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PubMed1998

Mitochondrial DNA analysis of Sporothrix schenckii in North and South America.

Mitochondrial DNA (mtDNA) types based on restriction fragment length polymorphism (RFLP) patterns with HaeIII were investigated in clinical isolates of Sporothrix schenckii in North and South America. In addition to 14 mtDNA types (Types 1-14) so far reported, six new mtDNA types, Types 15-20 were found in this study. Type 3 was divided into two subtypes, Subtype 3A and Subtype 3B based on RFLP with Msp1. Type 14 was also divided into three subtypes, Subtype 14A, Subtype 14B and Subtype 14C based on RFLP with Hha1. Nineteen isolates in the United States consisted of 1 isolate of Type 1, 12 of Type 2, 2 of Type 4, 3 of Type 14 (1 of Subtype 14B and 2 of Subtype 14C) and 1 of Type 15. Twenty nine isolates in Venezuela consisted of 13 of Type 3 (Subtype 3B), 6 of Type 4, 1 of Type 18, 3 of Type 19 and 6 of Type 20. Thirteen isolates in Argentina consisted of 2 of Type 3 (Subtype 3A), 4 of Type 4, 4 of Type 16 and 3 of Type 17. One isolate in Brazil was Type 3 (Subtype 3A). Based on the phylogeny of 20 mtDNA types (Types 1-20) constructed by estimating sequence divergences of mtDNA, mtDNA types were clustered into two groups: Group A (Types 1-3, Type 11 and Types 14-19) and Group B (Types 4-10, Types 12-13 and Type 20). These results suggest that S. schenckii isolates in North and South America mainly belong to Group A.

DNA, Fungal↗
PubMed1982

Comparative DNA analysis of three South American marsupials.

Published information on marsupials DNA is limited to a group of species belonging to only one genus. No previous reports have been written on South American species. In this paper we characterize the DNA of three out of the four marsupials found in Uruguay. Analytical and preparative ultracentrifugations in neutral CsCl gradients, including four intercalating agents and in Cs2SO4 gradients in presence of increasing amounts of Hg++ ion did not allow us to separate any satellite fraction. The buoyant density of the unique peak measured in CsCl gradients was in every case 1.697 g/cc with a G-C content of 37.7%. Digestion of total DNA with 11 restriction endonucleases produced a different pattern of bands for the three species, although some possible homologies could be established. Hybridization with 32P-rRNA of Southern blots of the gels containing digested DNAs demonstrated that the repeated sequences evidenced do not correspond to the ribosomal cistrons.

Animals↗
PubMed1982

The binding of gyrase to DNA: analysis by retention by nitrocellulose filters.

Three distinct Escherichia coli DNA gyrase complexes with DNA can be identified using a nitrocellulose filter-binding assay. One complex consists of an ensemble of two subunit A and two subunit B protomers bound noncovalently to specific sequences of DNA. High levels of each subunit alone are inactive but a single gyrase molecule binds DNA to a filter. At 23 degrees, the complex has a dissociation constant of approximately 10(-10) M and a half-time of decay of about 60 h. It is sufficiently stable that it can be purified by gel filtration and retain full supercoiling activity. Gyrase binds preferentially to relaxed DNA over supercoiled DNA by a factor of about 10. On addition of oxolinic acid, a second complex is formed that is distinguished by its stability in high ionic strength solutions and by efficient conversion to a third form upon addition of protein denaturants. The first and second complexes require Mg++ for optimal formation. The third form has been shown previously to contain denatured A protomers covalently linked to DNA that is broken at the site of attachment.

Collodion↗
PubMed2004

Nucleation-synchronized strand displacement for highly sensitive DNA analysis.

Nucleation process is a rate-limiting step of DNA strand exchange reactions between double helical DNA and its complementary DNA. Here we designed the double stranded probes that have single stranded portion. The single stranded portion is responsible for the nucleation process and mismatch recognition. A single base mismatch in 25mer DNAs could be detected in a few minutes at ambient temperatures. Using this method, various types of SNPs are rapidly and reliably detected without any aids of enzymes or a special instrument.

Base Pair Mismatch↗
PubMed1989

DNA analysis by flow cytometry in cutaneous T-cell lymphomas.

DNA histograms of skin and blood specimens from 64 patients with known or suspected cutaneous T-cell lymphoma (CTCL) have been examined and compared with normal blood mononuclear cells and skin biopsy samples from 50 patients with various benign cutaneous conditions (i.e. patch test infiltrates, eczema, psoriasis, lichen planus, atopic dermatitis) in an attempt to establish whether DNA measurements by flow cytometry may improve the early recognition of CTCL. The results indicate that right-skewed G0/G1 peaks are seen frequently in both benign disorders and known and suspected CTCL. Such peaks may reflect increased stainability of DNA due to chromatin dispersion during cell activation and/or cell proliferation and do not constitute reliable evidence of malignancy. In contrast, discrete aneuploid DNA peaks are confined to malignant lesions, but are seen almost exclusively in the advanced stages in which the diagnosis can be established easily based on routine histological criteria. These data indicate that DNA measurements by flow cytometry is of only limited help in the early recognition of CTCL and support the view that the lymphoid infiltrate in early CTCL may be reactive (rather than neoplastic) or alternatively may contain only minor reactive (rather than neoplastic) or alternatively may contain only minor populations of abnormal (malignant) cells which cannot be detected by currently available DNA measurement techniques.

Adult↗
PubMed1998

Characterization of Vibrio vulnificus isolated from cockles (Anadara granosa): antimicrobial resistance, plasmid profiles and random amplification of polymorphic DNA analysis.

Antibiotic susceptibility, plasmid profiles and random amplification of polymorphic DNA (RAPD) were used to study strains of Vibrio vulnificus isolated from cockles (Anadara granosa). Thirty-six isolates were analyzed. The prevalent biotypes were 1 (72.2% of the isolates) and 2 (27.8%). Among these, 21 strains of biotype 1 and two strains of biotype 2 contained plasmid DNA bands ranging in size from 1.4 to 9.7 MDa. Thirty-one (83.3%) were found to be resistant to one or more of the antimicrobial agents tested, however no specific correlation between antimicrobial resistance patterns and a single biotype was found. In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility. Two primers produced polymorphisms in all strains tested, producing bands ranging from 0.25 to 2.7 kb, indicating a high variability among both biotype 1 and biotype 2 of the V. vulnificus strains investigated. RAPD identity across biotypes was also observed among Vibrio vulnificus strains.

Animals↗
PubMed2003

Molecular fingerprinting of fish-pathogenic Lactococcus garvieae strains by random amplified polymorphic DNA analysis.

In this work, we used the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in Lactococcus garvieae, an important pathogen for fish. Fifty-seven strains with different hosts and geographical origins, including Japan and several countries of the Mediterranean area such as Spain, Portugal, France, Italy, England, and Turkey, were analyzed. Two primers, oligonucleotides 5 and 6 (Pharmacia Biotech) were utilized; primer 5 was the most discriminative, since allowed us to differentiate 10 RAPD -types related to the origin of the strains. Regardless of the oligonucleotide primer employed, the 57 isolates of L. garvieae studied were separated into three genetic groups, composed of the Spanish, Portuguese, English, and Turkish strains (group A), the Italian and French strains (group B), and the Japanese strains (group C). The similarity of isolates within each group, estimated on the basis of the Dice coefficient, ranged from 75 to 100%. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen.

Animals↗
PubMed1995

The use of DAPI as a replacement for ethidium bromide in forensic DNA analysis.

Ethidium bromide is used extensively for the visualization of DNA in forensic applications. This is usually accomplished by the addition of dye to the gel and buffer before electrophoresis or by staining the gel after the run. In both procedures considerable ethidium bromide containing buffer is generated that must be decontaminated. We demonstrate that another fluorescent DNA binding dye, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), can be used in place of ethidium bromide for yield and restriction gels, and to stain the visual marker of analytical gels. A low concentration of DAPI is added directly to the DNA. Most of the stain is immediately bound to the DNA and remains bound during electrophoresis.

DNA↗
PubMed1998

Hb Tak confirmed by DNA analysis: not expressed as thalassemia in a Hb Tak/Hb E compound heterozygote.

Hemoglobin variants in which a frameshift results in chain elongation are unusual and also have a low population frequency. Hb Tak was previously characterized by amino acid analysis, and it was assumed to be due to an insertion of the dinucleotide CA into codon 146 [CAC-->CA(CA)C] which abolishes the normal stop codon at position 147. This insertion causes a frameshift which results in elongation of the beta chain by 11 amino acids. This variant has previously been described in a few Thai families. We report the DNA sequence of Hb Tak in an individual of Cambodian descent who is a Hb E/Hb Tak compound heterozygote. In contrast with extended variants of the alpha-globin chain that are expressed as alpha-thalassemias, the hematologic effect of Hb Tak/Hb E is a mild polycythemia. The combination of Hb Tak/Hb E is not expressed as a thalassemia.

Adult↗
PubMed2003

DNA analysis indicates that Asian elephants are native to Borneo and are therefore a high priority for conservation.

The origin of Borneo's elephants is controversial. Two competing hypotheses argue that they are either indigenous, tracing back to the Pleistocene, or were introduced, descending from elephants imported in the 16th-18th centuries. Taxonomically, they have either been classified as a unique subspecies or placed under the Indian or Sumatran subspecies. If shown to be a unique indigenous population, this would extend the natural species range of the Asian elephant by 1300 km, and therefore Borneo elephants would have much greater conservation importance than if they were a feral population. We compared DNA of Borneo elephants to that of elephants from across the range of the Asian elephant, using a fragment of mitochondrial DNA, including part of the hypervariable d-loop, and five autosomal microsatellite loci. We find that Borneo's elephants are genetically distinct, with molecular divergence indicative of a Pleistocene colonisation of Borneo and subsequent isolation. We reject the hypothesis that Borneo's elephants were introduced. The genetic divergence of Borneo elephants warrants their recognition as a separate evolutionary significant unit. Thus, interbreeding Borneo elephants with those from other populations would be contraindicated in ex situ conservation, and their genetic distinctiveness makes them one of the highest priority populations for Asian elephant conservation.

Alleles↗
PubMed1995

Double-stranded DNA analysis by capillary electrophoresis with laser-induced fluorescence using ethidium bromide as an intercalator.

We have developed a rapid and sensitive method to analyze double-stranded DNA by capillary electrophoresis equipped with a laser-induced fluorescence detector. Using ethidium bromide as an intercalator in polyacrylamide gel-filled capillaries along with a green He/Ne laser source (excitation: 543 nm; emission: 600 nm), we found the detection sensitivity for a single DNA-ethidium bromide complex was in the picogram level with a resolution of one base pair separation between two DNA fragments. In the case of the separation of a phi X174 DNA-HaeIII digest, sizes from 72 to 1353 bp were well resolved within 12 min. For the analysis of PCR-amplified DNA, low levels of amplified DNA fragments could be detected that could not be visualized in agarose gel after standard ethidium bromide staining. Considering the high resolving power and sensitivity, we believe that this method can be a useful tool for the analysis of double-stranded DNA especially for PCR-amplified DNA fragments.

Base Sequence↗
PubMed2003

[Mitochondrial DNA analysis of Sporothrix schenckii].

Restriction fragment length polymorphism (RFLP) in mitochondrial DNA (mtDNA) of clinical and environmental isolates of Sporothrix schenckii was investigated. Isolates of S. schenckii were classified into 24 mtDNA types (Types 1-24) based on mtDNA RFLP patterns with HaeIII and clustered into two major groups by phylogeny, Group A and Group B. Group A isolates are predominant in South Africa, North America, Central America and South America, while Group B isolates are predominant in Australia, Japan and China. Based on the mtDNA-RFLP patterns with HaeIII, most environmental isolates morphologically identified as S. schenckii were confirmed to be species distinct from S. schenckii and S. schenckii isolates were few, while all of more than 500 clinical isolates were confirmed as S. schenckii. Therefore, RFLP analysis of mtDNA is essential for the identification of environmental, but not clinical isolates of S. schenckii.

DNA, Mitochondrial↗
PubMed1999

Flow cytometric DNA analysis and chromosomal aberrations in malignant glioblastomas.

In this study we combined flow cytometry with fluorescence in situ hybridization to detect numerical aberrations in chromosomes. Fifty-nine human malignant gliomas were examined by flow cytometry for DNA-content and cell cycle analysis and for numerical aberrations of chromosome 1 by in situ hybridization using a chromosome specific centromere probe. Of the gliomas analysed, 42% were diploid and 58% showed aneuploid tumour cell populations. The DNA index was heterogeneous ranging from 1.0 to 2.3. The S-phase analysis showed proliferation activity from a very low range of 0.7% up to 17.0%. In general, diploid gliomas exhibited a lower S-phase activity than aneuploid gliomas. Of the aneuploid gliomas, 15% showed a peridiploid pattern with a DNA index mean of 1.1. In these peridiploid tumours a trisomy of chromosome 1 could be detected by fluorescence in situ hybridization (FISH). The frequency of trisomic chromosome 1 in malignant gliomas reflects a very slight increase in DNA index from diploid to peridiploid (DNA index 1.1). Comparison of chromosome numbers and DNA content gave good correlation. Also important, the results reflects the cell cycle, specifically the extent of S-phase activity. In general, cell proliferation of diploid and peridiploid gliomas is much less than in higher aneuploid gliomas. The analysis of DNA content may thus yield results with respect to the biological behaviour of tumours in general.

Chromosome Aberrations↗
PubMed2006

MiniX-STR multiplex system population study in Japan and application to degraded DNA analysis.

We sought to evaluate a more effective system for analyzing X-chromosomal short tandem repeats (X-STRs) in highly degraded DNA. To generate smaller amplicon lengths, we designed new polymerase chain reaction (PCR) primers for DXS7423, DXS6789, DXS101, GATA31E08, DXS8378, DXS7133, DXS7424, and GATA165B12 at X-linked short tandem repeat (STR) loci, devising two miniX-multiplex PCR systems. Among 333 Japanese individuals, these X-linked loci were detected in amplification products ranging in length from 76 to 169 bp, and statistical analyses of the eight loci indicated a high usefulness for the Japanese forensic practice. Results of tests on highly degraded DNA indicated the miniX-STR multiplex strategies to be an effective system for analyzing degraded DNA. We conclude that analysis by the current miniX-STR multiplex systems offers high effectiveness for personal identification from degraded DNA samples.

Asian People↗
PubMed1990

Flow cytometric DNA analysis of parathyroid tumors with special reference to its diagnostic and prognostic value in parathyroid carcinoma.

The nuclear DNA content of paraffin-embedded parathyroid tumors from 49 patients with proven primary hyperparathyroidism was determined by flow cytometric analysis. The lesions included 14 primary and 11 locally recurrent or metastatic lesions from 16 carcinoma patients, 28 single adenomas from 28 patients, and 15 hyperplastic glands from five patients with familial multiple endocrine neoplasia type 1. No abnormal DNA stemline was found in any of the hyperplastic glands. One (3.6%) of the adenomas was aneuploid. There was no difference in ploidy patterns between the primary and recurrent lesions of the carcinomas and five (31%) of the carcinomas expressed aneuploidy. Four of the five patients with aneuploid carcinoma had recurrences including pulmonary metastases. One of them died of this disease 12 years after the initial operation, and all except one of the others are hypercalcemic even after removal of the successive recurrent or metastatic tumors. Of the 11 patients with diploid carcinoma, four had either local recurrence or pulmonary metastasis. Two of them are living with normocalcemia 3 and 6 years, respectively, after removal of the recurrent tumors and the others are alive with mild hypercalcemia. The remaining seven patients with diploid carcinoma, however, have no recurrence 2 to 5 years after the initial operation. Thus aneuploid parathyroid carcinomas are likely to show more malignant behavior than those with a diploid DNA pattern. All of the patients with adenoma and hyperplasia have been normocalcemic after a mean follow-up interval of 37 months. This study indicates that flow cytometric analysis of nuclear DNA content is a valuable adjunct to histologic examination in the diagnosis of parathyroid carcinoma and the prediction of the clinical outcome.

Adenoma↗