Characterization of six rat strains (Rattus norvegicus) by mitochondrial DNA restriction fragment length polymorphism

Restriction fragment length polymorphism (RFLP) was used to examine the extent of mtDNA polymorphism among six strains of rats (Rattus norvegicus) - Wistar, Wistar Munich, Brown Norway, Wistar Kyoto, SHR and SHR-SP. A survey of 26 restriction enzymes has revealed a low level of genetic divergence among strains. The sites of cleavage by EcoRI, NcoI and XmnI were shown to be polymorphic. The use of these three enzymes allows the 6 strains to be classified into 4 haplotypes and identifies specific markers for each one. The percentage of sequence divergence among all pairs of haplotypes ranged from 0.035 to 0.33%, which is the result of a severe population constriction undergone by the strains. These haplotypes are easily demonstrable and therefore RFLP analysis can be employed for genetic monitoring of rats within animal facilities or among different laboratories.

Análise de restrição de DNA ribossomal amplificado (ARDRA) pode diferenciar Fusarium solani f. sp. phaseoli de F. solani f. sp. glycines

Métodos moleculares têm sido utilizados para caracterizar a diversidade entre isolados de Fusarium spp. patogênicos e não patogênicos a uma cultura e, para determinar relações genéticas entre formae speciales. Testes de patogenicidade realizados em soja (Glycine max) e feijoeiro (Phaseolus vulgaris) com 17 isolados de Fusarium solani não demonstraram especificidade de hospedeiros. Utilizou-se a técnica ARDRA (Amplified Ribosomal DNA Restriction Analysis) para analisar a região ITS1 - 5,8S rDNA - ITS2, amplificada com os primers ITS5 e ITS4. Os produtos amplificados foram digeridos com as enzimas de restrição Hae III e Msp I. Os padrões de bandas gerados pela digestão com a enzima Hae III permitiram diferenciar três grupos entre os isolados de F. solani, sendo um grupo específico para isolados de F. solani f. sp. phaseoli com 100% de similaridade entre os 11 isolados. Entre os isolados de F. solani f. sp glycines foram observados dois padrões distintos de restrição. A técnica de ARDRA utilizando a enzima Hae III apresenta, portanto, potencial para utilização como um marcador para diferenciação entre as formae specialesphaseoli e glycines, dentro do complexo F. solani.

Genetic and Antigenic Analysis of Adenovirus Type 3 Strains Showing Intermediate Behavior in Standard Seroneutralization Test

During an epidemiological survey of acute respiratory infection in Rio de Janeiro, among 208 adenovirus isolates, we found two strains that we were not able, by a standard neutralization procedure, to distinguish between type 3 or 7. However, DNA restriction pattern for the two strains with different enzymes were analyzed and showed a typical Ad3h profile. Using a cross-neutralization test in which both Ad3p and Ad7p antisera were used in different concentration against 100 TCID50 of each adenovirus standard and both isolates, we were able to confirm that the two isolates belong to serotype 3. An hemagglutination inhibition test also corroborated the identification of both strains as adenovirus type 3. Comparing Ad3h and Ad3p genome, we observed 16 different restriction enzyme sites, three of which were located in genomic regions encoding polypeptides involved in neutralization sites

Typing of Pseudomonas aeruginosa from hospitalized patients: a comparison of susceptibility and biochemical profiles with genotype

Typing techniques are essential for understanding hospital epidemiology, permitting the elucidation of the source of infection and routes of bacterial transmission. Although DNA-based techniques are the "gold standard" for the epidemiological study of Pseudomonas aeruginosa, antibiotic profiles and biochemical results are used because they are easy to perform and to interpret and relatively inexpensive. Antibiotypes (susceptibility profiles) and biotypes (biochemical profiles) were compared to genotypes established by DNA restriction enzyme analysis in 81 clinical isolates of P. aeruginosa from three hospitals in Porto Alegre, Brazil. The epidemiological relationship among patients was also evaluated. Susceptibility and restriction profiles were discrepant in more than 50% of the cases, and many antibiotypes were observed among isolates from the same genotype. Furthermore, susceptibility profiles did not allow the distinction of isolates from unrelated genotypes. Since a large number of isolates (63%) yielded the same biochemical results, only 10 biotypes were detected, showing that this typing method has a low discriminatory power. On the other hand, DNA restriction enzyme typing allowed us to establish 71 distinct types. Epidemiological data about the relation among P. aeruginosa isolates were not conclusive. The results of the present study indicate that the only method that can establish a clonal relation is DNA restriction enzyme typing, whereas the other methods may cause misleading interpretations and are inadequate to guide proper infection control measures.

Identification and in vitro production of Lactobacillus antagonists from women with or without bacterial vaginosis

Lactobacilli isolated from the vaginal tract of women with and without bacterial vaginosis (BV) were identified and characterized for the production of antagonists. Bacterial samples were isolated from healthy women (N = 16), from patients with clinical complaints but without BV (N = 30), and from patients with BV (N = 32). Identification was performed using amplified ribosomal DNA restriction analysis. Production of antagonistic compounds was evaluated by the double-layer diffusion technique using Gram-positive (N = 9) and Gram-negative bacteria (N = 6) as well as yeast (N = 5) as indicator strains. Of a total of 147 isolates, 133 were identified as pertaining to the genus Lactobacillus. Lactobacillus crispatus was the species most frequently recovered, followed by L. johnsonii and L. jensenii. Statistical analysis showed that L. crispatus was more frequent in individuals without BV (P < 0.05). A higher production of antagonists was noted in L. crispatus isolates from healthy women (P < 0.05). More acidic local pH and higher H2O2 production by isolated lactobacilli from healthy women suggest these mechanisms as the possible cause of this antagonism. In conclusion, a significant correlation was detected between the presence and antagonistic properties of certain species of Lactobacillus and the clinical status of the patients.

Restriction fragment length polymorphism of 195 bp repeated satellite dna of Trypanosoma cruzi supports the existence of two phylogenetic groups

The restriction fragment length polymorphism of the 195 bp repeated DNA sequence of Trypanosoma cruzi was analyzed among 23 T. cruzi stocks giving a reliable picture of the whole phylogenetic variability of the species. The profiles observed with the enzymes Hinf I and Hae III were linked together and supported the existence of two groups. Group 1 shows a 195 bp repeated unit (Hinf I) and high molecular weight DNA (Hae III), while group 2 presents a ladder profile for each enzyme, which is a characteristic of tandemly repeated DNA. The two groups, respectively, clustered stocks pertaining to the two principal lineages evidenced by isoenzyme and RAPD markers. The congruence among these three independent genomic markers corroborates the existence of two real phylogenetic lineages in T. cruzi. The specific monomorphic profiles for each major phylogenetic lineage suggest the existence of ancient sexuality and cryptic biological speciation.

Restriction site heteroplasmy in the mitochondrial DNA of Brycon opalinus (Cuvier, 1819) (Characiformes, Characidae, Bryconiae)

Homoplasmy is a feature usually found in the mtDNA of higher animal taxa. On the other hand, the presence of two classes of mtDNA in the same cell or organism is rare and may appear in length or site variation. Data from mtDNA RFLP analysis of Brycon opalinus populations (Cuvier, 1819; Characiformes, Characidae, Bryconinae) revealed site heteroplasmy from endonuclease NheI digestion. Southern blotting hybridization was used to survey a total of 257 specimens with 24 restriction enzymes. Three different restriction fragment patterns of mtDNA were obtained from NheI digestion. Two individuals from hatchery broodstock were found to have two of them. NheI digests of heteroplasmic individuals yielded two fragments of approximately 1180 and 1260 bp. Despite the low frequency of this type of heteroplasmy in the whole B. opalinus population, the presence of site heteroplasmy in this species supports the evidence of this phenomenon in lower vertebrate groups.

Polymerase chain reaction and restriction fragment length polymorphism analysis of the ITS2 region for differatiation of Brazilian Biomphalaria intermediate hosts of the Schistosoma mansoni

We sequenced the internal transcribed spacer 2 of the ribosomal DNA (ITS2-DNAr) from the three Schistosoma mansoni intermediate hosts in Brazil: Biomphalaria glabrata, Biomphalaria tenagophila and Biomphalaria straminea. Analysis of a restriction map from those sequences allowed us to select putative restriction enzymes able to identify the snail species under study. Four restriction enzymes were used and HpaII provided simple species-specific profiles easily visualized in polyacrylamide gels. The use of ITS2 is advantageous as it provides a small fragment of 460 bp which may be easily amplified by PCR. In the current work, we showed that the amplification of ITS2-DNAr together with HpaII enzyme restriction is an auxiliary molecular tool for the morphological identification of such snails as well as for taxonomic and phylogenetic studies of neotropical planorbids.

Restriction enzyme analysis of the human cytomegalovirus genome in specimens collected from immunodeficient patients in Belém, State of Pará, Brazil

INTRODUCTION: Human cytomegalovirus is an opportunistic betaherpesvirus that causes persistent and serious infections in immunodeficient patients. Recurrent infections occur due to the presence of the virus in a latent state in some cell types. It is possible to examine the virus using molecular methods to aid in the immunological diagnosis and to generate a molecular viral profile in immunodeficient patients. The objective of this study was to characterize cytomegalovirus genotypes and to generate the epidemiological and molecular viral profile in immunodeficient patients. METHODS: A total of 105 samples were collected from immunodeficient patients from the City of Belém, including newborns, hemodialysis patients, transplant recipients and HIV+ patients. An IgG and IgM antibody study was completed using ELISA, and enzymatic analysis by restriction fragment length polymorphism (RFLP) was performed to characterize viral genotypes. RESULTS: It was observed that 100% of the patients had IgG antibodies, 87% of which were IgG+/IgM-, consistent with a prior infection profile, 13% were IgG+/IgM+, suggestive of recent infection. The newborn group had the highest frequency (27%) of the IgG+/IgM+ profile. By RFLP analysis, only one genotype was observed, gB2, which corresponded to the standard AD169 strain. CONCLUSIONS: The presence of IgM antibodies in new borns indicates that HCMV continues to be an important cause of congenital infection. The low observed genotypic diversity could be attributed to the small sample size because newborns were excluded from the RFLP analysis. This study will be continued including samples from newborns to extend the knowledge of the general and molecular epidemiology of HCMV in immunodeficient patients.

Molecular characterization of Mycobacterium tuberculosis isolates from Tehran, Iran by restriction fragment length polymorphism analysis and spoligotyping

Abstract: INTRODUCTION Characterization of Mycobacterium tuberculosis (MTB) isolates by DNA fingerprinting has contributed to tuberculosis (TB) control. The aim of this study was to determine the genetic diversity of MTB isolates from Tehran province in Iran. METHODS MTB isolates from 60 Iranian and 10 Afghan TB patients were fingerprinted by standard IS6110-restriction fragment length polymorphism (RFLP) analysis and spoligotyping. RESULTS The copy number of IS6110 ranged from 10-24 per isolate. The isolates were classified into 22 clusters showing ≥ 80% similarity by RFLP analysis. Fourteen multidrug-resistant (MDR) isolates were grouped into 4 IS6110-RFLP clusters, with 10 isolates [71% (95% CI: 45-89%)] in 1 cluster, suggesting a possible epidemiological linkage. Eighteen Iranian isolates showed ≥ 80% similarity with Afghan isolates. There were no strains with identical fingerprints. Spoligotyping of 70 isolates produced 23 distinct patterns. Sixty (85.7%) isolates were grouped into 13 clusters, while the remaining 10 isolates (14.2%) were not clustered. Ural (formerly Haarlem4) (n = 22, 31.4%) was the most common family followed by Central Asian strain (CAS) (n = 18, 25.7%) and T (n = 9, 12.8%) families. Only 1strain was characterized as having the Beijing genotype. Among 60 Iranian and 10 Afghan MTB isolates, 25% (95% CI: 16-37) and 70% (95% CI: 39-89) were categorized as Ural lineage, respectively. CONCLUSIONS A higher prevalence of Ural family MTB isolates among Afghan patients than among Iranian patients suggests the possible transmission of this lineage following the immigration of Afghans to Iran.

Development of nuclear DNA probes for the typing of Trypanososma cruzi

We have developed and tested a new way of typing Trypanosoma cruzi, mamely the use of cloned nuclear DNA fragments as genetic markers. Restriction fragment length polymorphisms were verified on Soutern blots hybridized to random probes. Fragment patterns were analyzed and dendrograms constructed. Our results on well characterized laboratory strains correlate well to published isoenzyme studies. Some of the probes were also hybridized to chromosomes separated by pulse field gel electrophoresis a higher degree of heterogeneity was observed at this level.

Detection of Babesia bigemina infection: use of a DNA probe - a review

The development of a repetitive DNA probe for Babesia bigemina was reviewed. The original plasmid (p(Bbi)16) contained an insert of B. bigemina DNA of approximately 6.3 kb. This probe has been evaluated for specificityand analytical sensitivity by dot hybridization with isolates from Mexico, the Caribbean region and Kenya. A partial restriction map has been constructed and insert fragments have been subcloned and utilized as specific DNA probes. A comparison of 32P labelled and non-radioactive DNA probes was presented. Non-radioctive detection systems that have been used include digoxigenin dUTP incorporation, and detection by colorimetric substrate methods. Derivatives from the original DNA probe have been utilized to detect B. bigemina infection in a) experimentally inoculated cattle, b) field exposed cattle, c) infected Boophilus microplus ticks, and d) the development of a PCR amplification system.

Protein-DNA associations in a gender-specific gene of Schistosoma mansoni: characterization by UV cross-linking, DNase I footprinting and band shift assays

Protein extracts obtained from male and female shistosomes were incubated with a gender-specific gene, F-10, transcribed only in adult females and encoding a major egg-shell protein. The protein/DNA interaction was measured using the band shift, DNase-I-footprinting and UV cross-linking techniques. The results showed a clear band shift when a 302 bp restriction fragment containing the 3'end of the gene was incubated with either female or male proteins. This fragment also contained a putative steroid hormone regulatory element (HRE). In contrast, only the male proteins produced a shift with the 495 bp fragment corresponding to the middle region of the gene. DNase I footprinting showed that proteins from males and females interacted with the F-10 gene by binding to multiple adjacent sites along the DNA, thus generatingrelatively long protected fragments of approximately 100 bp. This result suggested that the adjacent binding of several moles of proteins occured at the 5'end of the gene. UV cross-linking between schistosome proteins and a 21 bp synthetic oligonucleotide the F-10 HRE, evidence proteins having MWS of 30,45 and 65 kDNA. These proteins are presumably involved in the regulation of transcription of the F-10 gene.

Comparative Analysis by Polymerase Chain Reaction Amplified Minicircles of Kinetoplast DNA of a Stable Strain of Trypanosoma cruzi from São Felipe, Bahia, its Clones and Subclones: Possibility of Predominance of a Principal Clone in this Area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicicles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment lenght polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%.This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.