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 DNA polymerases of Leishmania mexicana

Two previously isolated DNA polymerases from the parasitic protozoan Leishmania mexicana were further characterized by exposure to inhibitors of mammalian DNA polymerases. DNA polymerase A, a high molecular mass enzyme, and DNA polymerase B, a beta-like DNA polymerase were compared to each other and to their mammalian counterparts regarding pH optimum, utilization of templates, and response to various inhibitors and ionic strengths. The results suggest that the DNA polymerases from L. mexicana differ from the host enzymes and may offer a target for chemotherapeutic intervention.     

Analysis of chromosomal integration and deletions of yeast plasmids.

Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6.19, 6.06 and 5.97 kilobases as referenced to sequenced phiX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA. Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique HpaI site and by having small overlapping deletions in the same region. The HpaI site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA.     

Integration of the DNA of filamentous bacteriophage Cflt into the chromosomal DNA of its host.

It was demonstrated for the first time that filamentous bacteriophage Cflt, which contains single-stranded DNA, can incorporate its genome into that of its host. Evidence in support of the incorporation was obtained from a Southern blot hybridization analysis of DNA isolated from Cflt-lysogenized cells. DNAs from different Cflt-lysogenized cells were purified, and the integration patterns were compared. Because all integration patterns were identical and only one fragment in Cflt replicative-form DNA was missing, it appears that the integration was site specific. Only one complement of viral DNA was integrated per host chromosome. To determine the attachment site on the viral DNA, the physical map of EcoRI, XhoI, SstII, and BglII on Cflt DNA was constructed. Based on this physical map and a Southern blot hybridization analysis of lysogen DNA with these restriction endonucleases, we demonstrated that DNA sequences from all regions of the Cflt genome were represented in the integrated viral sequences. The attachment site on the viral genome was located at 69.2 to 73.8 min on the Cflt DNA.     

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.     

Molecular cloning of the genome of human spumaretrovirus

DNA of human spumaretrovirus (HSRV) was cloned from both cDNA and from viral DNA into phage lambda and bacterial plasmid vectors. The recombinant plasmids harboring viral DNA were characterized by Southern blot hybridization and restriction mapping. Physical maps were constructed from cDNA and found to be colinear with the restriction maps obtained from viral DNA. The recombinant clones isolated contained viral DNA inserts which range in size from 2.2 kb to 15.4 kb. The recombinant clones allowed to construct a physical map of the complete HSRV provirus of 12.2 kb.     

Identification of Phytophthora citrophthora with Cloned DNA Probes

Two different DNA fragments, one of 2.9 kilobases and the other of 5.1 kilobases, were cloned from Phytophthora citrophthora and showed no homology with DNA from plants and other related fungi. These DNA probes hybridized with DNA from 12 different P. citrophthora isolates obtained from a variety of hosts but did not hybridize with DNA from 6 P. citrophthora isolates obtained from cacao. Southern blot analysis revealed that the probes contained repetitive DNA, and restriction fragment length polymorphisms were identified among several P. citrophthora isolates. Of the isolates tested, two major groups were observed whose genetic similarity correlated with geographical distribution. One of the DNA probes was used to detect P. citrophthora growing from infected citrus roots incubated on semiselective medium. P. citrophthora was not detected by a hybridization assay of total DNA extracted directly from infected roots.     

Studies on T4-head maturation. 2. Substrate specificity of gene-49-controlled endonuclease

The substrate specificity of 49+-enzyme was investigated in vitro. The enzyme showed a marked preference for rapidly sedimenting T4 DNA (greater than 1000 S) when helix-destabilizing proteins from Escherichia coli or phage T4 were added to the reaction. Regular replicative T4 DNA (200-S DNA) or denatured T4 DNA was not cleaved by the enzyme in the presence of these proteins but if they were omitted from the reaction both DNAs become good substrates for the enzyme. 200-S DNA was cleaved at its natural sites of single strandedness which occur at one-genome intervals. Gaps in T4 DNA which were constructed by treatment of a nicked DNA with exonuclease III were also cleaved by 49+-enzyme in the absence of helix-destabilizing proteins. Single-stranded T4 DNA was extensively degraded and up to 50% of the material was found to be acid-soluble in a limit digest. The degradation products were predominantly oligonucleotides of random size. No preference for a 5'-terminal nucleotide was observed in material from a limit digest with M13 DNA. Double-stranded DNA was nicked upon exposure to 49+-enzyme and double-strand breakage finally occurred by an accumulation of single-strand interruptions. No acid-soluble material was produced from native T4 DNA. The introduction of nicks in native DNA did not improve its properties as a substrate for the enzyme. Double-stranded DNA was about 100-fold less sensitive to the enzyme than single-stranded DNA.     

Production of antibodies specific for double stranded antigen DNA cloned from immune complexes in plasma of a SLE patient.

The antigenicity of antigen DNA isolated from immune complexes in plasma of a SLE patient was examined. DDY mice were immunized with the cloned antigen DNA carrying a sequence homologous with a part of bacteriophage f1 (KS8 DNA) by the coupling method, and the antibody response was estimated by radioimmunoassay. Antibodies specific to double stranded DNA were elicited. Moreover, the antibodies showed preferential binding to KS8 DNA than other DNA derived from Escherichia coli. These results suggest that KS8 DNA has a significant antigenicity in mice.     

Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage.

Uracil DNA N-glycosylase is a repair enzyme that releases uracil from DNA. A major function of this enzyme is presumably to protect the genome from pre-mutagenic uracil resulting from deamination of cytosine in DNA. Here, we report that human uracil DNA N-glycosylase also recognizes three uracil derivatives that are generated as major products of cytosine in DNA by hydroxyl radical attack or other oxidative processes. DNA substrates were prepared by gamma-irradiation of DNA in aerated aqueous solution and incubated with human uracil DNA N-glycosylase, heat-inactivated enzyme or buffer. Ethanol-precipitated DNA and supernatant fractions were then separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results demonstrated that human uracil DNA N-glycosylase excised isodialuric acid, 5-hydroxyuracil and alloxan from DNA with apparent K(m) values of approximately 530, 450 and 660 nM, respectively. The excision of these uracil analogues is consistent with the recently described mechanism for recognition of uracil by human uracil DNA N-glycosylase [Mol,C.D., Arval,A.S., Slupphaug,G., Kavil,B., Alseth,I., Krokan,H.E. and Tainer,J.A. (1995) Cell, 80, 869-878]. Nine other pyrimidine- and purine-derived products that were identified in DNA samples were not substrates for the enzyme. The results indicate that human uracil DNA N-glycosylase may have a function in the repair of oxidative DNA damage.     

Molecular cloning of the complete Epstein-Barr virus genome as a set of overlapping restriction endonuclease fragments.

A complete collection of fragments of Epstein-Barr virus DNA, obtained by cleavage with restriction endonuclease Eco RI, has been cloned. Fourteen different internal fragments of the virus genome, derived from linear virion DNA of the B95-8 strain, and sequences corresponding to the terminal regions of virion DNA, derived from intracellular circular EBV DNA isolated from 895-8 cells, were cloned. Sizes of fragments were determined by agarose gel electrophoresis and their sum leads to an estimated molecular weight of 110 x 10(6) for virion DNA. Large Eco RI DNA fragments of special interest were also cloned in cosmids using another source of EBV DNA, that is, to circular viral DNA derived from Raji cells. In order to provide a set of overlapping sequences, all the 29 internal Bam HI fragments of B95-8 virion DNA were cloned in pBR322. The map location within the viral genome of each cloned DNA fragment was identified by hybridizing to blots of virion DNA cleaved with several different restriction endonucleases.     

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.     

Isolation and characterization of cloned human DNA fragments carrying reiterated sequences common to both autosomes and the X chromosome.

Several recombinants were identified and purified from a cloned library of human DNA by virtue of their homology to DNA from a mouse-human hybrid cell line containing a single human chromosome, the X, and their lack of homology to mouse DNA. Three recombinants were characterized in detail, and all were homologous to reiterated DNA from the human X chromosome. These recombinants also were homologous to reiterated sequences on one or more human autosomes and, therefore, were not X chromosome specific. The recombinant DNA fragments homologous to human reiterated X DNA were the same fragments homologous to human reiterated autosomal DNA. Digestion of genomic DNAs with several restriction enzymes revealed that the pattern of fragments homologous to one recombinant, lambda Hb2, was the same on autosomes as on the X chromosome, suggesting that the molecular organization of these elements on the X is not distinct from their organization on autosomes.     

Differential replication of satellite DNA in polyploid tissues of Drosophila virilis

Satellite DNA amounts were examined in adult tissues of Drosophila virilis, a species whose DNA contains three prominent satellites. Satellite amounts in DNA from six of the seven tissues were lower than in DNA from diploid (adult brain) tissue. Satellite amounts in adult ovary DNA, however, were equivalent to or greater than diploid levels. When DNA from pupal ovaries was examined, a 30% increase in satellite amounts over diploid levels was found. An RNA-DNA hybridization experiment showed that the ribosomal RNA genes in pupal ovary DNA were under-replicated relative to diploid DNA levels.     

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.     

Complete enzymatic synthesis of DNA containing the SV40 origin of replication

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.     

PCR-DGGE研究微生物种群中多条带产生原因分析

鸡肠道微生物菌群经PCR-DGGE分析,回收PCR-DGGE分析胶上的一条DNA片段,回收的DNA片段再重复进行2次PCR-DGGE分析,以及分别用PCR反复循环扩增和PCR高保真酶扩增后再进行DGGE分析等方法研究PCR-DGGE分析中多条带产生原因。结果显示PCR-DGGE分析中多条带产生原因可能是作PCR扩增模板的DNA混杂有少量其他DNA片段,多条带现象不易被消除。DGGE分析胶上的DNA片段测序时,将该DNA片段回收、PCR扩增后克隆,提取多个阳性克隆菌的质粒DNA片段,分别与其原目的DNA片段进行DGGE分析,在DGGE分析胶上选取与原目的DNA片段处于同一电泳位置的质粒DNA测序,提高测序的准确性。     

Genotyping accuracy for whole-genome amplification of DNA from buccal epithelial cells.

We compared the accuracy of genotyping for DNA extracted from lymphocytes to that of DNA amplified from buccal epithelial cells. Amplification was via a rolling circle/phi29 DNA polymerase commercial kit. Paired buccal and lymphocyte DNA samples were available from 30 individuals. All samples were genotyped for 12 SNPs, 5 microsatellites and 2 VNTRs. The accuracy of genotyping (no-call proportions, reproducibility, and concordance) was similar for DNA from lymphocytes in comparison to amplified DNA from buccal samples. If used with caution, these data suggest that rolling-circle whole-genome amplification can be used to increase the DNA mass available for large-scale genotyping projects based on DNA from buccal cells.     

Characteristics of Deoxyribonucleic Acid Polymerase Isolated from Spores of Rhizopus stolonifer

Deoxyribonucleic acid (DNA)-dependent DNA polymerase was purified several hundredfold from germinated and ungerminated spores of the fungus Rhizopus stolonifer. The partially purified enzymes from both spore stages exhibited identical characteristics; incorporation of [(3)H]deoxythymidine monophosphate into DNA required Mg(2+), DNA, a reducing agent, and the simultaneous presence of deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxyadenosine triphosphate. Heat-denatured and activated DNAs were better templates than were native DNAs. The buoyant density of the radioactive product of the reaction was similar to that of the template DNA. The enzyme is probably composed of a single polypeptide chain with an S value of 5.12 and an estimated molecular weight of 70,000 to 75,000. During the early stages of purification, the enzyme fraction from ungerminated spores required exogenous DNA for maximum activity, whereas the corresponding enzyme fraction from germinated spores did not require added DNA. Apparently DNA polymerase from germinated spores was more tightly bound to endogenous DNA than was the enzyme from ungerminated spores.