Rapid preparation of vector-free hybridization probes suitable for screening recombinant libraries

A procedure is described to rapidly prepare radioactively labeled DNA inserts from crude recombinant plasmid DNA preparations. These probes can subsequently be used to identify homologous nucleotide sequences in bacteria containing recombinant plasmids by colony hybridization. In a single procedure, crude recombinant plasmid DNA is both ³²P-labeled and fragmented by nick-translation in the presence of sufficient pancreatic DNase I to produce radioactive DNA of about 0.2–0.3-kb single-strand length. At this DNA fragment length the majority of the vector and insert sequences are on different DNA fragments. The insert DNA can then be separated from vector and contaminating Escherichia colt host chromosomal DNA by the following method. The DNA fragment population is first denatured and renatured under conditions such that the recombinant plasmid DNA reassociates but host DNA does not. The renatured plasmid DNA fragments are separated from the denatured host DNA by hydroxylapatite chromatography. The plasmid DNA fragments are then denatured and renatured with an excess of insert-free vector DNA. Conditions are chosen such that the insert DNA remains single-stranded while the vector DNA becomes double-stranded. The single-stranded insert DNA can be separated from the double-stranded vector DNA on hydroxylapatite and used directly for colony hybridization.     

Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization Assay for Replication Origin Deoxyribonucleic Acid of Escherichia coli

Deoxyribonucleic acid (DNA)-DNA hybridization on nitrocellulose filters can be used to assay for replication origin DNA from Escherichia coli if the DNA attached to the filters is enriched for the replication origin sequences. Such DNA can be readily isolated from very rapidly growing cells. When low amounts of this DNA were attached to filters, radioactively labeled DNA from the replication origin hybridized 1.7 times as well as radioactive replication terminus DNA. Under identical conditions, radioactively labeled DNA from exponentially growing cells hybridized only 1.3 times as well as radioactive replication terminus DNA. The replication origin, replication terminus, and randomly labeled DNA hybridized with similar efficiencies to filters containing DNA isolated from cells incubated in the absence of required amino acids. This DNA appeared to have all sequences present at equal frequencies. The hybridization assay was used to demonstrate that the DNA synthesized shortly after the addition of amino acids to cells previously deprived of required amino acids was primarily from the replication origin and then rapidly became similar to DNA synthesized by exponentially growing cells.     

Induction of prophage SPO2 in Bacillus subtilis: isolation of excised prophage DNA as a covently closed circle.

Bacillus subtilis tryC2, thyA, thyB, lysogenic for the phage DNA polymerase negative mutant SPO2 susL244, was induced under conditions preventing phage and bacterial DNA synthesis. The biological activity of DNA from induced cells and from uninduced controls was assayed by transformation and transfection, respectively. About 50% of the phage DNA biological activity in DNA extracted from induced cells was resistant to exposure to pH 11.8 TO 11.9. This DNA was operationally defined as alkali-resistant phage DNA. Transforming bacterial DNA from uninduced or induced cells and transfecting DNA from uninduced cells were more than 95% inactivated after exposure to high pH. The alkali-resistant phage DNA was characterized by sucrose gradient centrifugation, by centrifugation in cesium chloride-propidium iodide, and by electron microscopy. It was found to consist of a majority of covalently closed circular DNA molecules. Length measurements of a few relaxed circular molecules indicate a molecular weight of these similar to that previously found for mature SPO2DNA. Attempts to isolate similar covalently closed circular phage DNA from induced bacteria lysogenic for SPO2 phage with a functional DNA polymerase gene were unsuccessful. The gene order in mature and prophage SPO2 was determined by rescue of single and double markers from the respective type of DNA. The data obtained show that prophage DNA is (genetically) permuted relative to mature DNA. The phage attachment site is suggested to be located between genes I and J.     

Dominant distribution of mitochondrial DNA from recipient oocytes in bovine embryos and offspring after nuclear transfer

In the process of nuclear transfer, heteroplasmic sources of mitochondrial DNA from a donor cell and a recipient oocyte are mixed in the cytoplasm of the reconstituted embryo. The distribution of mitochondrial DNA heteroplasmy in nuclear transfer bovine embryos and resultant offspring was investigated by measuring polymorphism in the displacement loop region of mitochondrial DNA using PCR-mediated single-strand conformation polymorphism. Most offspring (20 of 21 calves) from recipient oocytes of undefined mitochondrial DNA genotypes showed different genotypes from the mitochondrial DNA of donor cells. The single calf that was an exception showed heteroplasmy, including the donor mitochondrial DNA genotype. Six cloned calves were produced from oocytes of a defined mitochondrial DNA genotype. All of these clonal members and various tissues showed only the mitochondrial DNA genotype derived from the oocyte. The mitochondrial DNA from donor cells appeared to be eliminated during early embryonic development; it gradually decreased at the early cleavage stages and was hardly detectable by the blastocyst stage. These results indicate that the genotype of mitochondrial DNA from recipient oocytes may become the dominant category of mitochondrial DNA in calves resulting from nuclear transfer.     

Separation of the Herpesvirus Deoxyribonucleic Acid Duplex into Unique Fragments and Intact Strand on Sedimentation in Alkaline Gradients

Deoxyribonucleic acid (DNA) extracted from herpes simplex virions forms multiple partially overlapping bands upon denaturation and centrifugation in alkaline sucrose density gradients. The most rapidly sedimenting DNA corresponds to an intact strand 48 x 10(6) daltons in molecular weight. In this study, we analyzed the DNA fragments generated in alkaline sucrose gradients with respect to size and uniqueness of base sequences. The distribution of sedimentation constants of the various fragments obtained in numerous gradients showed that the fragments smaller than the whole strand fall into six distinct classes ranging in molecular weight from 10 x 10(6) to 39 x 10(6) daltons. Four types of DNA strands can be reconstructed from the whole strand and six fragments on the basis of their molecular weights. DNA from each of the bands self-hybridizes to a lower extent than unfractionated viral DNA, indicating that each of the bands preferentially contains sequences from one unique strand. The data permit reconstruction of four possible types of DNA duplexes differing in the positions of the strand interruptions. Analysis of viral DNA extracted from nuclei of cells labeled with (3)H-thymidine for intervals from 3 to 120 min showed that nascent DNA is invariably attached to small fragments and that the fragments become elongated only upon prolonged incubation of cells. The experiments suggest that viral DNA replication begins at numerous initiation sites along each strand and that the elongation beyond the size of the replication unit involves repair or ligation, or both. Since newly made DNA yields more fragments than viral DNA extracted from mature virions, it is suggested that the fragmentation of mature DNA on denaturation with alkali arises from incomplete processing of specific initiation sites. Comparison of viral DNA extracted from nuclei with that extracted from mature cytoplasmic virions in cells labeled for 120 min indicates that packaged DNA is not randomly selected from among the nuclear DNA population but rather represents DNA molecules which in alkaline gradients yield a minimal number of fragments.     

Detection of Nitrosomonas spp. by polymerase chain reaction

Abstract A unique genomic DNA fragment was isolated from Nitrosomonas europaea ATCC 19718. Based on the sequence of this fragment, oligonucleotide primers for polymerase chain reaction amplification were prepared which amplify sequences of 775 and 658 bp. The predicted DNA fragments were both amplified from the genome of N. europaea and a Nitrosomonas spp. isolated from a local oxidation pond. The primers failed to amplify DNA from the genomes of the ammonia oxidiser Nitrosolobous multiformis , the nitrite oxidiser Nitrococcus mobilis as well as from the genomes of other unrelated heterotrophic bacteria. These DNA sequences could be amplified from 0.01 ng of N. europaea genomic DNA or from 100 intact cells, and it was possible to detect Nitrosomonas DNA in a DNA mixture extracted from water samples drawn from a local oxidation pond.     

The number of globin gene sequences in "cytoplasmic" DNA fragments.

DNA was isolated from the cytoplasm of primary cultures of mouse foetal liver cells. The proportion of globin genes was determined by two methods of cDNA-DNA hybridisation in globin complementary DNA excess. The proportion was similar in ‘cytoplasmic’ DNA and nuclear DNA. This argues against an informational role for this class of ‘cytoplasmic’ DNA, which has all of the characteristics of nuclear DNA arising from nucleosomes derived from chromatin.     

A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA.

A deoxyribonuclease specific for methylated DNA was isolated from Diplococcus pneumoniae. The enzyme, an endonuclease, degrades DNA for Escherichia coli to fragments of average molecular weight about half a million; it forms discrete fragments from phage lambda DNA. Methyl-deficient E. coli DNA is not attacked, neither is DNA from Micrococcus radiodurans, which contains no methylated adenine or cytosine. Nor is DNA from D. pneumoniae or phage T7 attacked. However, DNA from M. radiodurans, D. pneumoniae, and T7 is attacked after methylation with and E. coli extract. Methylated T7 DNA is degraded to discrete fragments. Although the genetic transforming activity of normal DNA from D. pneumoniae is not affected by the enzyme, transforming activity of methylated DNA is destroyed. The enzyme is designated endonuclease R Dpn I. Under certain conditions another enzyme of complementary specificity can be isolated. This enzyme, designated endonuclease R Dpn II, produces a similar pattern of fragments from the DNA of T7 without prior methylation of the DNA. It also degrades normal DNA for D. pneumoniae. It is suggested that this pair of enzymes plays a role in some unknown control process, which would involve a large fraction of the specific base sequences that are methylated in E. coli DNA and are present but not methylated in DNA from other sources.     

Patch homologies and the integration of adenovirus DNA in mammalian cells.

The hamster cell line HE5 has been derived from primary hamster embryo cells by transformation with human adenovirus type 2 (Ad2). Each cell contains 2-3 copies of Ad2 DNA inserted into host DNA at apparently identical sites. The site of the junction between the right terminus of Ad2 DNA and hamster cell DNA was cloned and sequenced. The eight [corrected] right terminal nucleotides of Ad2 DNA were deleted. The unoccupied cellular DNA sequence in cell line HE5 , corresponding to the site of the junction between Ad2 and hamster cell DNA, was also cloned; 120-130 nucleotides in the cellular DNA were found to be identical to the cellular DNA sequence in the cloned junction DNA fragment, up to the site of the junction. The unoccupied and the occupied cellular DNAs and the adjacent viral DNA exhibited a few short nucleotide homologies. Patch homologies ranging in length from dodeca - to octanucleotides were detected by computer analyses at locations more remote from the junction site. When the right terminal nucleotide sequence of Ad2 DNA was matched to randomly selected sequences of 401 nucleotides from vertebrate or prokaryotic DNA, similar homologies were observed. It is likely that foreign (viral) DNA can be inserted via short sequence homologies at many different sites of cellular DNA.     

Isolation of a human X chromosome-linked gene essential for progression from G1 to S phase of the cell cycle

The tsBN462 cell line, a temperature-sensitive (ts) mutant isolated from the hamster cell line, BHK21/13, cannot progress into S phase at 39.5 degrees C, following the release from isoleucine deprivation. The mutant cells were transfected with high molecular weight (HMW) DNA from human KB cells, and several human DNA bands were found to be conserved through three cycles of ts+ transformation. Conserved human DNA was isolated from the cosmid library of the secondary ts+ transformant (K-1-1), using 32P-labelled total human DNA as a probe. The isolated human DNA covers about 70 kb of human DNA flanked with hamster DNA, and originates from the human X chromosome. The middle part (56 kb) of the isolated human DNA was conserved through the primary, secondary and tertiary ts+ transformation, without gross rearrangement.     

Changes in Chloroplast DNA Levels during Development of Pea (Pisum sativum)

Determinations were made of the percentage of chloroplast DNA (ct DNA) in total cell DNA isolated from shoots of pea at different stages of development. Labeled pea ct DNA was reassociated with a high concentration of total DNA; the percentage of ct DNA was estimated by comparing the rate of reassociation of this reaction with that of a model reaction containing a known concentration of unlabeled ct DNA. The maximum change in ct DNA content was from 1.3% of total DNA in young shoots to 7.3% in fully greened shoots. Analyses were also performed on DNA from embryos, etiolated tissue, roots, and leaves. The first leaf set to develop in pea was excised over a growth period of 8 days during which leaf length increased from 4 to 12 millimeters. Young leaves contained about 8% ct DNA; in fully greened leaves the level of ct DNA approached 12%, equivalent to as many as 9,575 copies of ct DNA per cell. Root tissue contained only 0.4% ct DNA.     

Variation in p30-related proteins in gross virus-induced tumor cell lines derived from H-2 congenic mice

The distribution of DNA topoisomers in intracellular simian virus 40 DNA was analyzed by gel electrophoresis. The results suggested that DNA extracted from 70S chromatin had a different superhelical density distribution as compared with the DNA obtained from virions or virion assembly intermediates. The heterogeneity of simian virus 40 viral DNA superhelical density at a late time after infection was partly due to increased virion production and partly due to the intrinsic heterogeneity of the superhelical density of DNA extracted from virions. Using two-dimensional gel electrophoretic analysis we also showed that simian virus 40 DNA templates used for DNA replication have a higher average superhelical density than the bulk of intracellular viral DNA.     

Thermodynamic comparison of PNA/DNA and DNA/DNA hybridization reactions at ambient temperature

The thermodynamics of 13 hybridization reactions between 10 base DNA sequences of design 5'-ATGCXYATGC-3' with X, Y = A, C, G, T and their complementary PNA and DNA sequences were determined from isothermal titration calorimetry (ITC) measurements at ambient temperature. For the PNA/DNA hybridization reactions, the binding constants range from 1.8 x 10(6)M(-1)for PNA(TT)/DNA to 4.15 x 10(7)M(-1)for PNA(GA)/DNA and the binding enthalpies range from -194 kJ mol(-1)for PNA(CG)/DNA to -77 kJ mol(-1)for PNA(GT)/DNA. For the corresponding DNA/DNA binding reactions, the binding constants range from 2.9 x 10(5)M(-1)for DNA(GT)/DNA to 1.9 x 10(7)M(-1)for DNA(CC)/DNA and the binding enthalpies range from -223 kJ mol(-1)for DNA(CG)/DNA to -124 kJ mol(-1)for DNA(TT)/DNA. Most of the PNA sequences exhibited tighter binding affinities than their corresponding DNA sequences resulting from smaller entropy changes in the PNA/DNA hybridization reactions. van't Hoff enthalpies and extrapolated Delta G values determined from UV melting studies on the duplexes exhibited closer agreement with the ITC binding enthalpies and Delta G values for the DNA/DNA duplexes than for the PNA/DNA duplexes.     

一种快速提取细菌总DNA的方法研究

随着分子生物学技术应用于环境微生物研究的深入开展,占自然界微生物物种总数的90%以上的不能人工培养或培养困难的微生物已经可以借助分子生物学技术进行功能基因的开发和利用。而快速得到纯度较高,结构完整的细菌染色体DNA成为这一技术得以实现的前提。本文报道了利用高温处理和SDS的裂解作用相结合而建立的一种快速、简便的提取细菌染色体DNA的方法。经过脉冲电泳实验证明,利用本方法提取得到的几种革兰氏阳性和革兰氏阴性菌株的基因组DNA结构完整,并且无明显降解,无须经过纯化,可以直接进行PCR扩增和酶切等分子生物学操作,将此方法进一步应用于土壤环境DNA的提取方面,同样达到了快速得到大片段、高质量的环境微生物基因组的目的,为研究未培养的环境微生物多样性打下了坚实的基础,同时为环境基因组的提取提供了一个新的途径。     

Extraction of cellular DNA from human cells and tissues fixed in ethanol

DNA can be extracted from ethanol-fixed lymphoid cells and tissues. The fixation procedure is simple and rapid, and the DNA extraction itself is the same as that normally used for fresh tissue or cells. DNA extracted from ethanol-fixed material is indistinguishable from DNA extracted from fresh samples based on its purity, its ability to be digested with restriction endonucleases, and its ability to specifically hybridize to DNA probes. The capability to extract DNA from ethanol-fixed cells and tissues eliminates the need for stringent handling and storage requirements of fresh or frozen specimens.     

Herpesvirus saimiri DNA in tumor cells--deleted sequences and sequence rearrangements.

Herpesvirus saimiri DNA in continuous lymphoblastoid cell lines obtained from viral induced tumors in marmosets has been analyzed by gel electrophoresis of restricted DNA. Southern transfer to nitrocellulose filters, and hybridization to 32P-labeled viral DNA or DNA fragments. The viral DNA fragments EcoRI-G, -H, -D, and -I, KpnI-A, and BamHI-D and -E were not detected in Southern transfers of DNA from the nonproducing 1670 cell line. For each restriction endonuclease, a new fragment appeared, consistent with a 13.0-megadalton deletion of viral DNA sequences. This deletion encompassed 35 to 48 +/- 0.6 megadaltons from the left end of the unique DNA region. A sequence arrangement map is presented for the major population of H. saimiri DNA sequences in the 1670 cell line. Although H. saimiri DNA in the nonproducing 70N2 cell line can be distinguished from viral DNA in the 1670 cell line by several criteria, the same sequences were found to be deleted in the major population of viral DNA molecules. Unlike 1670 and 70N2 cells, restricted DNA from the virus-producing cell lines 77/5 and 1926 contained all of the DNA fragments present in the parental virion DNA. DNA from 1670, 70N2, and 77/5 cells contained additional viral DNA fragments that did not comigrate with any virion DNA fragments. Most of these unexplained fragments were confined to or highly enriched in partially purified circular or linear DNA fractions. DNA from tumor cells taken directly from a tumor-bearing animal contained viral DNA indistinguishable from the parental virion DNA by the assay conditions used. These results indicate that viral DNA sequence rearrangements can occur upon cultivation of tumor cells in vitro and that excision of DNA sequences from the viral genome may play a role in establishing the nonproducing state of some tumor cell lines.     

A DNA helicase from Xenopus laevis ovaries

A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.     

DNA isolation from forest soil suitable for PCR assays of fungal and plant rRNA genes

This protocol for DNA isolation from forest soil samples is advantageous because it uses only one liquid transference step and can process several samples with minimal time and equipment. The use of benzyl chloride early in the extraction protocol increases DNA yield and purity. The obtained DNA is useful for PCR amplification of nuclear and mitochondrial ribosomal related sequences from fungi and ribosomal DNA from plant chloroplasts. Isolated DNA can be used either undiluted or at low dilutions in PCR assays. A final glassmilk treatment of isolated DNA is useful to recover high molecular weight DNA fractions from agarose gel. DNA losses during glassmilk treatment can generate negative PCR results.     

Nucleotide Composition of Nuclear and Mitochondrial Deoxyribonucleic Acid of Fungi

The buoyant density of nuclear and mitochondrial deoxyribonucleic acid (DNA) from 14 species of fungi was determined by CsCl density gradient equilibrium centrifugation. The buoyant density of both types of DNA was the same for all three Mucorales analyzed. The buoyant density of mitochondrial DNA was significantly lower than that of the nuclear DNA for nine species of Ascomycetes and two species of Basidiomycetes. No simple correlation could be obtained from the comparison of the two types of DNA. Mitochondrial DNA represented a very small proportion of total DNA. Heat-denatured mitochondrial DNA renatured more readily than nuclear DNA.     

Molecular cloning of unintegrated and a portion of integrated moloney murine leukemia viral DNA in bacteriophage lambda.

A covalently closed circular form of unintegrated viral DNA obtained from NIH 3T3 cells freshly infected with Moloney murine leukemia virus (M-MLV) and a port of the endogenous M-MLV from the BALB/Mo mouse strain have been cloned in bacteriophage lambda. The unintegrated viral DNA was cleaved with restriction endonuclease HindIII and inserted into the single HindIII site of lambda phage Charon 21A. Similarly high-molecular-weight DNA from BALB/Mo mice ws cleaved sequentially with restriction endonucleases EcoRI and HindIII and separated on the basis of size, and one of the two fractions which reacted with an M-MLV-specific complementary DNA was inserted into the HindIII site of Charon 21A. Recombinant clones containing M-MLV-reacting DNA were analyzed by restriction endonuclease mapping, heteroduplexing, and infectivity assays. The restriction endonuclease map of the insert derived from unintegrated viral DNA, lambda x MLV-1, was comparable to published maps. Electron microscope analysis of the hybrid formed between lambda x MLV-1 DNA and 35S genomic M-MLV RNA showed a duplex structure. The molecularly cloned lambda x MLV-1 DNA contained only one copy of the long terminal repeat and was not infectious even after end-to-end ligation of the insert DNA. The insert DNA derived from endogenous M-MLV, lambda x MLVint-1, contained a DNA stretch measuring 5.4 kilobase pairs in length, corresponding to the 5' part of the genomic viral RNA, and cellular mouse DNA sequences measuring 3.5 kilobase pairs in length. The viral part of the insert showed the typical restriction pattern of M-MLV DNA except that a single restriction site, PvuII, in the 5' long terminal repeat was missing. Reconstructed genomes containing the 5' half derived from the integrated viral DNA and the 3' half derived from the unintegrated viral DNA were able to induce XC plaques after transfection in uninfected mouse fibroblasts.