Deletions of specific regions of the simian sarcoma-associated virus genome are found in defective viruses and in the simian sarcoma virus.

Extrachromosomal DNA was isolated from tissue culture cells that were acutely infected with simian sarcoma virus (SSV) and its associated helper (simian sarcoma-associated virus [SSAV]). Two sizes of closed circular viral genomic DNA intermediates were isolated, cleaved at the single EcoRI site, and ligated to the Charon 21A phage lambda vector. Cloned molecules of the larger size all represented the full-length (9.0-kilobase [kb]) SSAV molecule. A heterogeneous group of clones was derived from the smaller DNA circles. These included the SSV genome and SSAV deletion mutants. When two SSV clones were compared with the helper, they contained the following three characteristic deletions: (i) a 250-base pair deletion in the gag gene about 1.0 kb from the 5' end of the genome; (ii) a 1.85-kb deletion in the pol gene; and (iii) a 1.9-kb deletion at the 3' end, which included part of the env gene. This latter deletion was the site of the onc gene substitution. Six other clones of the smaller molecules represented the following variants of the SSAV genome: (i) two clones of the entire genome containing only one long terminal repeat unit; (ii) one clone with the 1.85-kb deletion of the pol gene observed in SSSV; and (iii) three clones having a deletion of the 3' end of the SSAV genome. In each of the latter clones, the 5' border of the deletion was indistinguishable from the 5' border of the onc substitution in SSV. The fidelity of genetic deletions observed suggested that certain regions of the SSAV genome were deleted at a high frequency. In certain cases, these deletions may have been accompanied by a substitution of cellular sequences to generate SSV.     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Zhikong Scallop, Chlamys farreri Jones et Preston, and Identification of BAC Clones Containing the Genes Involved in Its Innate Immune System

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.     

Integration of SV40 DNA into the cell genome and viral mutagenesis

Integration of DNA of a temperature-sensitive SV40 mutant (tsA239) into the cell genome was studied. The viral A gene (the oncogene) encodes the tumour T antigen which is ts in the mutant and is devoid of mutagenic and transforming activity under non-permissive conditions (40 degrees C). Clones of Chinese hamster cells infected by tsA239 mutant were analysed. Those infected by wild-type SV40 served as controls. As shown by dot-hybridization, SV40 DNA was detected in cells of 14 out of 18 clones infected by tsA mutant and incubated at 40.5 degrees C, and in all 20 clones infected by tsA mutant and incubated under permissive conditions (33 degrees C), the difference between the two groups being insignificant (p greater than 0.05). By means of blot-hybridization it was established that viral DNA was integrated into the cell genome of all 12 clones analysed, belonging to the three experimental series: infection by tsA mutant, incubation at 40.5 and 33 degrees C, infection by wt SV40, incubation at 40.5 degrees C. The number of integration sites ranged from one to four in different clones. Integration of SV40 DNA in tandems was observed. The data presented allow to conclude that integration per se does not play a crucial role in determining the mutagenic and transforming effect of the virus. Obviously, what matters is the activity of viral oncogene product - the T antigen.     

An integrated map of Arabidopsis thaliana for functional analysis of its genome sequence.

The genome of the model plant species Arabidopsis thaliana has recently been sequenced. To accelerate its current genome research, we developed a whole-genome, BAC/BIBAC-based, integrated physical, genetic, and sequence map of the A. thaliana ecotype Columbia. This new map was constructed from the clones of a new plant-transformation-competent BIBAC library and is integrated with the existing sequence map. The clones were restriction fingerprinted by DNA sequencing gel-based electrophoresis, assembled into contigs, and anchored to an existing genetic map. The map consists of 194 BAC/BIBAC contigs, spanning 126 Mb of the 130-Mb Arabidopsis genome. A total of 120 contigs, spanning 114 Mb, were anchored to the chromosomes of Arabidopsis. Accuracy of the integrated map was verified using the existing physical and sequence maps and numerous DNA markers. Integration of the new map with the sequence map has enabled gap closure of the sequence map and will facilitate functional analysis of the genome sequence. The method used here has been demonstrated to be sufficient for whole-genome physical mapping from large-insert random bacterial clones and thus is applicable to rapid development of whole-genome physical maps for other species.     

人神经营养因子-3重组逆转录病毒的包装与鉴定

在逆转录病毒载体pLXSN中引入人神经营养因子-3(hNT-3),构建成重组质粒pLXSN-NT3,转染包装细胞PA317后经G418筛选得到几个稳定产毒的细胞克隆.测定这几个克隆的病毒滴度,最高达到1.60×105,且产毒细胞株不产生复制型病毒.PCR和大乳鼠背根神经节活性检测证明hNT-3已整合到宿主细胞基因组并能稳定表达.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences.

A modular dihydrofolate reductase gene has been introduced into Chinese hamster ovary cells lacking dihydrofolate reductase. Clones capable of growth in the absence of added nucleosides contain one to five copies of the plasmid DNA integrated into the host genome. Upon stepwise selection to increasing methotrexate concentrations, cells are obtained which have amplified the transforming DNA over several hundredfold. A detailed analysis of the chromosomes in three clones indicated the appearance of cytologically distinct chromosomal regions containing the amplified plasmid DNA which differ in surrounding sequence composition, structure, and location. Two of the clones examined have extensive, homogeneously staining regions. The DNA in these homogeneously staining regions replicates in the early part of the S phase. The amplified plasmid DNA is found associated at or near the ends of chromosomes or on dicentric chromosomes. We propose that integration of DNA may disrupt telomeric structures and facilitate the formation of dicentric chromosomes, which may then undergo bridge breakage-fusion cycles. These phenomena are discussed in relation to DNA transfer experiments and modes of gene amplification and chromosome rearrangement.     

Clusters containing different mobile dispersed genes in the genome of Drosophila melanogaster.

Ten clones containing the actively transcribed mobile dispersed gene Dm255 and its flanking sequences were selected from the HindIII bank of the Drosophila melanogaster genome. The Dm225 sequences present in these clones were identical while the flanking sequences were different in all of the clones analysed. Four of them contained, in addition to Dm225, other DNA sequences binding high amounts of cytoplasmic poly(A) + RNA. The properties of these new genes are similar to those of Dm255: they are also actively transcribed, multiple in copies, scattered throughout the genome, and located at varying genome sites which also were scattered throughout the whole genome of D. melanogaster. Thus, different mobile dispersed genes often appear as closely apposing units forming gene clusters in the genome.