Restriction Pattern Analysis Research Articles

Genetic variation among Malaysian isolates of Salmonella typhi as detected by ribosomal RNA gene restriction patterns.

Genetic variation among Malaysian isolates of Salmonella typhi was determined by analysis of ribosomal RNA gene restriction patterns. Of the 20 isolates analyzed, eight different pattern combinations were detected. The amount of variation observed was also dependent upon the restriction endonuclease used; PstI produced more different patterns than did SmaI. The results suggested that disease activity was due to a number of different clones circulating simultaneously rather than a single strain. Further implications of the data are discussed.

Application of Chromosome Fingerprinting to the Differentiation of Brewing Yeasts

Chromosome fingerprinting was used to differentiate among a number of lager yeasts that were indistinguishable when examined by DNA restriction pattern analysis. Electrophoresis conditions were varied empirically to obtain optimal resolution of chromosomes in various size ranges. Details of the practical application of the technique are described with the investigation of putative mutants of a brewing yeast. This application illustrated the problems in differentiating brewery yeast and contaminants with the use of classical microbiological procedures. The technique also was used to verify the integrity of isolates of another brewing yeast obtained from outside sources.

Identification of Azospirillum Strains at the Genome Level with Total DNA Restriction Pattern Analysis

Summary Seventeen strains belonging to the genus Azospirillum were isolated from crop plant roots and characterized following routine morphological and physiological methods without obtaining a definite species assignment. DNA restriction pattern analysis was therefore performed on those strains and on 9 reference strains belonging to Azospirillum lipoferum, Azospirillum brasilense and Azospirillum amazonense. Fragments obtained with endonuclease BglII digestion were separated with PAGE and stained with silver nitrate. An internal standard was used to rescale the separation profiles, thereby permitting the comparison of samples from different gels. Each pattern was found to be unique, allowing the distinction of single strains. UPGMA analysis, yielding clusters of band patterns, was utilized to assign strains to a species. Restriction pattern analysis is therefore suggested for the routine identification of azospirilla.

RDNA fingerprinting as a tool in epidemiological analysis ofSalmonella typhiinfections

Characterization of 169 strains of Salmonella typhi of phage types C1, C4, D1 and D9 isolated in 1975-88 was carried out by rDNA gene restriction pattern analysis. Twenty-four isolates had been recovered during four large waterbone outbreaks in the last 20 years in Sicily; 145 strains, isolated from apparently sporadic cases of infection in Southern Italy in the same period of time, were also examined. Application of rRNA-DNA hybridization technique after digestion of chromosomal DNA with Cla I showed the identity of patterns of the epidemic strains of phage types C1 and D1, confirming attribution of the outbreaks to single bacterial clones. Patterns of the two available strains of lysotype D9 were slightly different, whilst the 12 epidemic strains of phage type C4 could be assigned to two distinct patterns scarcely related to each other and, consequently, to two different clones. A considerable heterogeneity was detected among all apparently sporadic isolates of the four phage types under study. This fingerprinting method appears a reliable tool to complement phage typing in characterizing isolates of S. typhi. In particular, epidemiological features of spread of this salmonella serovar in areas, where simultaneous circulation of indigenous and imported strains occurs, can be elucidated.

Prevalence of recombinational versus mutational events in damaged plasmid DNA containing regions of homology with the chromosome

Plasmid DNA modified by in vitro treatments was transformed in E. coli bacterial cells. A streptomycin-resistant strain, carrying the peculiar rpsL421 mutation, was used as a recipient for the cloning vector pNO1523, which carries the wild-type (streptomycin-sensitive) rpsL allele. Transformants were streptomycin-sensitive unless a change in plasmid sequence had occurred. The analysis of the MaeI restriction pattern of plasmids isolated from streptomycin-resistant transformants, together with the detection of the phenotype that they conferred to a streptomycin-dependent strain, allowed us to identify plasmids that had undergone recombination with the host chromosome. The number of these plasmids exceeded by far that of plasmids resulting from mutational events.

Discrimination of Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum by restriction pattern analysis of DNA adjacent to the hom gene

Different strains of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum were analysed for restriction fragment length polymorphism using the homoserine dehydrogenase gene (hom) as a probe. The hybridization patterns obtained PvuII- or Asp700-restriction of chromosomal DNA were specific and distinguishable for each of the three species and identical for the different strains of each species. Thus, the method employed allows rapid distinction of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum. The former species could also be discriminated from the latter two by its resistance to 0.5 g/l of the methionine analog ethionine.

A species-specific DNA probe obtained from Streptococcus salivarius subsp. thermophilus detects strain restriction polymorphism

Genomic polymorphism in Streptococcus salivarius subsp. thermophilus was revealed by DNA restriction pattern analysis. A 4.2-kb variable DNA fragment was cloned from strain NST7 and hybridised with the DNA of 25 strains allowing an easy detection of intraspecific RFLP. Strong and weak hybridisation signals were observed and the latter were specifically revealed by a 2.1-kb fragment of the probe. Probe specificity was demonstrated by the absence of homology with DNA of strains belonging to 10 other species, with the exception of S. salivarius subsp. salivarius, confirming a close relationship between S. salivarius and S. thermophilus.

A species-specific DNA probe obtained fromStreptococcus salivariussubsp.thermophilusdetects strain restriction polymorphism

Genomic polymorphism in Streptococcus salivarius subsp. thermophilus was revealed by DNA restriction pattern analysis. A 4.2-kb variable DNA fragment was cloned from strain NST7 and hybridised with the DNA of 25 strains allowing an easy detection of intraspecific RFLP. Strong and weak hybridisation signals were observed and the latter were specifically revealed by a 2.1-kb fragment of the probe. Probe specificity was demonstrated by the absence of homology with DNA of strains belonging to 10 other species, with the exception of S. salivarius subsp. salivarius, confirming a close relationship between S. salivarius and S. thermophilus.

Leishmania: Genus identification based on a specific sequence of the 18S ribosomal RNA sequence

The analysis of PvuII restriction patterns of Leishmania spp. and Trypanosoma spp. genomic DNA showed genus distinctive profiles. A specific PvuII site was detected in the 5′ domain of 18S ribosomal DNA of Leishmania. A 20-mer oligonucleotide encompassing this PvuII region was synthesized. This sequence, when utilized as probe, on short exposures of dot tests, detected 10 3 whole promastigotes of all Leishmania species analyzed but did not hybridize with T. cruzi or human nucleic acids. Two other oligonucleotides were synthesized to be used as primers for amplification through polymerase chain reaction of the 18S ribosomal DNA region containing the PvuII site. The probes described may be useful for the detection of Leishmania spp. under clinical and epidemiological trials.

Identification of coagulase-negative staphylococci by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and rRNA restriction patterns

A total of 1,417 staphylococcal and micrococcal strains were collected from the beards and scalps of 10 subjects over a period of 8 months. Sixteen strains identified as Staphylococcus epidermidis with an API system had distinctive yellow colonies on nutrient agar plates and sodium dodecyl sulfate-polyacrylamide gel electrophoresis whole-cell polypeptide profiles similar to those of Staphylococcus capitis; this identification was confirmed by analysis of rRNA gene restriction patterns.

Evaluation of Recent Techniques Used to Identify Individual Strains ofSaccharomycesYeasts

Three techniques (yeast chromosome fingerprinting, DNA restriction pattern analysis, and total soluble protein profiling) were compared for their abilities to differentiate strains of bakers' and b...

Detection of molecular rearrangements in Prader‐Willi syndrome patients by using genomic probes recognizing four loci within thePWCR

The Prader-Willi chromosome region (PWCR) in Prader-Willi syndrome patients was analyzed by using genomic DNA probes mapping to 15q11.2-q12. The present report includes analysis of dosage by RFLP and densitometric studies, and analysis of restriction patterns. Twelve Prader-Willi syndrome (PWS) patients were studied: 5 had deletions of 15q11-q13, one had an unbalanced translocation, and 6 were karyotypically normal. Four genomic DNA probes were used: pML34 (D15S9); pTD3-21 (D15S10); IR4-3 (D15S11), a subclone of IR4; and IR10-1 (D15S12), a subclone of IR10 (Donolon et al.: Proc Natl Acad Sci USA 83:4408-4412, 1986). The results presented demonstrate that molecular rearrangements have occurred in 10 of the 12 PWS patients investigated and that the specific rearrangements differ from patient to patient. Patients with apparently similar cytogenetic deletions differ at the molecular level with deletions and/or duplications of various loci. The present study reports molecular alterations within the PWCR in PWS patients reported as cytogenetically normal. However, the 6 karyotypically normal patients are a heterogeneous group with molecular rearrangements ranging from none detected to deletions and/or duplications. These molecular studies suggest that a physical disruption of the PWCR causes the PWS not only in those patients reported to have a cytogenetic aberration but also in those identified as apparently karyotypically normal. The question remains as to whether the PWS patients in whom a molecular abnormality has not been detected have an autosomal recessive form of PWS, a molecular disruption which has not yet been detected, or another mechanism producing an apparently identical phenotype. The order of the 4 loci on chromosome 15 is hypothesized to be cen----D15S9----D15S12----D15S11----D15S10.

Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome

This paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from the Escherichia coli chromosome on the restriction map established by Kohara et al. (1987). The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Kohara et al. (1987). Therefore, the sequenced genes available in the EMBL library may be localized on the E. coli chromosome restriction map. This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions. Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between the E. coli K12 strains used by the various laboratories involved in DNA sequencing. The program is versatile enough to be used with other genomes.

Nosocomial Outbreak of Systemic Candidosis Associated With Parenteral Nutrition

AbstractEight patients in two surgical units developed systemic candidosis during a 40-day period from June 5 to July 13, 1987 (in five cases Candida albicans was identified). Three of them died. All cases belonged to a group of 27 patients receiving parenteral nutrition (PN), while among the 108 patients who did not receive PN, no cases were observed (p = .000001). Candida was cultured from two PN bags administered to the cases. A specialized nutrition nurse was responsible for the PN compounding and for maintaining administration sets in the two wards involved.An epidemiological investigation in which 19 uninfected patients who had had PN were used as controls, showed no significant difference between cases and controls except that lipids were more frequently added to bags administered to cases (p = .0005). Furthermore, the bags administered to cases contained a higher average number of multidose constituents (p = .0008) when the comparison was focused on the two days before the onset of symptoms.Given the favorable medium provided by lipids, even a low level contamination of PN solutions during compounding and/or administration could have been responsible for the outbreak. The finding of a more frequent exposure of cases to multidose vials suggests, although not conclusively, that an extrinsic contamination occurred during compounding. Six isolates of C albicans were available from four cases. C albicans was cultured from the pharyngeal swabs of two physicians and three nurses, including the specialized nutrition nurse. According to DNA restriction pattern analysis, a single strain was responsible for all the cases of systemic candidosis, with only the specialized nutrition nurse harboring the same strain. DNA fingerprinting provided a reliable system for typing C albicans strains. This was the first outbreak of C albicans systemic infection associated with PN.

Physiological, chemical, morphological, and plant infectivity characteristics of Frankia isolates from Myrica pennsylvanica: correlation to DNA restriction patterns.

The filter exclusion method was used to isolate Frankia strains from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. A total of 16 isolates from five locations were cultured. The isolates were characterized by morphological, chemical, physiological, and plant infectivity criteria and compared with genomic DNA restriction pattern data, which were used to assign the isolates into gel groups (see accompanying paper). The isolates from M. pennsylvanica evaluated in this study were characteristic of Frankia physiological group B strains and were indistinguishable on the basis of whole-cell wall chemistry and diaminopimelic acid isomer analysis. Distinct differences in the spectrum of utilized organic acids and carbohydrates were observed among the isolates and were the only phenotypic criteria by which the isolates could be separated and assigned into separate groups. In general, isolates within a restriction pattern gel group had identical utilization patterns, whereas intragroup isolates had different utilization patterns. Correlation of these phenotypic characteristics with the results of molecular analysis revealed an exclusive carbohydrate and organic acid utilization pattern for each gel group as established by restriction pattern analysis.

DNA restriction patterns and DNA-DNA solution hybridization studies of Frankia isolates from Myrica pennsylvanica (bayberry).

Sixteen Frankia strains were isolated from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. Restriction pattern analysis of total genomic DNA was used to group the isolates into gel groups, and the genetic relatedness among the isolates was evaluated by DNA-DNA solution hybridization studies. Restriction pattern analysis provided a distinctive reproducible fingerprint for each isolate. Isolates fell into nine separate groups (strain types). More than one strain type was isolated from most sites. Isolates from two different gel groups were found in 3 of 10 nodules examined. Of the 16 isolates, 10 contained extrachromosomal DNA. Six different extrachromosomal DNA banding patterns were found. Genomically similar isolates carried related, but different, banding patterns. DNA hybridization studies indicated that isolates from a single plant species can be minimally related as determined by total genome homology. Homology ranged from 12 to 99%. Highly divergent strains were isolated from the same plant and found to cohabit the same nodule. Thus, this study demonstrated that Frankia strains which infect the same host plant are not only phenotypically different but also genetically diverse.

Ribosomal RNA Gene Restriction Patterns Provide Increased Sensitivity for Typing Salmonella typhi Strains

To date, epidemiologic associations among strains of Salmonella typhi are based exclusively on phage typing, which may be of limited value if a common phage type is involved. Analysis of ribosomal RNA gene restriction patterns allows separation of most independently isolated strains of identical phage types. The sensitivity of the method is dependent on the restriction enzymes used to digest chromosomal DNA. It was highest for PstI, which separated 16 of 20 strains that belonged to 8 phage types including 3 untypable strains. Three strains differed in their phage types but had identical ribosomal RNA gene restriction patterns. Also, two pairs of strains indistinguishable by phage typing exhibited identical patterns; however, two of these strains were expected to be identical because they were isolated from two patients who were likely exposed to the same source. Ribosomal RNA gene restriction patterns appear to be stable. Thus, the method may complement phage typing and aid in further differentiation of strains.

Evaluation of evolutionary divergence in the genus Naegleria by analysis of ribosomal DNA plasmid restriction patterns

Ribosomal DNA (rDNA) plasmid restriction maps of 10 strains and rDNA hybridisation patterns of 61 additional strains have been used to assess inter- and intra-specific diversity and phylogenetic relationships in the genus Naegleria. The results obtained by this method largely confirm those of previous studies based on a variety of other criteria. They indicate that very little variation exists within the pathogenic species Naegleria fowleri despite its worldwide distribution and that it is closely related to the non-pathogenic Naegleria lovaniensis. Naegleria gruberi is most likely a polyphyletic grouping and care should be taken when using one strain as a reference point for this species. In addition, the two subspecies of the pathogenic Naegleria australiensis arose separately from within the range of variability encompassed by N. gruberi, as did the species Adelphamoeba galeacystis which should probably be assigned to the genus Naegleria. The species Naegleria jadini and Naegleria andersoni are not closely related to any other in the genus based on their rDNA patterns.

Characterisation of methicillin-resistant Staphylococcus aureus isolates by restriction endonuclease digestion of chromosomal DNA.

Clinical isolates of Staphylococcus aureus from the London Hospital were characterised by genetic analysis of antibiotic-resistance determinants and by restriction endonuclease digestion of chromosomal DNA and compared with isolates from elsewhere in the UK. Restriction enzyme digestion of chromosomal DNA confirmed that a single strain of methicillin-resistant S. aureus (MRSA) persists at the London Hospital, although its antibiotic-resistance profile and plasmid carriage are not constant. Methicillin-sensitive isolates, on the other hand, each had readily distinguishable and unique DNA restriction patterns. The DNA restriction digest pattern of the London Hospital MRSA isolates was identical to that of "epidemic" (E) MRSA isolates from the Thames regions. By contrast, other MRSA isolates had DNA restriction patterns which differed from those of EMRSA isolates and from each other. These results confirm the discriminatory value of restriction pattern analysis as a typing method.

Diploid Brassica napus somatic hybrids: characterization of nuclear and organellar DNA

Five somatic hybrids between Brassica campestris and B. oleracea were obtained. Molecular, morphological and cytological information all suggest that the resynthesized B. napus plants were hybrids. All five plants were diploid (2n=38) and had mainly bivalents at meiosis. Seedset was low after selfing but normal after crossing with B. napus. Molecular proof of the hybrid nature of these plants was obtained by hybridization of a rDNA repeat to total DNA. Analysis of chloroplast DNA restriction patterns revealed that all hybrids had chloroplasts identical to the B. oleracea parent. The analysis of mitochondrial DNA indicated that three hybrids had restriction patterns identical to those of B. campestris, and the other two had restriction patterns similar to those of B. oleracea. The 11.3 kb plasmid present in mitochondria of the B. campestris parent was also found in mitochondria of all five hybrids. This suggests that the plasmid from a B. campestris type of mitochondria was transferred into mitochondria of a B. oleracea type.