DNA Banding Patterns Research Articles

Definitive Differentiation between Single and Mixed Mycobacterial Infections in Red Deer (Cervus elaphus) by a Combination of Duplex Amplification of p34 and f57 Sequences and Hpy188I Enzymatic Restriction of Duplex Amplicons

Severe emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis infection due to gross and microscopic similarities with lesions caused by Mycobacterium avium subsp. paratuberculosis or M. avium subsp. avium. The aim of this study was to improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiated M. bovis from M. avium subsp. paratuberculosis and M. avium subsp. avium infections, whereas the restriction step differentiated single M. avium subsp. paratuberculosis or M. avium subsp. avium infections from mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in all M. avium subsp. avium strains tested, regardless of their origin and the results of IS901 PCR. In contrast, the restriction site was abrogated in all M. avium subsp. paratuberculosis strains tested, independent of their origin, Mycobactin J dependency, and IS900 PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to exclude M. bovis infection and to identify single (M. avium subsp. paratuberculosis or M. avium subsp. avium) or mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of M. avium subsp. paratuberculosis, the absence of the Hpy188I restriction site from the us-p34 amplicon.

The occurrence of rare rpoB mutations in rifampicin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait

Mutations associated with rifampicin (RIF) resistance in two regions of the rpoB gene were studied by line probe (INNO-LiPA Rif. TB) assay (LiPA) and/or DNA sequencing in 32 RIF-resistant and 21 RIF-susceptible Mycobacterium tuberculosis strains isolated from 53 tuberculosis patients in Kuwait. The LiPA identified all susceptible strains as RIF sensitive, and the DNA sequences of the 81 bp rifampicin resistance-determining region (RRDR) and the N-terminal region of the rpoB gene from five isolates were identical to the wild-type sequences from M. tuberculosis H 37Rv. The LiPA identified 24 of 32 (75%) phenotypically documented RIF-resistant M. tuberculosis isolates as RIF resistant, with specific detection of mutation in 19 isolates, whilst 8 strains were identified as RIF susceptible. DNA sequencing of the RRDR confirmed the results for the 19 RIF-resistant isolates and identified precise mutations in five isolates for which specific base changes could not be determined by LiPA, as well as identifying a mutation (insertion 514TTC) in three isolates and no mutation in five of the eight isolates that were detected as RIF susceptible by LiPA. Two of the latter five isolates contained a V146F mutation in the N-terminal region of the rpoB gene and were recovered from patients of Middle Eastern origin. These analyses identified 11 different mutations in two regions of the rpoB gene. The majority of the isolates carrying identical or no mutations in the rpoB gene exhibited unique DNA banding patterns in fingerprinting studies. The occurrence of rare mutations in the rpoB gene, namely insertion 514TTC in 3 (9%) and V146F in 2 (6%) of 32 RIF-resistant M. tuberculosis isolates from Kuwait, is the highest reported so far.

Morphological and Molecular Characterization of Common Bean Landraces and Cultivars from the Caribbean

Little information is available regarding the relationship of Caribbean bean landraces with the Andean and Mesoamerican gene pools of common bean (Phaseolus vulgaris L.) While small‐seeded Mesoamerican black beans are prevalent in parts of the Caribbean, many of the regionally preferred red mottled and medium‐to‐large‐seeded bean landraces found there are postulated to belong to the Andean gene pool. To test this hypothesis, we analyzed the morphological characteristics, phenological traits, phaseolin status, and random amplified polymorphic DNA (RAPD) banding patterns of 54 red mottled or large‐seeded bean landraces and cultivars from the Caribbean (16 from the Dominican Republic, 14 from Haiti, 1 from Jamaica and 23 from Puerto Rico) compared with 11 Andean bean lines from other regions. To estimate phylogenetic relationships among the lines, distances were calculated and dendrograms constructed for morphological and/or phenological and molecular characteristics by the complete clustering method. The landraces were grouped into two clusters morphologically: one with Mesoamerican characteristics, which included all the red mottled lines from Haiti and three landraces from the Dominican Republic collected near the Haitian border and the other with Andean characteristics, which included all the lines from Puerto Rico and the remaining lines from the Dominican Republic. RAPD and phaseolin polymorphisms identified three groups, one corresponding to the genotypes with Mesoamerican morphologies, one to those with Andean morphologies, and another that had Andean phenotypes but proximity to the Mesoamerican group, suggesting possible introgression between the gene pools. Phaseolin variability agreed with other molecular and morphological data allowing the assignment of genotypes to a Mesoamerican or Andean gene pool. Knowledge of this variability may provide useful information concerning the potential value of Caribbean landraces to Andean bean breeding programs.

New Anastomosis Groups, AG-T and AG-U, of Binucleate Rhizoctonia spp. Causing Root and Stem Rot of Cut-Flower and Miniature Roses

ABSTRACT Root and stem rot of cut-flower roses (Rosa spp.) was observed in commercial glasshouse-grown roses in 10 prefectures of Japan from 1998 through 2001. Binucleate-like Rhizoctonia spp. were isolated mainly from the disease plants. In all, 670 isolates were divided into two types based on cultural appearance; 168 isolates of light brown to brown type and 502 isolates of whitish type. A hyphal anastomosis reaction using representative isolates from each type revealed that the light brown to brown type belonged to anastomosis group G (AG-G), whereas the whitish type (AG-CUT) failed to anastomose with tester strains of binucleate Rhizoctonia AG-A through AG-S. Neither isolates of AG-G nor AG-CUT anastomosed with tester strains of a previously reported unknown AG (AG-MIN) of binucleate Rhizoctonia spp. collected from miniature roses. In pathogenicity tests, randomly selected isolates of the three groups caused root and stem rot on cut-flower and miniature roses. To differentiate AG-CUT and AG-MIN from known AGs of binucleate Rhizoctonia spp., restriction fragment length polymorphism (RFLP) and sequence analyses of a ribosomal (r)DNA internal transcribed spacer (ITS) region were conducted. Among the eight restriction enzymes used, HaeIII produced DNA banding patterns for AG-CUT that differed from those of tester strains and AG-MIN. Additionally, restriction profiles of AG-MIN differed from those of all tester strains. AG-G isolates from cut-flower roses had the same RFLP pattern as the tester strains of AG-G. Based on the results of hyphal anastomosis and RFLP and sequence analysis of an rDNA-ITS region, we propose that AG-CUT be designated AG-T and AG-MIN be designated AG-U, two new AGs of binucleate Rhizoctonia spp. The phylogenetic tree based on the sequence data of the rDNA-ITS region showed that isolates of AG-MIN were in a distinct clade from other AGs, whereas isolates of AG-CUT were in the same clade as those of AG-A. More detailed phylogenetic analysis besides rDNA-ITS region might be necessary for AG classification of binucleate Rhizoctonia spp.

First Report of Gray Mold in Tomato Caused by Botrytis cinerea in Baja California, Mexico.

San Quintin Valley, a 60-mile-long coastal plain (30°30'N, 116°W) in the Baja California Peninsula, is one of the major fresh tomato (Lycopersicon esculentum Mill.) production areas in Mexico with more than 8,000 ha. During the last 10 years, the valley's tomato production has declined because of gray mold and stem canker diseases. Flower rot, reddish brown margins on the leaves and stems, and fruit with a gray mold were observed on field-grown tomato plants (Roma type cv. Tequila) in the autumn of 2003. Severity ranging from 55 to 60% was observed at harvest. Infected tissues were sampled and disinfested by immersion in 1% NaOCl for 1 min, rinsed in sterile water, and placed on malt extract agar at 22°C. Fungal conidia were then transferred to 2% potato dextrose agar (PDA). The resulting fungal colonies were definitively identified as Botrytis cinerea Pers.:Fr. The colonies of B. cinerea were first hyaline and white and became dark gray after 96 h. Mycelia were septate with dark branched conidiophores. Conidia were unicellular, ellipsoid, and ranged from 5 to 8 × 8 to 14 μm. Profuse black sclerotia developed in 7-day-old cultures. Infection site analyses in diseased flowers at different stages during the bloom were done with scanning electron microscopy. Fungal hyphae were located predominantly on the receptacle areas, whereas conidia were located in the ovaries as described previously (3). The identity of B. cinerea was confirmed by a restriction digest with ApoI of the 413-kb polymerase chain reaction amplification product obtained with BA2f/BA1r primers (1) and random amplified polymorphic DNA banding patterns (2). Pathogenicity tests were done by spray inoculation of 1-ml aqueous conidial suspension (106 CFU/ml) on 20 healthy plants during the blossom stage. An equal number of plants sprayed with sterile water was used as the control. Plants were incubated at 20 ± 2°C for 5 days. The fungus was reisolated from diseased flowers and peduncles after surface disinfestation (2.5% NaOCl) and plating on PDA. No symptoms were observed in the noninoculated controls. To our knowledge, this is the first report of B. cinerea causing gray mold disease on tomato in Baja California.

Utilization of intergeneric somatic hybrids as an index discriminating taxa in the genus Citrus and its related species

Embryogenic protoplasts of ‘Shogun’ mandarin (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts from Citropsis gabunensis Swing. & M. Kell, and two green embryoids were regenerated after 3 months of culture. Two months after transfer to the regeneration medium, numerous plantlets were obtained from the embryoids. These plants grew vigorously, had well-developed root systems, and exhibited leaf characteristics intermediate to those of the parents. The absolute nuclear genome size of the regenerated plant SH2 (1.75 pg/4C) was the sum of those of the ‘Shogun’ mandarin (0.75 pg/2C) and C. gabunensis (0.97 pg/2C). The chromosome counts of the young leaves revealed that they were tetraploids (2n=4x=36). Random amplified polymorphic DNA (RAPD) analysis of the two lines (SH1 and SH2) verified their hybridity. Cytoplasmic genome analysis using universal primers reveal that their chloroplast (cp) DNA banding pattern is identical to that of C. gabunensis, while the banding pattern of the mitochondrial (mt) DNA is identical to that of the ‘Shogun’ mandarin. These somatic hybrids are important materials for investigating the phylogenetic relationships between these two genera in the subfamily Aurantioideae.

In vitro mutation and selection of doubled-haploid Brassica napus lines with improved resistance to Sclerotinia sclerotiorum

This paper describes a new protocol to develop doubled-haploid (DH) Brassica napus lines with improved resistance to Sclerotinia sclerotiorum. In this protocol, haploid seedlings derived from microspore cultures of B. napus were used to produce haploid calli for in vitro mutation-selection. For routine screening, mutation was induced by EMS (ethylmethane sulfonate) or occurred spontaneously, and screening for resistant mutants occurred on media with added oxalic acid (OA) as a selection agent. In tests with selected lines, the optimal concentration of EMS for mutation was determined to be 0.15%, and the optimal concentration of OA for in vitro screening was 3 mmol/l (half lethal dose was 3.1 mmol/l) for the first cycle of screening. There was an accumulated effect of OA toxicity on calli over two cycles of screening, but the growth and capacity of the surviving calli for regenerating seedlings were not affected by OA. Of the 54 DH lines produced from the in vitro mutation-selection, two DH lines of resistant mutants, named M083 and M004, were selected following seedling and glasshouse tests. The resistance of M083 and M004 to S. sclerotiorum following tests with both mycelial inoculum and OA was greater than that of their donor lines and the resistant control Zhongyou 821. In both glasshouse and field disease nurseries, disease indices on M083 and M004 were less than 50% of those of the control. The time required for M083 and M004 to mature was 14 days and 10 days shorter, respectively, than that of their donor lines. Furthermore, M083 had more pods per inflorescence, a greater 1,000 seed weight and higher yield than its donor line. Random amplified polymorphic DNA characterisation showed that M083 had DNA band patterns that differed from its donor line.

Clustering of Brown Planthopper Biotype Based on RAPD-PCR

Random amplified polymorphic DNA (RAPD) technique was used to differentiate two brown planthopper biotypes, that were multiplied in the greenhouse. Ten selected random decamer RAPD primers produced unique DNA band patterns for each individual brown planthopper. However, no single primer produced DNA band that could differentiate the two brown planthopper biotype populations. Nonetheless, analysis of the DNA band patterns was able to cluster the majority of the individual samples according to their respective biotypes. Molecular data analysis also indicated greater genetic variation within biotype population than among biotypes.

Pengelompokan Biotipe Wereng Cokelat Berdasarkan Hasil PCR-RAPD

Random amplified polymorphic DNA (RAPD) technique was used to differentiate two brown planthopper biotypes, that were multiplied in the greenhouse. Ten selected random decamer RAPD primers produced unique DNA band patterns for each individual brown planthopper. However, no single primer produced DNA band that could differentiate the two brown planthopper biotype populations. Nonetheless, analysis of the DNA band patterns was able to cluster the majority of the individual samples according to their respective biotypes. Molecular data analysis also indicated greater genetic variation within biotype population than among biotypes.

Outbreaks of Infection Caused by Community‐Acquired Methicillin‐Resistant Staphylococcus aureus in a Canadian Correctional Facility

Methicillin-resistant Staphylococcus aureus (MRSA) has been identified in prison settings in the United States. The present study investigated two clusters of skin and soft tissue infection caused by community-acquired (CA) MRSA in a correctional facility in southern Ontario. Outbreak investigations were conducted by the responsible public health authority. Strain relatedness was assessed through comparison of pulsed-field gel electrophoresis and antibiograms. Two distinct outbreaks of CAMRSA-associated disease occurred in 2002 and 2004. Most patients presented with abscesses in the lower extremities. All isolates had identical DNA banding patterns on pulsed-field gel electrophoresis. One-half of the affected inmates resided in a cellblock with one other affected inmate. No other risk factors were identified. One of the first outbreaks of CAMRSA infections in a correctional facility in Canada is documented. Taken in conjunction with outbreaks elsewhere, this suggests that residence in correctional facilities may be a risk factor for CAMRSA infection.

Detection of mitochondrial DNA mutations using temporal temperature gradient gel electrophoresis.

Mitochondrial disorders are a group of clinically and genetically heterogeneous diseases. Common recurrent mitochondrial DNA (mtDNA) point mutations account for the molecular defects of a small proportion of patients. In order to identify mtDNA mutations, comprehensive mutational analysis of the entire mitochondrial genome is necessary. We developed the temporal temperature gradient gel electrophoresis (TTGE) method to screen for mutations in mtDNA. The entire mitochondrial genome was amplified using 32 pairs of overlapping primers followed by TTGE analysis of the DNA fragments. TTGE method was first validated on 200 DNA fragments containing known mutations or polymorphisms. On TTGE, homoplasmic nucleotide substitutions show a single band shift and heteroplasmic mutations show multiple banding patterns. The known mutations or polymorphisms were correctly identified. TTGE was then used to screen for unknown mutations in the mitochondrial genome. DNA banding patterns, deviated from wild-type, suggestive of either homoplasmic or heteroplasmic mutations, were followed by direct DNA sequencing to identify the mutations. Numerous mutations and polymorphisms were detected. The results demonstrated that TTGE detects and distinguishes heteroplasmic mutations from homoplasmic polymorphisms. It also detects heteroplasmic changes in the background of a homoplasmic polymorphism. Overall, TTGE was proven to be a simple, rapid, sensitive, and effective mutation detection method.

A survey of the composition and diversity of bacterial populations in bleached kraft pulp-mill wastewater secondary treatment systems.

Bacterial community compositions from 10 pulp- and paper-mill treatment systems were compared using both traditional and molecular techniques. 16S-RFLP (Random Fragment Length Polymorphisms) analysis was used to examine the genotypic profiles of the whole bacterial community of each treatment system. Although all the communities shared approximately 60% of their DNA band pattern, as determined by computer-assisted cluster analysis, each community displayed a unique profile that was stable over time under normal operating parameters. Reverse Sample Genome Probing (RSGP) and 16S-RFLP were used to compare the culturable bacterial communities of several geographically separated pulp-mill biotreatment system communities. There was little overlap in the composition of the culturable community between mills at the genus level. Furthermore, RSGP variation was almost as high within a mill as between mills. Partial sequences of the 16S rRNA genes from culturable isolates identified Bacillus spp., Pseudomonas spp., and Xanthobacter as some of the dominant species. Finally, several 16S rRNA genes from two whole community 16S RNA gene libraries were partially sequenced and identified as similar to unknown alpha-, beta-, and gamma-Proteobacteria, Ralstonia, Alcaligenes, Nitrospira, Firmicutes, and clones representing the new Holophaga/Acidobacterium phylum. These findings suggest that although these pulp- and paper-mill biotreatment communities perform similar functions, they are populated by unique mixtures of species.

GISH, AFLP and PCR-RFLP analysis of an intergeneric somatic hybrid combining Goutou sour orange and Poncirus trifoliata.

Intergeneric somatic hybrids combining Goutou sour orange (Citrus aurantium L.) with trifoliate orange Poncirus trifoliata (L.) Raf] were produced by electrofusion and their genetic inheritance analyzed by amplified fragment length polymorphism (AFLP), genomic in situ hybridization (GISH), and PCR-restriction fragment length polymorphism (PCR-RFLP). Sixteen mini-calluses were obtained after 20 days of culture; they all developed into embryoids on EME500 medium. Following several subcultures on shoot induction medium for a total culture period of 6 months, shoots regenerated. The plants grew vigorously with a well-developed root system and exhibited the trifoliate leaf character of P. trifoliata. Ploidy analysis verified that all of the regenerates were tetraploids (2 n=4 x=36) as expected. GISH analysis confirmed that 18 chromosomes came from trifoliate orange and the remaining 18 from Goutou sour orange, as with most symmetric somatic hybrid plants; moreover, chromosome translocations were also observed in one plant. AFLP analysis of 16 regenerates and their fusion parents indicated that all of the somatic hybrids except one were genetically uniform. Analysis of the somatic hybrid cytoplasmic genomes with universal primers revealed that their chloroplast DNA (cpDNA) banding patterns were identical to those of the mesophyll parent trifoliate orange, while their mitochondria (mt) genomes were of the callus parent sour orange. The potential of GISH in Citrus somatic hybrid analysis is discussed.

Which genetic marker for which conservation genetics issue?

Conservation genetics focuses on the effects of contemporary genetic structuring on long-term survival of a species. It helps wildlife managers protect biodiversity by identifying a series of conservation units, which include species, evolutionarily significant units (ESUs), management units (MUs), action units (AUs), and family nets (FNs). Although mitochondrial DNA (mtDNA) evolves 5-10 times faster than single-copy nuclear DNA (scnDNA), it records few traces of contemporary events. Thus, mtDNA can be used to resolve taxonomic uncertainties and ESUs. Variable number of tandem repeats (VNTRs) evolve 100-1000 times faster than scnDNA and provide a powerful tool for analyzing recent and contemporary events. VNTR analysis techniques include polymerase chain reaction (PCR)-based microsatellite assays and oligonucleotide probing. Size homoplasy problems in PCR-based microsatellite assays can strongly affect the inference of recent population history. The high homozygosity in endangered species is reflected in a relatively low number and level of variability in microsatellite loci. This combined with "allelic dropout" and "misprinting" errors contributes to the generation of highly biased genetic data following analyses of natural populations. Thus, in conservation genetics, microsatellites are of limited use for identifying ESUs, MUs, and AUs. In contrast to PCR-based microsatellite analysis, oligonucleotide probing avoids errors resulting from PCR amplification. It is particularly suitable for inferring recent population history and contemporary gene flow between fragmented subpopulations. Oligonucleotide fingerprinting generates individual-specific DNA banding patterns and thus provides a highly precise tool for monitoring demography of natural populations. Hence, DNA fingerprinting is powerful for distinguishing ESUs, MUs, AUs, and FNs. The use of oligonucleotide fingerprinting and fecal DNA is opening new areas for conservation genetics.

Application of PLS and Back-Propagation Neural Networks for the estimation of soil properties

Two different multivariate calibration methods, Partial Least Squares (PLS) and Back-Propagation Neural Networks (BP-ANN), were applied to microbial community DNA to predict soil properties (%Sand, %Silt, %Clay, %Nitrogen, %Organic Carbon, DNA) in environmental soil samples. The microbial community DNA was extracted from 48 environmental soil samples. After amplification of bacterial ribosomal RNA by polymerase chain reaction (PCR), the products were separated by gel electrophoresis. Characteristic complex band patterns were obtained, indicating high bacterial diversity. Two hundred and fifty-six DNA band patterns were used to predict the soil properties. Based on the brightness of the bands, densitometric curves of DNA band patterns were extracted from the gel images. The curves were smoothed using the Savitsky–Golay method and scaled to DNA standard markers. The predictive powers of the two methods (PLS and BP-ANN) are presented and compared. The Back-Propagation Neural Networks method combined with principal component analysis (PCA) was found to have the most predictive power with the independent test set.

Genetic identification and host range of the Spanish strains of Echinococcus granulosus

Three strains of Echinococcus granulosus have been previously identified in Spain (namely ‘sheep’, ‘horse’ and ‘pig’), but these Spanish strains have not been properly genotyped yet. The aim of the present research was to identify the genotype to which they correspond to. Cyst isolates were obtained from different host species, and the strain to which each belonged was established by analysis of its random amplified polymorphic DNA (RAPD) banding patterns. These results were compared to those obtained with the analysis of two mitochondrial fragment sequences (cytochrome oxidase 1 (CO1) and NADH dehydrogenase 1 (ND1)) from each isolate. The Spanish ‘sheep’ strain corresponded with the genotype 1 (G1) of the parasite, infecting Spanish sheep, cattle, goat, pig, wild boar and human; the Spanish ‘horse’ strain corresponded with the genotype 4 (G4), only infecting Spanish horses; and the Spanish ‘pig’ strain corresponded with the genotype 7 (G7), infecting Spanish goat, pig and wild boar. Goat, pig and wild boar can be infected by two genotypes, G1 and G7. This circumstance, and especially the possibility of sylvatic intermediate hosts serving as reservoirs of the G1 genotype of the parasite, must be taken into consideration by authorities in order to develop and evaluate effective anti-hydatidosis programmes.

Chamber with modifiable inner width for performing transversal alternating field electrophoresis in variable numbers of minigels.

We present a transversal alternating field electrophoresis chamber that allows modifiable inner widths to accommodate low- or high-throughput formats, with 7.8 cm opposite electrode separation and 30.4 cm electrode length. Removable slotted sheets divide the chamber into four smaller compartments, each one supporting a minigel of 3.85 cm in length and 7.1 cm in width. Replacements of slotted sheets with solid dielectric blocks with the sizes and shapes of compartments permit to occlude chamber compartments, changing from 4 to 1 the numbers of minigels per run, from 88 to 13 the maximum numbers of samples, and from 1688 to 422 mL the volume of buffer poured into the chamber. Saccharomyces cerevisiae chromosomes gave its characteristic DNA band pattern in all compartments, whereas migrations of DNA molecules are not affected by the occlusion of compartments.

Multiplication and germination of somatic embryos of American ginseng derived from suspension cultures and biochemical and molecular analyses of plantlets

Seedlings from 11 seed sources (lines) of American ginseng from different geographic regions were evaluated on Murashige and Skoog medium (MS) containing 10 μM α-naphthaleneacetic acid (NAA) and 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for callus development and somatic embryo formation. Leaf and stem explants callused at a frequency of 18.2–100%, while somatic embryos were produced from these calluses at a frequency of 25–87.5% after 5 mo. Suspension cultures of nine lines were established by transferring embryogenic callus to MS liquid medium with NAA and 2,4-D at 2.5 and 2.25 μM, respectively, and maintained by subcultures every 8 wk. Globular somatic embryos from these cultures were germinated on half-strength MS containing 1% activated charcoal, and roots >5 mm in length developed within 3 wk. A 7-d exposure to 3 μM gibberellic acid and 5 μM 6-benzylaminopurine significantly enhanced shoot development and promoted further root development. The chromosome number, profiles of the common triterpenoid saponins (ginsenosides), and random amplified polymorphic DNA (RAPD) banding patterns in plantlets derived from suspension culture were compared to those of zygotic seedlings. The chromosome number in root tip cells and suspension cultured cells was 48. Patterns of the six major ginsenosides, determined by thin-layer chromatography, in leaves of tissue culture-derived plantlets were identical to those in seedlings. RAPD patterns among plantlets originating from the same tissue-cultured line were mostly identical; however, altered patterns were observed in some lines that had been maintained in suspension culture for almost 4 yr. The results from this study indicate that propagation of desired ginseng genotypes in suspension culture can be achieved, and that biochemical and molecular markers can be used for authentication of resulting plantlets.

Contribution of AGC to ACC and other mutations at codon 315 of the katG gene in isoniazid-resistant Mycobacterium tuberculosis isolates from the Middle East

The presence of ACC and other mutations at codon 315 in the katG gene was detected by PCR amplification followed by restriction fragment length polymorphism (PCR-RFLP) generated with restriction enzymes Msp I and MspA1 I in 37 isoniazid-resistant and 22-susceptible Mycobacterium tuberculosis isolates from Kuwait obtained in 2001. The mutation AGC to ACC was detected in 22 (60%) isolates while any mutation at codon 315 of the katG gene was present in 24 (65%) of 37 isoniazid-resistant isolates. The typing studies showed that majority of the isolates carrying mutations at codon 315 exhibited unique DNA banding patterns. The results were extended by additional analysis of 67, 28 and 17 isoniazid-resistant and 18, seven and six-susceptible M. tuberculosis isolates from Kuwait, Dubai and Beirut, respectively, that were analyzed previously for ACC mutation alone. These studies showed that one of 21, one of 10 and two of 11 isolates (all recovered from patients of Middle Eastern origin) with no AGC to ACC mutation from Kuwait, Dubai and Beirut, respectively, contained other mutations at codon 315 of the katG gene. None of the susceptible strains contained any mutation at codon 315. The PCR-RFLP with MspA1 I that detects all mutations at codon 315, compared with Msp I that detects only ACC mutation, identified more isoniazid-resistant strains with mutations at codon 315 in the katG gene. The data also showed that mutations other than AGC to ACC at codon 315 in the katG gene occur frequently in M. tuberculosis isolates recovered from Middle Eastern patients and should be incorporated in a rapid screen for the detection of mutations for isoniazid-resistance in the katG gene from this ethnic group.

Morphology, genetic diversity, temperature tolerance and toxicity of Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) strains from Thailand and Japan

Cylindrospermopsis raciborskii is a planktonic, nostocalean cyanobacterium, which produces an alkaloid heptatoxin, cylindrospermopsin. We performed morphological observations, 16S rDNA sequence analysis, PCR fingerprint analysis of short tandemly repeated repetitive (STRR) sequences, temperature tolerances and toxin analysis to characterize 24 strains of this toxic cyanobacterium isolated from Thailand and Japan. All strains shared common morphological traits characteristic of C. raciborskii and showed high 16S rDNA sequence similarity, forming a defined cluster together with the reference strains from Australia. In particular, some of the Thai strains shared 99.9% to 100% similarity with the Australian strains. Various combinations of STRR primers revealed different and unique DNA band patterns among strains of C. raciborskii. The phylogenetic tree revealed two main clusters of C. raciborskii strains, the Thai/Japan-Shinobazugaike cluster (cluster I) and the Japan-Gonoike cluster (cluster II). Cluster I was further divided into two subclusters, A (only Thai strains) and B (one Thai strain and the Japan-Shinobazugaike strains). Thus, the results from 16S rDNA and STRR analyses showed no clear geographical distinction between Japanese and Thai strains and between Thai and Australian strains. Thai strains were separated into adaptive and non-adaptive groups to low temperature (15 and 17.5 °C) and Japanese strains were composed of only low-temperature-adaptive ones. The toxin cylindrospermopsin was detected in some strains of cluster I-A and in one strain of cluster II. We conclude that C. raciborskii is a species that has recently begun to invade, and a species with different physiological strains or ecotypes in temperature tolerance; the toxin is synthesized without any relation to phylogenetic or genetic clusters and to geography.