The sensitivity of phage DNA and plasmid DNA for a restriction enzyme from Staphylococcus aureus

In Staphylococcus aureus transduction of different tetracycline and chloramphenicol plasmids with a group I/III modification was possible to group I and III strains. Group II strains, containing a restriction endonuclease, had a restriction both for the phage and the plasmids: two restriction-deficient group II strains were good acceptors for these plasmids.     

Construction of a Fibrobacter succinogenes Genomic Map and Demonstration of Diversity at the Genomic Level

The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies. Received: 23 October 1996 / Accepted: 31 December 1996     

L3T4 but not LFA-1 participates in antigen presentation by Ak-positive L-Cell transformants

Genomic DNA was isolated from 29 t strains and 4 congenic lines of mice, digested with restriction endonucleases, and hybridized with a probe representing the complement component 4 (C4) gene. All but one of the enzymes revealed restriction fragment length polymorphism in this sample of C4-related genes. Double digestion analysis suggested the presence of three C4 gene copies in some of the t chromosomes and two copies in others. The enzymes distinguished 16 different haplotypes among the 33 strains tested. Based on their restriction fragment length patterns, the t strains could be divided into four groups with strains in each group more closely related to each other with respect to their C4-region genes than strains belonging to different groups. At least three of these four groups represent different branches of the evolutionary tree constructed for the t chromosomes. The C4-related genes of the chromosomes are in strong linkage disequilibrium with the class II genes of the H-2 complex. Typing for the Ss and Slp allotypes of C4 has revealed the presence of the Ss¹ phenotype in two t strains and of the Slp^a phenotype in one strain.     

Restriction and modification systems of ruminal bacteria

A high frequency of type II restriction endonuclease activities was detected inSelenomonas ruminantium but not in other rumen bacteria tested. Eight different restriction endonucleases were characterized in 17 strains coming from genetically homogeneous local population. Chromosomal DNA isolated fromS. ruminantium strains was found to be refractory to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases. The presence of Dam methylation was detected inS. ruminantium strains as well as in several other species belonging to theSporomusa subbranch of low G+C Gram-positive bacteria (Megasphaera elsdenii, Mitsuokella multiacidus).     

Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis

The utility of sequence polymorphisms in the dense granule antigen GRA6 gene as typing markers for Toxoplasma gondii was investigated. The coding region of GRA6 was amplified, sequenced and compared for 30 Toxoplasma strains from eight different zymodemes (Z1-Z8). Sequence alignment identified nucleotide polymorphisms at 24 positions out of 690 bp, which correlated with murine-virulence. Types I, II, and III could be distinguished from each other on the basis of three, 10, and six variable positions, respectively. Two deletions of 15 bp and 3 bp existed in the avirulent (type II) strains. With one exception, all polymorphic positions resulted in amino acid substitutions, and the two gaps of 15 bp and 3 bp caused the deletion of six amino acids in type II strains. Intra-specific polymorphisms were also found in the virulent group. A high degree of sequence polymorphism correlating with the phenotypes of T. gondii strains points to the GRA6 gene being a good marker for strain characterisation and typing of the isolates of this apicomplexan. The large variety of amino acid changes supports the view that the GRA6 protein plays an important role in the antigenicity and pathogenicity of T. gondii. The existence of polymorphic restriction sites for endonuclease MseI was used to develop a PCR-RFLP method which could simply differentiate the three different groups (types I, II, III) of T. gondii.     

Restriction enzyme analysis of mitochondrial DNAs of petite mutants of yeast: Classification of petites, and deletion mapping of mitochondrial genes

Summary We have analyzed the restriction digest patterns of the mitochondrial DNA from 41 cytoplasmic petite strains of Saccharomyces cerevisiae, that have been extensively characterized with respect to genetic markers. Each mitochondrial DNA was digested with seven restriction endonucleases (EcoRI, HpaI, HindIII, BamHI, HhaI, SalI, and PstI) which together make 41 cuts in grande mitochondrial DNA and for which we have derived fragment maps. The petite mitochondrial DNAs were also analyzed with HpaII, HaeIII, and AluI, each of which makes more than 80 cleavages in grande mitochondrial DNA. On the basis of the restriction patterns observed (i.e., only one fragment migrating differently from grande for a single deletion, and more than one for multiple deletions) and by comparing petite and grande mitochondrial DNA restriction maps, the petite clones could be classified into two main groups: (1) petites representing a single deletion of grande mitochondrial DNA and (2) petites containing multiple deletions of the grande mitochondrial DNA resulting in rearranged sequences. Single deletion petites may retain a large portion of the grande mitochondrial genome or may be of low kinetic cimplexity. Many petites which are scored as single continuous deletions by genetic criteria were later demonstrated to be internally deleted by restriction endonuclease analysis. Heterogeneous sequences, manifested by the presence of sub-stoichiometric amounts of some restriction fragments, may accompany the single or multiple deletions. Single deletions with heterogeneous sequences remain useful for mapping if the low concentration sequences represent a subset of the stoichiometric bands. Using a group of petites which retain single continuous regions of the grande mitochondrial DNA, we have physically mapped antibiotic resistance and mit^- markers to regions of the grande restriction map as follows: C (99.3-1.4 map units)-OXI-1 (2.5-15.7)-OXI-2 (18.5-25)-P (28.1-34.2)-OXI-3 (32.2-61.2)-O_II (60-62)-COB (64.6-80.8)-O_I (80.4-85.7)-E (95-98.9).Supported by USPHS Training Grant 5-T01-GM-00090-19.Supported by USPHS Training Grant T32-GM-07197.The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U.S. Energy Research and Development Administration under Contract EY-76-C-02-0069.     

Discrimination of species and strains of basidiomycete genusCoprinus by random amplified polymorphic DNA (RAPD) analysis

All five examined strains ofCoprinus cinereus could be clearly discriminated from the strains of five otherCoprinus species by RAPD patterns with 12 of 13 primers. Also one specimen of unknownCoprinus strain was identified to beC. cinereus by this method. The RAPD patterns were similar among the strains in the same species; many common DNA fragments were recognized as well as some strain-specific DNA fragments. Thus all seven strains ofC. cinereus and all four strains ofC. angulatus examined could be distinguished individually. Diakryotic strains showed the combined RAPD patterns of the two monokaryotic strains constituting the dikaryon. The combined RAPD markers observed in the dikaryons were segregated in their basidiospore progeny. All 18 randomly picked progeny showed different combinations of RAPD markers from the parental strains.     

Genetic characterization of weedy rice (Oryza sativa L.) based on morpho-physiology, isozymes and RAPD markers

 Weedy rice (Oryza sativa L.) is an important resource for breeding and for studying the evolution of rice. The present study was carried out to identify the genetic basis of the weedy rices distributed in various countries of the world. One hundred and fifty two strains of weedy rice collected from Bangladesh, Brazil, Bhutan, China, India, Japan, Korea, Nepal, Thailand and the USA were tested for variations in six morpho-physiological characteristics and in 14 isozyme loci. Twenty six weedy strains selected from the above materials were assayed for the Est-10 locus, six RAPD loci of the nuclear genome, and one chloroplast locus. From the results of multivariate analysis based on the morpho-physiological characteristics and the isozymes, weedy rice strains were classified into indica and japonica types, and each type was further divided into forms resembling cultivated and wild rice. Thus, four groups designated as I, II, III and IV were identified. Weedy strains of group I (indica-type similar to cultivars) were distributed mostly in temperate countries, group II (indica-type similar to wild rice) in tropical countries, group III (japonica-type similar to cultivars) in Bhutan and Korea, group IV ( japonica-type similar to wild rice) in China and Korea. In group I, classified as indica, several strains showed japonica-specific RAPD markers, while some others had japonica cytoplasm with indica-specific RAPD markers in a heterozygous state at several loci. One weedy strain belonging to group II showed a wild rice-specific allele at the Est-10 locus. However, in groups III and IV, no variation was ound either for the markers on Est-10 or for the RAPD loci tested. Judging from this study, weedy rice of group I might have originated at least partly from gene flow between indica and japonica, whereas that of group II most probably originated from gene flow between wild and cultivated indica rice. Weedy rice of group III is thought to have originated from old rice cultivars which had reverted to a weedy form, and that of group IV from gene flow between japonica cultivars and wild rice having japonica backgrounds. Received: 2 May 1996 / Accepted: 30 August 1996     

Molecular characterization of lactic acid bacteria recovered from natural fermentation of beet root and carrot Kanji

The lactic acid bacteria (LAB) play an important role in the fermentation of vegetables to improve nutritive value, palatability, acceptability, microbial quality and shelf life of the fermented produce. The LAB associated with beetroot and carrot fermentation were identified and characterized using different molecular tools. Amplified ribosomal DNA restriction analysis (ARDRA) provided similar DNA profile for the 16 LAB strains isolated from beetroot and carrot fermentation while repetitive extragenic palindromic PCR (rep-PCR) genotyping could differentiate the LAB strains into eight genotypes. Thirteen strains represented by five genotypes could be clustered in five distinct groups while three LAB strains exhibiting distinct genotypes remained ungrouped. These genotypes could be identified to be belonging to L. plantarum group by 16S rDNA sequencing. The recAnested multiplex PCR employing species-specific primers for the L. plantarum group members identified the LAB strains of six genotypes to be L. paraplantarum and the other two genotypes to be L. pentosus. Three genotypes of L. paraplantarum were consistently found on the third and sixth day of beetroot fermentation whereas a distinct genotype of L. paraplantarum and L. pentosus appeared predominant on the tenth day. From carrot Kanji two distinct genotypes of L. paraplantarum and one genotype of L. pentosus were identified. REP-PCR DNA fingerprinting coupled with 16S rDNA sequencing and recA-nested multiplex PCR could clearly identify as well as differentiate the diverse L. plantarum group strains involved in the fermentation.     

RFLP analysis of the ribosomal internal transcribed spacers and the 5.8S rRNA gene region of the genus Saccharomyces: a fast method for species identification and the differentiation of flor yeasts

The PCR amplification and subsequent restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was applied to the identification of yeasts belonging to the genus Saccharomyces. This methodology has previously been used for the identification of some species of this genus, but in the present work, this application was extended to the identification of new accepted Saccharomyces species (S. kunashirensis, S. martiniae, S. rosinii, S. spencerorum, and S. transvaalensis), as well as to the differentiation of an interesting group of Saccharomyces cerevisiae strains, known as flor yeasts, which are responsible for ageing sherry wine. Among the species of the Saccharomyces sensu lato complex, the high diversity observed, either in the length of the amplified region (ranged between 700 and 875 bp) or in their restriction patterns allows the unequivocal identification of these species. With respect to the four sibling species of the Saccharomyces sensu stricto complex, only two of them, S. bayanus and S. pastorianus, cannot be differentiated according to their restriction patterns, which is in accordance with the hybrid origin (S. bayanus × S. cerevisiae) of S. pastorianus. The flor S. cerevisiae strains exhibited restriction patterns different from those typical of the species S. cerevisiae. These differences can easily be used to differentiate this interesting group of strains. We demonstrate that the specific patterns exhibited by flor yeasts are due to the presence of a 24-bp deletion located in the ITS1 region and that this could have originated as a consequence of a slipped-strand mispairing during replication or be due to an unequal crossing-over. A subsequent restriction analysis of this region from more than 150 flor strains indicated that this deletion is fixed in flor yeast populations.     

Diversity of DNA methylation pattern and total DNA restriction pattern in symbiotic Nostoc

Attempts were made to use total DNA restriction patterns and the response of purified DNA to treatment with restriction endonucleases to characterize several symbiotic Nostoc strains which had been isolated from different host plants cultivated in Italy. Among 27 restriction endonucleases tested, several did not cut any DNA and no significant variation in the susceptibility of the genomes to DNA restriction was seen among the strains. Therefore the Nostoc strains could not be separated into groups based on their different susceptibilities to the action of restriction endonucleases. However, in studies of total DNA restriction patterns, the restriction endonucleases BfrI and HpaI gave unique band patterns for each cyanobacterial isolate. Different profiles were even found in strains isolated from host plants belonging to the same species. The results do not support any definition of symbiotic Nostoc genomic groups or species and show that a tight specificity between the host plant and the cyanobacterium might not exist in the symbiotic associations involving Nostoc.     

Heterogeneity of Strains Assigned to Gluconobacter frateurii Mason and Claus 1989 Based on Restriction Analysis of 16S-23S rDNA Internal Transcribed Spacer Regions

Twenty-three strains, which were assigned to Gluconobacter frateurii and maintained at Culture Collection NBRC, were re-identified at the species level on the basis of restriction analysis of 16S-23S rDNA ITS regions by digestion with six restriction endonucleases: Bsp1286I, MboII, AvaII, TaqI, BsoBI, and BstNI. The strains examined were divided into six groups, Group III-1, Group III-2, Group III-3, Group III-4, Group III-5, and Group IV. Group III-1 and Group III-4 respectively were divided into two subgroups, Subgroup III-1a, Subgroup III-1b and Subgroup III-4a, Subgroup III-4b. Gluconobacter frateurii NBRC 3264^T was included in Group III-2, along with strains NBRC 3265 and NBRC 3270, and G. thailandicus BCC 14116^T was included in Group III-3, along with strains NBRC 3254, NBRC 3256, NBRC 3258, NBRC 3255, and NBRC 3257. These groupings were supported by a phylogenetic tree based on 16S-23S rDNA ITS sequences. Strains of group III-2 and Group IV were unequivocally re-identified as G. frateurii, but strains of Group III-3, Group III-4, and Group III-5 were not necessarily re-identified as G. frateurii. The results obtained indicate that the 23 strains have a taxonomically heterogeneous nature, and they are referred to as the G. frateurii complex.     

Differentiation of Chlamydia Species by Combined Use of Polymerase Chain Reaction and Restriction Endonuclease Analysis

To differentiate Chlamydia spp., a primer pair designed to generate a genus-specific region of the major outer membrane protein (MOMP) gene was used in a PCR to amplify a single DNA fragment of 245-259 bp. In the PCR, the expected single DNA fragment was amplified from strains of Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, respectively. By restriction endonuclease analysis with AluI and PvuII, the amplified products exhibited four distinct patterns, corresponding to the four species. It is, therefore, concluded that one-step PCR followed by restriction endonuclease analysis as described in this study could be a valuable method for the detection and differentiation of Chlamydia species.     

Toxoplasma gondii: genotyping of strains from Brazilian AIDS patients with cerebral toxoplasmosis by multilocus PCR-RFLP markers

This study investigated the genetic characteristics of the Toxoplasma gondii strains isolated from 87 patients with cerebral toxoplasmosis and AIDS, treated in Sao Paulo State, Brazil. The laboratorial diagnosis of cerebral toxoplasmosis was based on positive serological exams and PCR of blood and/or cerebrospinal fluid. Four markers (5'-SAG2, 3'-SAG2, SAG3 and GRA6) were chosen to analyze the samples. Each having clear resolution to distinguish the three clonal lineages after PCR amplified targets were treated with restriction enzyme digestion (PCR-RFLP). The genotyping provided the following results: 40 patients (46%) were infected with strains classified as type I; 4 (4%), as type III; 13 (15%) were infected with polymorphic strains (unusual genotype); 6 patients with type I or II alleles; and 15 (17%) patients had strains not classified for any marker. PCR-RFLP, also classified 9 (11%) clinical isolates as type II, which is uncommon in South America. However, the sequencing of the nested-PCR products (of SAG3 marker) of type II and polymorphic isolates (of 5'-SAG2, SAG3 and GRA6 markers) showed a nucleotide polymorphism compared with the archetypal clonal genotypes (types I, II and III) and these isolates were considered as polymorphic strains. The markers used here were inappropriate to distinguish the most isolates considered as polymorphic strains. These data confirm other studies showing the high rate of genetic polymorphism in T. gondii strains isolated in Brazil.     

Genetic linkage mapping in Acacia mangium 1. Evaluation of restriction endonucleases, inheritance of RFLP loci and their conservation across species

Random genomic probes were used to assess levels of restriction fragment length polymorphism (RFLP) in two 2-generation outbred pedigrees of Acacia mangium Willd. Probes were evaluated for their ability to detect polymorphic loci in each pedigree and to determine the relative efficiency of different restriction enzymes in revealing polymorphisms. Sixty two percent of the probes which detected single- or low-copy number sequences revealed polymorphisms with at least one restriction enyzme. HpaII was the most efficient in detecting polymorphism among first-generation individuals. The recognition sequence of HpaII contains a CpG dimer, suggesting that cytosines in the CpG sequence may be hotspots for mutation in plant genomes, as previously reported in bacterial and mammalian genomes. Mendelian inheritance of 230 loci was demonstrated based on single-locus segregation in second-generation individuals. Less than 5% of loci showed evidence of segregation distortion. The proportion of fully informative loci (15%) was lower than previously reported in eucalypts reflecting the lower level of genetic diversity in A. mangium. The RFLP probes are suitable for the construction of a high-density genetic linkage map in A. mangium. Cross-hybridisation of the A.mangium RFLPs to DNA from species representing the three subgenera of the genus Acacia indicates that these markers could be used in breeding programs of other diploid acacias, for comparative studies of genome organisation, and for phylogenetic studies. Received: 5. June 1999 / Accepted: 30 July 1999     

Highly polymorphic region of the human prothrombin (F2) gene

Summary We have found a highly polymorphic region in the human prothrombin gene. Our sequence differed from that previously reported at as many as 6 positions in a 225-bp stretch spanning exon 6 and its flanking regions; four of these positions were related to endonuclease restriction sites for AluI, HpaII(MspI), MboII, and NcoI. AluI and HpaII digested all alleles of the Japanese tested. MboII and NcoI restriction fragment length polymorphisms are highly heterozygous and not in linkage disequilibrium; they thus serve as good human DNA markers     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells

Strains of species in the Bacillus cereus group are potentially enterotoxic. Thus, the detection of all B. cereus group strains is important. As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the B. cereus group, we explored the potential of the groEL gene as a phylogenetic marker. A phylogenetic analysis of the groEL sequences of 78 B. cereus group strains revealed that the B. cereus group strains were split into two major clusters, one including six B. mycoides and one B. pseudomycoides (cluster II) and the other including two B. mycoides and the rest of the B. cereus group strains (cluster I). Cluster I was further differentiated into two subclusters, Ia and Ib. The sodA gene sequences of representative strains from different clusters were also compared. The phylogenetic tree constructed from the sodA sequences showed substantial similarity to the tree constructed from the groEL sequences. Based on the groEL sequences, a PCR assay for detection and identification of B. cereus group strains was developed. Subsequent restriction fragment length polymorphism (RFLP) analysis verified the PCR amplicons and the differentiation of the B. cereus group strains. RFLP with MboI was identical for all the B. cereus group strains analyzed, while RFLP with MfeI or PstI classified all B. cereus and B. thuringiensis strains into two groups. All cluster II B. mycoides and B. pseudomycoides strains could be discriminated from other B. cereus group bacteria by restriction analysis with TspRI.     

The T4336C Variant Is a Marker of Mitochondrial Subhaplogroup H1, a Common Component of the Russian and German Gene Pools

Analysis of mitochondrial DNA (mtDNA) restriction polymorphism carried out in a sample of Russians from Magadan (n= 150) showed that the frequency of the +4332AvaII variant (a T–C transition at nucleotide position 4336) in this population was 4.7%. All +4332AvaII types of mtDNA belonged to the mitochondrial group H. They were characterized by a back of the AluI restriction endonuclease site at position 7025. According to hypervariable segment 1 sequencing data, they contained the 16304C variant, and thus belong to the subgroup H1. Thus, the +4332AvaII (T4336C) variant is a marker of the mitochondrial subgroup H1, chiefly occurring in German-speaking populations. Utilization of the H1-mtDNA markers for the investigation of the genetic history and the origin of Slavs is discussed.