DNA-DNA Reassociation Kinetics Research Articles

INVITED REVIEW: Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances

Microbial ecology examines the diversity and activity of micro-organisms in Earth's biosphere. In the last 20 years, the application of genomics tools have revolutionized microbial ecological studies and drastically expanded our view on the previously underappreciated microbial world. This review first introduces the basic concepts in microbial ecology and the main genomics methods that have been used to examine natural microbial populations and communities. In the ensuing three specific sections, the applications of the genomics in microbial ecological research are highlighted. The first describes the widespread application of multilocus sequence typing and representational difference analysis in studying genetic variation within microbial species. Such investigations have identified that migration, horizontal gene transfer and recombination are common in natural microbial populations and that microbial strains can be highly variable in genome size and gene content. The second section highlights and summarizes the use of four specific genomics methods (phylogenetic analysis of ribosomal RNA, DNA-DNA re-association kinetics, metagenomics, and micro-arrays) in analysing the diversity and potential activity of microbial populations and communities from a variety of terrestrial and aquatic environments. Such analyses have identified many unexpected phylogenetic lineages in viruses, bacteria, archaea, and microbial eukaryotes. Functional analyses of environmental DNA also revealed highly prevalent, but previously unknown, metabolic processes in natural microbial communities. In the third section, the ecological implications of sequenced microbial genomes are briefly discussed. Comparative analyses of prokaryotic genomic sequences suggest the importance of ecology in determining microbial genome size and gene content. The significant variability in genome size and gene content among strains and species of prokaryotes indicate the highly fluid nature of prokaryotic genomes, a result consistent with those from multilocus sequence typing and representational difference analyses. The integration of various levels of ecological analyses coupled to the application and further development of high throughput technologies are accelerating the pace of discovery in microbial ecology.

Frequent Association of Epstein-Barr Virus in Japanese Patients with Burkitt's Lymphoma

Seven Japanese patients with Burkitt's lymphoma (BL), residing in Hokkaido, were studied during the period, 1979-1991. Immunological analyses of their lymphoma cells showed all to express surface and/or cytoplasmic immunoglobulins. Chromosomal analysis was performed in five cases. Four of the five lymphomas had the chromosomal translocation, t(8;14), and one had t(2;8). Three patients had extremely high IgG antibody titers to Epstein-Barr virus (EBV) viral capsid antigen (VCA) and to EBV early antigens (EA). Two patients had positive antibodies to EA, and two others had normal antibody patterns comparable to those of EBV-seropositive age- and sex-matched healthy controls. Four of the seven lymphomas (57.1%) were positive for EBV-determined nuclear antigen (EBNA) by anticomplement immunofluorescence and/or EBV DNA using DNA-DNA reassociation kinetics, and/or Southern blot analysis. The frequency of EBV positivity in BL patients residing in Hokkaido was higher than that of cases previously reported in Japan. Three of the four EBV genome-positive BL patients responded well to chemotherapy, radiotherapy and/or surgical treatment, with no significant relapse being observed during the study period. In contrast, EBV genome-negative patients had poor prognoses despite having similar levels of clinical staging at the time of diagnosis.

Genetic relatedness within and between formae speciales of Fusarium oxysporum as measured by DNA-DNA reassociation kinetics.

Genetic relatedness among 18 formae speciales within Fusarium oxysporum was determined on the basis of DNA-DNA reassociation kinetics. DNA homology values were high (more than 95%) between isolates within the same forma specialis, while isolates of different formae speciales showed lower values (63 to 86%) of DNA homology. Comparatively high homology values (90 to 95%) were observed within isolates of different formae speciales causing disease on the Cruciferae or the Cucurbitaceae plants. Isolates of F. oxysporum consistently showed low homology values (36 to 48%) with isolates of F. solani. These results suggest that formae speciales of F. oxysporum can be differentiated genetically from each other, indicating that the reassociation analysis of DNA may be a useful tool in studies involving classification and phylogeny of subspecific taxa in this species.

Importance of Aeromonas sobria in Aeromonas bacteremia.

On the basis of DNA-DNA reassociation kinetics [1], core oligosaccharide analysis [2], and the electrophoretic mobility of selected cellular enzymes [3], recent taxonomic studies of motile spp. indicate that the group commonly referred to as the Aeromonas hydrophila complex is actually composed of nine or more distinct species, three of which are currently identifiable by using simple biochemical tests [4, 5]. Several studies have suggested that separating these groups may be of clinical as well as taxonomic interest because most enterotoxin-producing isolates are either A. hydrophila or sobria [6] and because differences in antimicrobial susceptibility between the three species have been noted [7, 8]. In addition to the above-mentioned properties, we have previously found [5] that ^90^0 of all blood isolates of submitted to our laboratory for biotyping belonged to either the A. sobria or A. hydrophila groups, with A. sobria predominating. Furthermore, studies conducted by Daily et al. [9] on the relative virulence of aeromonads in mice found A. sobria to be more pathogenic than A. hydrophila, although caviae was not separated from the latter group. We have subsequently confirmed the studies of Daily et al. [9] and have additionally demonstrated the relative avirulence of A. caviae [10]. To determine whether these biologic properties ascribed to each species in vitro correlate with manifestation of clinical disease, we have analyzed our recent experiences with bacteremia in our institution.

An established avian fibroblast cell line without mitochondrial DNA.

An established avian fibroblast cell line (LSCC-H32) has been found to be inherently resistant to the growth-inhibitory effect of ethidium bromide, when supplied with exogenous uridine. After long-term exposure to ethidium bromide (90 days), the cell population has been transferred to drug-free medium for 60 days, and then seeded at low cell density. Three clones have been isolated and propagated in drug-free medium for 5, 6, and more than 12 months, respectively. It was found that none of these cell lines had detectable cytochrome c oxidase activity and that they were virtually devoid of cytochromes aa3 and b. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 300 copies of mitochondrial DNA per cell was found in LSCC-H32 cells. Analysis of DNA extracted from cell populations exposed to ethidium bromide for 90 days and then transferred to drug-free medium for long periods of time revealed no mitochondrial DNA molecules by reassociation kinetics or by Southern blot hybridization of HindIII-or AvaI-digested total cellular DNA.

Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts.

Chicken embryo fibroblasts in uridine-containing medium are inherently resistant to the growth-inhibitory effect of ethidium bromide. The drug was found to inhibit the incorporation of [3H]thymidine into mitochondrial DNA circular molecules. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 604 copies of mitochondrial DNA per cell was found. This number decreased progressively in cells exposed to ethidium bromide, and by day 13 ca. one copy of mitochondrial DNA was detected per cell. When the cells were then transferred to drug-free medium, the number of copies increased very slowly as a function of time. On the other hand, analyses of DNA extracted from cell populations exposed to ethidium bromide for 20 or more days, with or without subsequent transfer to drug-free medium, revealed very little or no mitochondrial DNA by reassociation kinetics or by Southern blot hybridization of AvaI- or HindIII-digested total cellular DNA. As a result of the elimination of mitochondrial DNA molecules, the establishment of cell populations with a respiration-deficient phenotype was confirmed by measuring cytochrome c oxidase activity as a function of the number of cell generations and the absorption spectrum of mitochondrial cytochromes.

Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts.

Chicken embryo fibroblasts in uridine-containing medium are inherently resistant to the growth-inhibitory effect of ethidium bromide. The drug was found to inhibit the incorporation of [3H]thymidine into mitochondrial DNA circular molecules. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 604 copies of mitochondrial DNA per cell was found. This number decreased progressively in cells exposed to ethidium bromide, and by day 13 ca. one copy of mitochondrial DNA was detected per cell. When the cells were then transferred to drug-free medium, the number of copies increased very slowly as a function of time. On the other hand, analyses of DNA extracted from cell populations exposed to ethidium bromide for 20 or more days, with or without subsequent transfer to drug-free medium, revealed very little or no mitochondrial DNA by reassociation kinetics or by Southern blot hybridization of AvaI- or HindIII-digested total cellular DNA. As a result of the elimination of mitochondrial DNA molecules, the establishment of cell populations with a respiration-deficient phenotype was confirmed by measuring cytochrome c oxidase activity as a function of the number of cell generations and the absorption spectrum of mitochondrial cytochromes.

DNA base sequence homology in Rhizoctonia solani Kuehn. VII. Genetic relatedness between AG-BI and other anastomosis groups.

DNA base sequence homology between isolates of AG-BI and other anastomosis groups in Rhizoctonia solani was determined by DNA-DNA reassociation kinetics. A comparatively high homology was observed between AG-BI and AG-2. AG-BI isolates showed 36.1 to 48.0% homology with isolate of AG-2-1 and 34.0 to 46.4% homology with isolates of AG-2-2. While, AG-BI isolates showed 18.7 to 30.2% homology with isolates in AG-3 and AG-6, but consistently low homology (0 to 14.4%) with isolates in the other anastomosis groups. These results suggest that AG-BI and AG-2 should not be considered as separate groups. The phylogenetical relationship between AG-BI and the groups of AG-2, AG-3 and AG-6 is discussed.

DNA base sequence homology in Rhizoctonia solani Kuehn. VI. Genetic relatedness among seven anastomosis groups.

DNA base sequence homology among isolates of different anastomosis groups (AG-1, AG-2, AG-3, AG-4, AG-5, AG-6 and AG-7) in Rhizoctonia solani was determined by DNA-DNA reassociation kinetics. DNA homology among isolates of these anastomosis groups showed consistently low values (0 to 33.7%). These results suggest that each of these anastomosis groups is a genetically isolated group. The significance of classification of R. solani into groups on the basis of anastomosis behaviours is stressed.

Characterization of human B-cell lines harbouring both adult T-cell leukaemia (ATL) virus and Epstein-Barr virus derived from ATL patients.

Human B-cell lines, designated ATLB cell lines, were spontaneously established from peripheral blood of adult T-cell leukaemia (ATL) patients. The cell lines consistently expressed ATL-associated antigen (ATLA) and Epstein-Barr virus-associated nuclear antigen (EBNA). A cloned ATLB line, designated ATLB 2, showed that both ATLA and EBNA antigens were present in the same B-cell clone. In this study, we have further characterized ATLV and EBV in the cloned ATLB 2 cell line by biochemical techniques. The ATLA antigens in these clones, initially shown by immunofluorescence, were studied by immunoprecipitation with human sera from ATL patients and the Western blotting technique using a mouse monoclonal antibody (GIN-14). We identified ATLV core polypeptides 24K and 19K in the ATLB cell extracts. Furthermore, total cellular DNA and poly(A) RNA from the ATLB cell lines were analysed for the presence of viral genomes with molecularly cloned DNA probes containing the ATLV and EBV sequence, respectively. The results showed that all ATLB 2 clones contained highly conserved ATLV proviral DNA irrespective of whether or not they expressed ATLA. They also contained several copies of EB virus DNA and DNA-DNA reassociation kinetics studies clearly showed that most of the EBV DNA sequences were present in ATLB cells. ATLV-related mRNA was detected in only ATLA-positive clones (ATLB 2-3 and 2-21) but not in a negative clone (ATLB 2-45).

DNA base sequence homology in Rhizoctonia solani Kuehn. IV. Genetic relatedness within AG-4.

DNA base sequence homology among AG-6 isolates of Rhizoctonia solani was determined by DNA-DNA reassociation kinetics. Five of 10 isolates examined shared high DNA homology ranging from 91.8 to 97.7%. This group was classified as AG-6 homogeneous group I (HG-I). DNA homology among the remaining 5 isolates was 55.4 to 66.2%. They showed only 47.5 to 62.9% DNA homology with AG-6 HG-I isolates. These isolates were designated AG-6 genotypic variation (GV) isolates. Each of them appeared to have gene structures that differed by nucleotide substitutions. The genetic divergence among AG-6 isolates was also clearly demonstrated by the electrophoretic patterns of proteins and appearances of the cultures. These results suggest that AG-6 consists of strains that are phylogenetically related but have significant genetic polymorphism.

DNA base sequence homology in Rhizoctonia solani Kuehn. V. Genetic relatedness within AG-6.

DNA base sequence homology among AG-6 isolates of Rhizoctonia solani was determined by DNA-DNA reassociation kinetics. Five of 10 isolates examined shared high DNA homology ranging from 91.8 to 97.7%. This group was classified as AG-6 homogeneous group I (HG-I). DNA homology among the remaining 5 isolates was 55.4 to 66.2%. They showed only 47.5 to 62.9% DNA homology with AG-6 HG-I isolates. These isolates were designated AG-6 genotypic variation (GV) isolates. Each of them appeared to have gene structures that differed by nucleotide substitutions. The genetic divergence among AG-6 isolates was also clearly demonstrated by the electrophoretic patterns of proteins and appearances of the cultures. These results suggest that AG-6 consists of strains that are phylogenetically related but have significant genetic polymorphism.

DNA base sequence homology in Rhizoctonia solani Kuehn III. Genetic relatedness within AG-3,AG-5,AG-7 and AG-BI.

DNA base sequence homology among isolates within each anastomosis group of Rhizoctonia solani, AG-3, AG-5, AG-7 and AG-BI, was investigated on the basis of DNA-DNA reassociation kinetics to examine their genetic relatedness. DNA homology value of isolates within each anastomosis group ranged from 97.6 to 100% with AG-3 (potato type, IV), from 94.0 to 98.2% with AG-5, from 95.6 to 98.8% with AG-7, and from 91.4 to 100.2% with AG-BI. These results suggested that each of these anastomosis groups is genetically homogenous.

Bromodeoxyuridine-DNA interactions associated with arrest of rat odontogenesis in vitro

To better characterize the molecular mechanism responsible for the bromodeoxyuridine (BrdU)-mediated arrest of mammalian odontogenesis in vitro, the nature of nuclear DNA-analogue interactions was determined. Bioactive doses of the radiolabelled analogue were added to the tissue culture medium of 16-day old embryonic rat incisor primordia. Control rudiments were similarly exposed to equimolar, radiolabelled thymidine. After 16–18 h, DNA was isolated and purified from the labelled organ cultures. Following sedimentation to equilibrium through neutral CsCl density gradients, [ 3H]-BrdU-labelled DNA revealed a buoyant density indicative of a 12–15 per cent level of substitution in place of thymidine. Furthermore, similar centrifugation of DNA through alkaline density gradients suggested that the substitution was localized predominantly within a single-strand. DNA-DNA reassociation kinetics subsequently revealed that disproportionately more radiolabelled BrdU was concentrated within repetitive DNA nucleotide sequences in contrast to the more random distribution of [ 3H]-thymidine moieties. Thus it is likely that BrdU exerts its inhibitory effects on odontogenic differentiation through a relatively small proportion of rat embryo nuclear DNA.

DNA base sequence homology in Rhizoctonia solani Kuehn I. Genetic relatedness within anastomosis group 1.

DNA base sequence homology among AG-2 group isolates of Rhizoctonia solani was investigated on the basis of DNA-DNA reassociation kinetics, and the genetic relationship among pathological types was examined. DNA homology value among isolates within each single type was 100.3% with the winter crops type (II), 98.1% with the rush type (IIIB) and 100.1% with the root rot type (IV). Comparatively high values of DNA homology ranging from 68.6 to 71.5% were observed between rush type isolates and root rot type isolates, both belonging to AG-2-2. DNA homology values of winter crops type isolates of AG-2-1 with rush type isolates of AG-2-2 was 37.6 to 40.1%, and 43.5 to 49.4% with root rot type isolates of AG-2-2. These results suggested that each pathological type in AG-2 is genetically homogenous, and that between the rush type and the root rot type of AG-2-2 there is a small but significant partial heterogeneity. AG-2-1 and AG-2-2 may form genetically distinct groups.

Analysis of BK virus-transformed cells and BK virus-induced tumors by DNA-DNA reassociation kinetics.

BK virus (BKV)-transformed cells and BKV-induced tumors as well as in vitro derived tumor cell lines were all found to contain BKV DNA sequences when analysed by DNA-DNA reassociation kinetics. In transformed cells the number of viral genome equivalents per diploid cell genome (CG divided by VG) decreased with increasing generations. Likewise, single cell clones had a lower CG divided by VG than parental transformed cells. BKV-induced tumors had a high CG divided by VG. Tumor cells cultivated in vitro and their clones had a lower CG divided by VG than BKV-induced tumors from which they were derived, suggesting a multiclonal origin of tumors. Hamster tumors induced by subcutaneous inoculation of BKV-transformed cells or tumor cell lines had a higher CG divided by VG than cells producing them. Variation in CG divided by VG is discussed in terms of cell selection depending on different in vitro or in vivo conditions of cell growth. In some cases, however, the decrease in CG divided by VG most likely depends on loss on free viral sequences from transformed cells or tumors.

DNA synthesis and template activity in a mutant of polyoma virus with altered expression of late viral proteins.

The 3049 strain of polyoma virus overproduces late RNA and proteins. The synthesis and accumulation of virus DNA was measured in cells infected with 3049 and a wild-type virus, lpS, to assess the possible role of gene dosage in this phenomenon. The rate of incorporation of [3H]thymidine into the DNA of 3049 and lpS virus was shown to be identical. The number of genome copies per cell, determined by DNA-DNA reassociation kinetics, was found to be similar in either whole cell or Hirt extracts of cells infected with the two viruses. Viral transcription complexes were isolated by Sarkosyl extraction, and the specific transcriptional activity was measured by incorporation of [3H]UTP. The incorporation of [3H]UTP and the DNA content of transcription complexes were indistinguishable. These results suggest that overproduction of late RNA (and protein) by the 3049 virus is not due to either an increased number of viral genomes per cell or an increased fraction of genomes involved in transcription. These and other data (unpublished) support the conclusion that the altered phenotype is due to a posttranscriptional mechanism.

Cytomegalovirus: detection in human colonic and circulating mononuclear cells in association with gastrointestinal disease.

The specificity and strength of the reported association between cytomegalovirus (CMV) and colonic adenocarcinoma were tested by analysis of a consecutive series of surgically resected colons for: CMV-DNA by a DNA-DNA reassociation kinetics (hybridization) procedure; latent virus by co-cultivation of fresh tissue with indicator fibroblasts; and CMV viral antigens by immunofluorescence. Ten of 13 cancer patients whose colonic tissue was able to be examined by all techniques showed evidence of active or prior CMV infection. Four cancer specimens were CMV-DNA (hybridization)- positive; an additional specimen from a cancerous colon was culture-positive. In six instances, CMV DNA was detected in mucosal cells adjacent to colon adenocarcinoma. In tissue from one of three patients with ulcera tive colitis and two of seven patients with other non-neoplastic colonic disease, CMV DNA was also detected. No fresh colonic tissues were demonstrated to have CMV surface or nuclear antigens when examined by immunofluorescence. Culture of peripheral lymphocytes was positive for CMV in three of 14 cancer patients. A CMV specific defect in humoral immunity could not be documented in that most cancer patients, as well as cancer-free patients, exhibited circulating specific antibody to CMV and had a normal capacity for CMV-specific antibody-dependent cellular cytotoxicity. We conclude that CMV, probably in a latent form, is readily detectable in colonic cells of man, including those derived from malignant, pre-malignant and non-malignant tissues. Neither preferential replication in damaged tissue nor carriage of CMV by peripheral lymphocytes homing to gut appear to explain the presence of CMV in colon cells.

Somatic cell hybrids between human lymphoma and human myeloid leukemia cells I. Induction of resident viral genome transcription and viral antigen formation by IUDR and TPA

We have determined the number of resident EBV DNA copies per cell and the extent of viral DNA transcription in two hybrid cell lines. The Putko line was derived from the fusion of a Burkitt lymphoma line (P3HR-1) and a myeloid leukemia line (K562) [Klein et al., J. Nat. Cancer Inst. 64, 725-735 (1980)]. Dutko is a hybrid of the Daudi lymphoma line and K562 (Klein, Zeuthen, and Ber, in preparation). These two cell lines differ significantly in their inducibility by IUDR or TPA (Klein et al., 1980; Zeuthen, Klein, and Ber, in preparation). Only Dutko can be induced to synthesize early antigen (EA), whereas Putko is noninducible altogether. Putko and Dutko contained 25 and 84 copies of EBV DNA per cell, respectively, as shown by DNA-DNA reassociation kinetics. The expression of the resident EBV genomes was restricted in both lines. Putko cells contained viral RNA transcribed from about 10% of the EBV DNA, whereas in Dutko cells at least 20% of the viral DNA was transcribed. After iododeoxyuridine (IUDR) exposure, viral RNA increased from 10 to 25% in Putko, and from 20 to more than 40% in Dutko cells. IUDR induction of Putko and Dutko cells did not increase the normally low level of viral transcripts found in the polysomal fraction. The tumor promoter, 12- 0-tetradecanoylphorbol-13-acetate (TPA) had no detectable effect on viral transcription. Possible molecular mechanisms that might be responsible for the differential inducibility of the two hybrid cell lines by IUDR are discussed.

Lack of association between BK virus and ependymomas, malignant tumors of pancreatic islets, osteosarcomas and other human tumors.

BK virus (BKV) DNA sequences were not detected in 142 human tumors analyzed by DNA-DNA reassociation kinetics and blot hybridization. The investigation was focused mainly on those rare types of human tumors (ependymomas, choroid plexus papillomas, tumors of pancreatic islets and osteosarcomas) that are induced with highest frequency by BKV in experimental animals. In addition, other tumors of the urinary apparatus and of the central nervous system were analyzed. BKV tumor (T) antigen was not detected in neoplastic tissues, and BKV T antibodies were not found in sera and cerebrospinal fluids from patients with neoplasms. Sequences homologous to BKV DNA were found in normal tissue from a kidney carrying a carcinoma. The neoplastic tissue from the same organ, however, had no sequences homologous to BKV DNA. Such DNA does not belong to BKV but probably to another papovavirus related to BKV.