DNA endonuclease activities associated with melanoma cell chromatin

Chromatin-associated DNA endonucleases, extracted from Cloudman mouse melanoma cell nuclei, were separated on isoelectric focusing into seven fractions in two widely separated groups pH 3.4–5.4 and 7.5–9.3, each active on calf thymus DNA. All fractions in the former group, pI's 3.4, 4.4 and 5.4, produced at least one single-strand scission per molecule on circular duplex phage PM2 DNA, and transformed circular single-stranded phage fd DNA into linear strands of uniform length. In the second group there was no detectable activity against PM2 DNA, but two fractions pI's 7.5 and 8.0 were active on fd DNA as above, whereas the other two, pI's 8.5 and 9.0 transformed fd DNA into a number of different sized, discrete segments. These results indicate that, even allowing for possible enzymatic identity of some of the isoelectrically separated forms, at least three different DNA endonucleases are associated with mouse melanoma cell chromatin.     

Fractionation of native and denatured transforming deoxyribonucleic acid from Bacillus subtilis

1. Native DNA from Bacillus subtilis was fractionated by stepwise elution from methylated albumin, the transforming activity being confined to two out of four fractions. Partial separation of DNA active in transformation for the arginine marker from that showing activity for the histidine and tryptophan markers was achieved. 2. Partial denaturation of DNA at 90 degrees and 93.5 degrees resulted in the preferential destruction of transforming activity for the histidine and tryptophan markers. 3. Denaturation of DNA at 100 degrees followed by chromatography on methylated albumin yielded five fractions, two of which exhibited residual activity. Redenaturation at 100 degrees resulted in the interconversion of four out of the five fractions. Redenaturation of fractions labelled with (15)N and (2)H suggested the presence of a specific component that did not readily take part in the interconversions.     

Nuclear DNA endonuclease activities on partially apurinic/apyrimidinic DNA in normal human and xeroderma pigmentosum lymphoblastoid and mouse melanoma cells

DNA endonuclease activities from nuclear proteins of normal human and xeroderma pigmentosum (XP), complementation group A, lymphoblastoid and Cloudman mouse melanoma cells were examined against partially apurinic/apyrimidinic (AP) DNA. Non-histone chromatin-associated and nucleoplasmic proteins, obtained from isolated nuclei, were subfractionated by isoelectric focusing and assayed for DNA endonuclease activity against linear, calf thymus DNA. All of the nine chromatin-associated and three of the nucleoplasmic fractions, which lacked DNA exonuclease activity, were tested for DNA endonuclease activity against both native and partially AP, circular, duplex, supercoiled PM2 DNA. In all three cell lines, four chromatin-associated, but none of the nucleoplasmic fractions, showed increased activity against DNA rendered AP by either heat/acid treatment or by alkylation with methyl methanesulfonate (MMS) followed by heat. One chromatin-associated activity, with pI 9.8, which was not active on native DNA, showed the greatest activity on AP DNA. AP activity was moderately decreased in XP cells and slightly decreased in mouse melanoma cells, as compared with normal cells, in the fraction at pI 9.8. Little or no increased activity was observed in any of the endonucleases from any of the cell lines on MMS alkylated DNA.     

cAMP对转化细胞中几种基因表达及CREB DNA结合活性的影响

 从癌基因、抑癌基因及转录因子 CREB(c AMP反应序列结合蛋白 )对 CRE DNA序列结合活性的相关性 ,对 db- c AMP处理的小鼠 C3H10 T1 /2转化细胞增殖抑制作用进行了研究 .实验结果表明 ,转化细胞中 PKA(蛋白激酶 A)活性显著低于正常细胞 ,而 PKC(蛋白激酶 C)活性则显著高于正常细胞 .斑点印迹和 Northern印迹分析显示转化细胞中 c- myc和 Ca M(钙调素 )基因表达明显高于正常细胞 ,而 p53基因和 Rb基因表达则明显低于正常细胞 ,这些差别与 C3H10 T1/ 2 转化细胞增殖失控有关 .转化细胞经 db- c AMP(1 mmol/L)处理后 ,细胞增殖受到明显抑制 ,db- c AMP处理0 .5h后 ,转化细胞中 PKA活性便明显增强 ,PKC活性则被显著抑制 ,处理 2 h后 ,c- myc和 Ca M基因表达下降 ,而 p53和 Rb基因表达则增强 ,这些变化与 c AMP抑制 C3H10 T1/ 2 转化细胞增殖有密切联系 .凝胶阻滞电泳分析显示 db- c AMP(1 mmol/L )处理短时间内 ,CREB对 CRE DNA序列无结合活性 ,1 2 h后开始出现较弱的结合活性 ,2 4 h后才明显加强 ,表明在 db- c AMP处理的早期 ,调控区中含有 CRE序列的基因不参与 db- c AMP对细胞增殖抑制的调节 ,即与 CREB磷酸化及其相应的 DNA结合活性无相关性 .     

DNA ligase activity in human cell lines from normal donors and Bloom''s syndrome patients.

DNA ligase activity was studied in several untransformed or virus-transformed human cell lines from normal donors and from Bloom's syndrome (BS) patients. This proneness genetic disease is characterized by several cytological abnormalities and cancer proneness and, recently, some transformed cell lines from these patients were described to present a reduced activity of DNA ligase I. Results presented in this work indicate that: (i) the total DNA ligase activity in crude extract from untransformed or transformed cell lines from several BS patients was significantly higher than in control cells; (ii) the partial purification of the enzyme after gel filtration on fast protein liquid chromatography of crude extracts from lymphoblastoid BS cells showed that the enzyme activity was eluted in a major 180 kDa form in which activity was higher than in control cells; (iii) the activity gel analysis of these enzyme fractions revealed that DNA ligase of human cells was correlated to a major 130 kDa polypeptide and, in BS cells, the extent of the activity of this band was equal or higher than that in control untransformed or transformed cells.     

Substrate specificity of RdgB protein, a deoxyribonucleoside triphosphate pyrophosphohydrolase

We have previously reported the identification of a DNA repair system in Escherichia coli for the prevention of the stable incorporation of noncanonical purine dNTPs into DNA. We hypothesized that the RdgB protein is active on 2'-deoxy-N-6-hydroxylaminopurine triphosphate (dHAPTP) as well as deoxyinosine triphosphate. Here we show that RdgB protein and RdgB homologs from Saccharomyces cerevisiae, mouse, and human all possess deoxyribonucleoside triphosphate pyrophosphohydrolase activity and that all four RdgB homologs have high specificity for dHAPTP and deoxyinosine triphosphate compared with the four canonical dNTPs and several other noncanonical (d)NTPs. Kinetic analysis reveals that the major source of the substrate specificity lies in changes in K(m) for the various substrates. The expression of these enzymes in E. coli complements defects that are caused by the incorporation of HAP and an endogenous noncanonical purine into DNA. Our data support a preemptive role for the RdgB homologs in excluding endogenous and exogenous modified purine dNTPs from incorporation into DNA.     

Transformation efficiencies and progeny analysis after varying different parameters of direct gene transfer of Nicotiana tabacum protoplasts

Nicotiana tabacum protoplasts were transformed by polyethylene glycol (PEG)-mediated uptake and electroporation, with circular and linear DNA, and with or without X-ray irradiation. We investigated the influence on the transient expression by these parameters as well as on the frequencies for stable transformation. Plants were regenerated and selfed, and the progenies of the transformed plants were analysed and used to compare the pattern of gene integration by these different variations in transformation methods. The results from the transient expression as judged by glucuronidase (GUS) activity, showed electroporation to give higher and more reproducible results than PEG-mediated uptake. Using linear instead of circular DNA increased the rate of stable transformation about 3 times. Including a mild X-ray treatment gave an increase in the same range. When the inheritance of the transferred trait was investigated, it was found that protoplasts transformed with linear DNA resulted in the highest number of plants with single-copy insertions. Protoplasts transformed with circular DNA showed the highest incidence of losing the trait, while plants in which the transformation included an X-ray treatment, had the highest frequency of multicopy insertion events.     

Transformation efficiencies and progeny analysis after varying different parameters of direct gene transfer of Nicotiana tabacum protoplasts

Nicotiana tabacum protoplasts were transformed by polyethylene glycol (PEG)-mediated uptake and electroporation, with circular and linear DNA, and with or without X-ray irradiation. We investigated the influence on the transient expression by these parameters as well as on the frequencies for stable transformation. Plants were regenerated and selfed, and the progenies of the transformed plants were analysed and used to compare the pattern of gene integration by these different variations in transformation methods. The results from the transient expression as judged by glucuronidase (GUS) activity, showed electroporation to give higher and more reproducible results than PEG-mediated uptake. Using linear instead of circular DNA increased the rate of stable transformation about 3 times. Including a mild X-ray treatment gave an increase in the same range. When the inheritance of the transferred trait was investigated, it was found that protoplasts transformed with linear DNA resulted in the highest number of plants with single-copy insertions. Protoplasts transformed with circular DNA showed the highest incidence of losing the trait, while plants in which the transformation included an X-ray treatment, had the highest frequency of multicopy insertion events.     

Transient expression of human neutral alpha-glucosidase AB (glucosidase II) in enzyme-deficient mouse lymphoma cells

To define new methods for gene isolation exploiting mutant mammalian cells we transformed a mutant mouse cell line deficient in glucosidase II with total human genomic DNA and detected transient expression of the human glucosidase II gene. Maximum gene expression was detected 48 h after addition of DNA as a 2.5-fold increase in neutral alpha-glucosidase activity (2.47 +/- 0.15, n = 4). When mutant mouse DNA was used for transformation, no increase in enzyme activity was seen. The increased enzyme activity was due to expression of the human gene product. Thus, by rocket immunoelectrophoresis, cells transformed with human DNA yielded a "rocket" which reacted with antibody to human but not to mouse glucosidase II and which hydrolyzed substrate in situ. Specific DNA sequences were required for expression of the enzyme activity, since digestion of DNA with EcoRI and SstI rendered the DNA ineffective for eliciting expression of the enzyme, while digestion of DNA with BamHI and XhoI did not affect the increase. Transfection with intact phage from a human genomic DNA library also resulted in transient expression of the human gene. These results demonstrate the feasibility of detecting, by enzymatic assay, transient expression of a human gene for an intracellular enzyme following DNA-mediated transformation both with total human DNA and with intact phage from a human recombinant library. This system could be used as an assay for isolation of a gene from a genomic library by sibling selection.     

大肠杆菌海藻糖合成酶基因的克隆和表达

利用Ｍｕ转座子细胞内克隆了大肠杆菌海藻糖合成酶 ｏｔｓＢＡ基因,克隆频率为１．４５ ｘ １０(－３)／ Ｋａｎ(ｒ)转导子。经遗传互补、酶切和部分序列分析表明ｏｔｓＢＡ基因位于克隆质粒。亚克隆 ２．８７ｋｂ ＤＮＡ片段至不同拷贝数表达质粒并分别转化大肠杆菌ｏｔｓＢＡ基因缺失株,转化株恢复 在０．５ｍｏｌ／Ｌ ＮａＣｌ培养基上生长的功能,高渗透压诱导实验表明,转化株能够合成克隆基因 产物海藻糖,但合成量不受克隆质粒拷贝数影响。海藻糖良好的抗高渗能力可能在农作物育 种方面发挥重要作用。为构建含有海藻糖合成酶基因的植物表达载体,并在农杆菌的介导下 转入植物,赋予其抗高渗、耐干旱能力奠定了重要的研究基础。     

Polyadenylated RNA complementary to repetitive DNA in mouse L-cells.

Complementary DNA, synthesized with L-cell polyadenylated RNA as template, renatured with total L-cell DNA to about 70%. About 30% complementary to unique sequence DNA and another 10 and 30% corresponded to sequences about 20- and 500-fold repetitive. Complementary DNA was fractionated after partial hybridization with total polyadenylated RNA to obtain preparations enriched or impoverished in complements of the most frequent polyadenylated RNA. Renaturation of these complementary DNA fractions with L-cell DNA revealed that most frequent RNAs are transcribed from repetitive DNA sequences, Complementary DNA, density labeled with bromodeoxyuridine, was fractionated by renaturation with L-cell DNA to yield fractions enriched in repetitive and unique sequence DNA. The denisty labeled complementary DNA was purified by equilibrium centrifiguation in an alkaline Cs2SO4 gradient. The complementary DNA representing mainly repetitive DNA sequences hybridized preferentially to frequent polyadenylated RNA.     

Regulation of transferrin receptor expression in concanavalin A stimulated and Gross virus transformed rat lymphoblasts

Expression of the cell surface receptor for the serum glycoprotein transferrin has been correlated with cellular proliferation in normal lymphocytes undergoing mitogen or antigen induced proliferative responses. In the present study, the expression of transferrin receptor in Concanavalin A stimulated rat lymphocytes or Gross virus transformed lymphoma cells has been examined with respect to the following questions: (1) is expression of receptor activity related to blastogenesis or to the subsequent IL-2 dependent DNA synthetic activity, and (2) is transferrin receptor expression regulated in similar fashion in both normal and malignant lymphoblasts? Scatchard analysis of saturation binding data illustrated that binding site number increased and subsequently decreased during the response while the receptor affinity for transferrin remained constant. These findings were confirmed by SDS-polyacrylamide gel electrophoretic analysis of radiolabeled cell surface proteins which specifically interact with transferrin. Examination of nonproliferating normal lymphoblasts (96 hr post Con A stimulation) compared with the same population of cells stimulated to reinitiate DNA synthesis with a partially purified preparation of Interleukin 2 (IL-2) showed that transferrin receptor expression was tightly linked to the IL-2 dependent stimulation of DNA replication. This coordinate regulation of receptor expression was markedly less stringent in retrovirus transformed thymic lymphoma cells.     

Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus.

Aflatoxins are extremely potent carcinogens produced by Aspergillus flavus and Aspergillus parasiticus. Cloning of genes in the aflatoxin pathway provides a specific approach to understanding the regulation of aflatoxin biosynthesis and, subsequently, to the control of aflatoxin contamination of food and feed. This paper reports the isolation of a gene involved in aflatoxin biosynthesis by complementation of an aflatoxin-nonproducing mutant with a wild-type genomic cosmid library of A. flavus. Strain 650-33, blocked in aflatoxin biosynthesis at the afl-2 allele, was complemented by a 32-kb cosmid clone (B9), resulting in the production of aflatoxin. The onset and profile of aflatoxin accumulation was similar for the transformed strain and the wild-type strain (NRRL 3357) of the fungus, indicating that the integrated gene is under the same control as in wild-type strains. Complementation analyses with DNA fragments from B9 indicated that the gene resides within a 2.2-kb fragment. Because this gene complements the mutated afl-2 allele, it was designated afl-2. Genetic evidence obtained from a double mutant showed that afl-2 is involved in aflatoxin biosynthesis before the formation of norsolorinic acid, the first stable intermediate identified in the pathway. Further, metabolite feeding studies with the mutant, transformed, and wild-type cultures and enzymatic activity measurements in cell extracts of these cultures suggest that afl-2 regulates gene expression or the activity of other aflatoxin pathway enzymes. This is the first reported isolation of a gene for aflatoxin biosynthesis in A. flavus.     

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.     

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.     

Cloning of genes related to exo-beta-glucanase production in Saccharomyces cerevisiae: characterization of an exo-beta-glucanase structural gene

The EXG1 gene of Saccharomyces cerevisiae was cloned and identified by complementation of a mutant strain (exg1-2) with highly reduced extracellular exo-beta-1,3-glucanase (EXG) activity. Two recombinant plasmids containing an overlapping region of 5.2 kb were isolated from a genomic DNA library and characterized by restriction mapping. The coding region was located by subcloning the original DNA inserts in a 2.7-kb HindIII-XhoI fragment. Exg+ strains and Exg- mutants transformed with yeast multicopy plasmids containing this DNA fragment showed an EXG activity 5- to 20-fold higher than for the untransformed Exg+ wild-type (wt) strains. The overproduced EXG had the same enzymic activity on different substrates, and showed the same electrophoretic behaviour on polyacrylamide gels and identical properties upon filtration through Sephacryl S-200 as those of the main EXG from Exg+ wt strains. The EXG1 gene transformed Schizosaccharomyces pombe, yielding extracellular EXG activity which showed cross-reactivity with anti-S. cervisiae EXG antibodies. A fragment including only a part of the EXG1 region was subcloned into the integrating vector YIp5, and the resulting plasmid was used to transform an Exg+ strain. Genetic and Southern analysis of several stable Exg- transformants showed that the fragment integrated by homology with the EXG1 locus. The chromosomal DNA fragment into which the plasmid integrated has a restriction pattern identical to that of the fragment on which we had previously identified the putative EXG1 gene. Only one copy of the EXG1 gene per genome was found in several strains tested by Southern analysis. Furthermore, two additional recombinant plasmids sharing a yeast DNA fragment of about 4.1 kb, which partially complements the exg1-2 mutation but which shows no homology with the 2.7-kb fragment containing the EXG1 gene, were also identified in this study. This 4.1-kb DNA fragment does not appear to contain an extragenic suppressor and could be related in some way to EXG production in S. cerevisiae.     

High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a approximately 6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use.     

Isolation and characterization of four adenovirus type 12-transformed human embryo kidney cell lines.

Four transformed cell lines were established from cultures of human embryo kidney (HEK) cells microinjected or transfected with cloned adenovirus 12 (Ad12) EcoRI-C DNA (0 through 16.5 map units of the left-hand end of the viral genome). Each cell line showed a different growth pattern. Southern blotting demonstrated that all of the cell lines contained Ad12-specific DNA sequences, but in the microinjected isolates these were at a much lower copy number than in the transfected isolate. Two cell lines (Ad12 HEK 1 and 3) appeared to contain tandemly repeated Ad12 EcoRI-C DNA fragments. Immunoprecipitation and Western blotting confirmed that Ad12 early region 1 (E1) proteins were being expressed by all four of the transformed cell lines, but indicated that E1A polypeptide expression was considerably less than E1B polypeptide expression. All of the Ad12-transformed HEK cell lines were tumorigenic when inoculated intracranially into athymic nude mice.     

DNA polymerase III holoenzyme of Escherichia coli. I. Purification and distinctive functions of subunits tau and gamma, the dnaZX gene products

Escherichia coli dnaZX, the gene which when mutant blocks DNA chain elongation, was cloned into a lambda PL promoter-mediated expression vector. In cells carrying this plasmid, the activity that complements a mutant dnaZ extract in replicating a primed single-stranded DNA circle was increased about 20-fold. Two polypeptides of 71 and 52 kDa were overproduced. Upon fractionation, two complementing activities were purified to homogeneity and proved to be the 71- and 52-kDa polypeptides. Immunoassays revealed their respective identities with the tau and gamma subunits of DNA polymerase III holoenzyme. The N-terminal amino acid sequences of the first 12 residues were identical in both subunits, as were their molar specific activities in dnaZ complementation. Thus, the tau subunit complements the defect in the mutant holoenzyme from the dnaZts strain as efficiently as does the gamma subunit. Inasmuch as the 71-kDa subunit (tau) can also overcome the enzymatic defect in a dnaX mutant strain, this polypeptide has dual replication functions, only one of which can be performed by the gamma subunit. Availability of pure tau and gamma subunits for study has provided the basis for proposing an asymmetry in the structure and function of a dimeric DNA polymerase III holoenzyme.