Restriction Fragment Length Polymorphism Studies Research Articles

Is There a Real Relationship between the Presence of Helicobacter pylori in Dental Plaque and Gastric Infection? A Genotyping and Restriction Fragment Length Polymorphism Study on Patient Specimens with Dyspepsia in Southwest Iran.

The oral cavity can act as an extra gastric reservoir for H. pylori, and the presence of the bacteria in the oral cavity is associated with a higher risk of dental caries development. This study aimed to determine the genotype and evaluate the association between the presence of H. pylori in dental plaque and gastric biopsy specimens in dyspeptic patients in Ahvaz, Southwest Iran. In this study, 106 patients with recruited dyspeptic complaints were selected, and from each patient, two gastric antral biopsy specimens and two dental plagues were examined. The presence of H. pylori was identified by the rapid urease test (RUT) and the amplification of ureAB and 16S rRNA genes. Also, to verify a hypothetical mouth-to-stomach infection route, the enzymatic digestions of three genes of cagA, vacA, and ureAB in H. pylori strains isolated from dental plaques and stomach samples were compared for each same case. H. pylori was found in the stomach of 52.8% (56/106) and the dental plaques of 17.9% (19/106) of the studied cases. On the other hand, H. pylori was recognized in the stomach of all 19 cases with oral colonization. Following a combination of restriction fragment lengths 21 polymorphism (RFLP) patterns of these three known genes on stomach and dental plague samples, 14 and 11 unique patterns were seen, respectively. However, for all H. pylori-positive cases (19), the comparison of RLFP patterns of these genes in dental plaque and gastric biopsy specimens was different for the same case. In this study, it seems that there is no significant association between the presence of H. pylori in dental plaque and the stomach of the same case.

Detection, Identification, and Molecular Characterization of a 16SrII-V Subgroup Phytoplasma Associated with Nicotiana plumbaginifolia.

Nicotiana plumbaginifolia Viviani, commonly known as curl-leaved tobacco, is an annual herbaceous plant belonging to Solanaceae family. This plant is native to Mexico, South America, and parts of the Caribbean and has been reported to be present in Taiwan since 2006. In March 2021, N. plumbaginifolia Viviani, found in Yunlin County, Taiwan, was observed to have phyllody, virescence, and witches'-broom, which is consistent with the disease symptoms caused by phytoplasma infection. Samples of the healthy and symptomatic plants were collected for analysis of the causal agent associated with the diseased N. plumbaginifolia Viviani. Under transmission electron microscopy, the phytoplasma-like pleomorphic bodies were found in the sieve tubes of the diseased plants. The 16S ribosomal RNA (rRNA)-based phylogenetic analysis and the iPhyClassifier-based virtual restriction fragment length polymorphism study demonstrated that the phytoplasma identified in this study can be classified into the 16SrII-V subgroup, which is similar to the peanut witches'-broom phytoplasma, a 'Candidatus phytoplasma aurantifolia'-related strain. Further identification of SAP54/PHYL1 and SAP11 homologs in the phytoplasma explain the disease symptoms of phyllody, virescence, and witches'-broom observed in diseased N. plumbaginifolia Viviani. The discovery of new phytoplasma plant hosts has gained scientific importance in light of the attempt to unravel an efficient strategy to fight the rapid spread of this disease, which poses a threat to the agricultural sector and food security in Taiwan.

Identification of 16SrII-V Phytoplasma Associated with Mungbean Phyllody Disease in Taiwan.

Mungbean (Vigna radiata (L.) R. Wilczek), an important legume crop in Asia, is primarily cultivated in the central-southern region of western Taiwan. In 2020, mungbean exhibiting typical phytoplasma-induced disease symptoms such as witches' broom, phyllody, virescence, and proliferation was observed in Yunlin County, Taiwan. Moreover, the seed harvested from diseased plants displayed premature germination. Transmission electron microscopy examination of leaf veins prepared from symptomatic mungbean demonstrated that the occlusion of sieve tubes resulted from the accumulation of phytoplasma-like bodies in sieve elements along with filament-like structures in sieve pores. The association of phytoplasma in symptomatic mungbean was confirmed by PCR analyses of the 16S ribosomal RNA (rRNA) and immunodominant membrane protein genes. Further analyses of the 16S rRNA-based phylogenetic tree and the iPhyClassifier-based virtual restriction fragment length polymorphism study demonstrated that the phytoplasma-associated mungbean phyllody disease identified in this study belongs to the 16SrII-V subgroup. BLAST analysis and the phylogenetic analysis indicated that the SAP11-like protein identified in mungbean phyllody disease is identical to peanut witches' broom phytoplasma SAP11, which explains the witches' broom phenotype observed in symptomatic mungbean. The results described in this report confirm that the 16SrII-V phytoplasma, a widely distributed phytoplasma associated with peanut witches' broom disease in Taiwan, has also infected mungbean. This is not only the first instance of mungbean phyllody disease found in Taiwan but also the first instance of mungbean phyllody disease caused by 16SrII-V subgroup phytoplasma.

The study of organelle DNA variability in alloplasmic barley lines in the NGS era.

Alloplasmic lines are a suitable model for studying molecular coevolution and interrelations between genetic systems of plant cells. Whole chloroplast (cp) and mitochondrial (mt) genome sequences were obtained by the MiSeq System (Illumina). Organelle DNA samples were prepared from a set of 12 alloplasmic barley lines with different cytoplasms of Hordeum vulgare ssp. spontaneum and H. vulgare ssp. vulgare, as well as from their paternal varieties. A bioinformatic approach for analysis of NGS data obtained on an organellar DNA mix has been developed and verified. A comparative study of Hordeum organelle genomes’ variability and disposition of polymorphic loci was conducted. Eight types of chloroplast DNA and 5 types of mitochondrial DNA were distinguished for the barley sample set examined. These results were compared with the previous data of a restriction fragment length polymorphism (RFLP) study of organelle DNAs for the same material. Formerly established data about a field evaluation of alloplasmic barley lines were revised in the light of information about organelle genomes gained after NGS. Totally 17 polymorphic loci were found at exons of chloroplast genomes. Seven of the SNPs were located in the genes of the Ndh complex. The nonsynonymous changes of nucleotides were detected in the matK, rpoC1, ndhK, ndhG and infA genes. Some of the SNPs detected are very similar in codon position and in the type of amino acid substitution to the places where RNA editing can occur. Thus, these results outline new perspectives for the future study of nuclear-cytoplasmic interactions in alloplasmic lines.

Restriction Fragment Length Polymorphism (RFLP) and Morphometric Studies of Two Morphologically Confused Crabs from Abu Quir, Alexandria, Egypt.

Restriction Fragment Length Polymorphism (RFLP) and Morphometric Studies of Two Morphologically Confused Crabs from Abu Quir, Alexandria, Egypt.

Isoliquiritigenin decreases the incidence of colitis-associated colorectal cancer by modulating the intestinal microbiota.

Imbalances in intestinal bacteria correlate with colitis-associated colorectal cancer (CAC). Traditional Chinese medicines have been used to adjust the gut microbiota, and isoliquiritigenin (ISL), a flavonoid extracted from licorice, has shown antitumor efficacy. In this study, the effects of ISL on CAC development and the gut microbiota were evaluated using an azoxymethane and dextran sulphate sodium (AOM/DSS)-induced mouse model of CAC (CACM). Histopathological analysis suggested that ISL reduced tumor incidence in vivo. Moreover, high-throughput sequencing and terminal restriction fragment length polymorphism (T-RFLP) studies of the bacterial 16S rRNA gene revealed that the structure of the gut microbial community shifted significantly following AOM/DSS treatment, and that effect was alleviated by treatment with high-dose ISL (150 mg/kg). Compared to the microbiota in the control mice (CK), the levels of Bacteroidetes decreased and the levels of Firmicutes increased during CAC development. ISL reversed the imbalance at the phylum level and altered the familial constituents of the gut microbiota. Specifically, the abundance of Helicobacteraceae increased after treatment with high-dose ISL, while the abundance of Lachnospiraceae and Rikenellaceae decreased. At the genus level, ISL reduced the abundance of opportunistic pathogens (Escherichia and Enterococcus), and increased the levels of probiotics, particularly butyrate-producing bacteria (Butyricicoccus, Clostridium, and Ruminococcus). Thus, ISL protects mice from AOM/DSS-induced CAC, and ISL and the gut microbiota may have synergistic anti-cancer effects.

Botryosphaeriaceae Species Associated With Cankers and Dieback Symptoms of Acacia mangium and Pinus caribaea var. hondurensis in Venezuela.

Several species in the Botryosphaeriaceae family cause wood stain, cankers, and dieback of trunks and branches in a wide range of forest tree species. The aim of this study was to characterize the botryosphaeriaceous fungi associated with decline symptoms observed in Acacia mangium and Pinus caribaea var. hondurensis, two economically important forest tree species grown in commercial plantations in Venezuela. Fungi isolated from symptomatic samples collected from both hosts in commercial sites were identified based on their morphology and DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2) and part of the β-tubulin and translation elongation factor 1-α genes. Lasiodiplodia theobromae and L. venezuelensis were routinely isolated from A. mangium and P. caribaea var. hondurensis. Additionally, the novel species Diplodia guayanensis was isolated and characterized from symptomatic and asymptomatic tissues of A. mangium. Multigene phylogenetic analyses along with restriction fragment length polymorphism studies further supported the identification of these species. A pathogenicity study was conducted under natural conditions and 12 weeks after inoculation all Botryosphaeriaceae spp. were shown to be highly virulent on A. mangium. Contrary, no lesions were observed in the wood of P. caribaea var. hondurensis when inoculated with L. theobromae and L. venezuelensis. However, both species were consistently reisolated from the asymptomatic tissue beyond the inoculation point. This study contributes to a better understand the role that species in the Botryosphaeriaceae play on disease symptoms and dieback of A. mangium and P. caribaea var. hondurensis from plantations in eastern Venezuela.

Assessment of Genetic Variability in the Coding Sequence of Melatonin Receptor Gene (MTNR1A) in Tropical Arid Sheep Breeds of India.

Seasonal behaviour in sheep, which varies in tropical and temperate environmental conditions, is a matter of study, because it can provide a clue to address the problem of seasonality in sheep. Melatonin receptor is the membrane-bound G-coupled receptor, sensing the message of photoperiodic cues thorough melatonin. Restriction fragment length polymorphism (RFLP) studies were carried out to assess the variability of gene at G612A and C606T SNPs in MTNR1A gene, which have been studied to be markers for out-of-season breeding. Allelic frequency distribution corresponded to higher frequency of GG and CC genotype, in tropical arid sheep breed in comparison with temperate region sheep breed. PCR amplification of MTNR1A gene of 30 animals was performed and single nucleotide polymorphisms (SNPs) identification was carried out using Lasergene software. Seven SNPs/mutations were identified, but most of them were synonymous, except the one G706A, leading to substitution of valine by isoleucine. Polyphen-2 analysis of G706A mutation revealed that it is a benign mutation. Two important SNPs C426T and G555A, which were identified in temperate sheep breeds, could not be traced in Magra and Marwari breeds of sheep. Thus, the Magra and Marwari breeds of tropical, arid region demonstrated the presence of both polymorphic SNPs markers G612A and C606T, associated with out-of-season breeding. GG and CC genotypes were having a higher prevalence in the studied population.

Phylogenetic diversity within the endemic brown trout Duero lineage: implications for conservation and management

Brown trout display great phenotypic and genetic variability. Use of mitochondrial DNA (mtDNA) variation has allowed the definition of seven different lineages in this species to date. One of them, the Duero (DU) lineage, was initially detected in the inner section of the Duero River in Spain, where it showed a parapatric distribution with the more widely distributed Atlantic (AT) lineage. Later mtDNA-RFLP (restriction fragment length polymorphism) studies detected the DU lineage in northern Spanish basins (Galicia). The aim of this work was to ascertain the origin and variability of these DU populations outside the Duero drainage. Using complete mtDNA control region sequencing, 11 novel DU haplotypes were identified. Several of them could be assigned to an endemic group in Galicia consistent with the long-time presence of the DU lineage outside the Duero River, and excluding a recent origin by human translocations. The DU haplotype group observed in north-western Iberian basins was estimated to diverge from that of the Duero River more than 100000 years ago. We therefore advocate for conservation strategies at regional and local scales rather than focussed in a single ESU as proposed in earlier works.

Transforming Growth Factor-Beta3 Gene Polymorphisms and Nonsyndromic Cleft Lip and Palate Risk: A Meta-Analysis

To investigate the association between transforming growth factor-beta3 (TGF-β3) genetic polymorphisms and nonsyndromic cleft lip and palate (NSCLP) risk. An extensive literature search for relevant studies was conducted on PubMed, Embase, Web of Science, Cochrane Library, and CBM databases from their inception through June 1st, 2013. Case-control studies addressing the correlation between TGF-β3 gene polymorphisms and NSCLP risk. The genotype distribution of the controls should conform to Hardy-Weinberg equilibrium. The quality of the included studies was assessed independently by two authors based on the Newcastle-Ottawa scale. All analyses were calculated using the STATA 12.0 software. The association between TGF-β3 gene polymorphisms and NSCLP risk was assessed. Eleven case-control studies were included with a total of 1601 NSCLP cases and 1463 healthy controls. Our meta-analysis results indicated that mutant variants of the TGF-β3 gene may be associated with an increased risk of NSCLP, especially among Asian populations. Further subgroup analyses also revealed significant associations between mutant variants of the TGF-β3 gene and an increased risk of NSCLP in the population-based and polymerase chain reaction-restriction fragment length polymorphism studies. Meta-regression analyses showed that ethnicity may be a major source of heterogeneity. Our meta-analysis suggests that TGF-β3 gene polymorphisms may contribute to NSCLP susceptibility, especially among Asian populations.

Genetic variability in Grapevine virus A from Vitis vinifera L. × Vitis labrusca L. in Sichuan, China

Grapevine virus A (GVA) has a quasispecies nature and is closely associated with the rugose-wood disease complex of Vitis vinifera L. In the different growing regions of table grape in Sichuan, southwest China, the incidence of GVA infection was found to be 16.9% by ELISA detection among 178 grape plants (Vitis vinifera L. × Vitis labrusca L.). The results of restriction fragment length polymorphism (RFLP) studies on the polymerase chain reaction (PCR) products of a total of 139 plasmids, cloned from 15 GVA isolates, suggested that the GVA isolates contained highly divergent variants. The plasmids from each GVA isolate that yielded different PCR-RFLP profiles were preferentially chosen for sequencing and were designated as variants. Furthermore, a phylogenetic study based on the analysis of the GVA coat protein genes and RNA silencing suppressor genes showed that the 40 variants obtained from the 15 GVA isolates represented 4 clades, designated as molecular groups I, II, III, and IV, respectively. The variant-specific PCR detection results indicated that the parts of the tested grape plants were specifically infected by mild variants.

Impact of 5,10-methylenetetrahydrofolate reductase gene polymorphism on neural tube defects

Neural tube defects (NTDs) are among the most common congenital malformations worldwide. Their etiology and exact mechanisms of development are incompletely understood. Many enzymes involved in folate metabolism and the genes encoding these enzymes have been studied as candidates in their etiology. A mutation in the methylenetetrahydrofolate reductase (MTHFR) gene--a C-->T transition at nucleotide 677--is one among them. The mutation results in substitution of alanine by valine at a functionally important site in the enzyme. It has been shown to be a risk factor for development of NTDs in certain populations. The present study was conducted to evaluate the role of MTHFR 677 C-->T mutation as a risk factor for NTD in the South Indian population and to determine the relative importance of the genotypes in the affected child and its mother. Blood samples were collected from the test and the control groups. The test group consisted of children with NTDs and their mothers, while the control group consisted of apparently healthy controls. MTHFR C677T polymorphism in the 3 groups was determined by polymerase chain reaction and restriction fragment length polymorphism studies. Comparison of polymorphism in the 3 groups was using the chi-square test. There was a significant difference in the prevalence of MTHFR 677 C-->T mutation among the 3 groups (p = 0.002). The risk conferred by the TT genotype in the child was statistically significant (OR 12.625, 95% CI 1.430-111.465). In the mothers, however, although there was an increased prevalence of the mutation compared with the control individuals, the difference was not statistically significant (p = 0.152). The MTHFR 677TT genotype is considered to be a definite risk factor for development of NTDs. It is the TT genotype status of the developing embryo, rather than the TT genotype status of its mother, that is the critical genetic determinant of MTHFR-related NTD risk.

Unusual clinical and immunologic manifestations of transplacentally acquired maternal T cells in severe combined immunodeficiency

The persistence of transplacentally transferred maternal T cells is common in infants with severe combined immunodeficiency (SCID), occurring in more than half of patients with SCID undergoing transplantation at our institution. These T cells respond poorly to mitogens in vitro but can cause cutaneous graft-versus-host disease; however, other effects of these cells are unknown. We describe 2 infants with SCID who had unusual problems associated with transplacentally transferred maternal T cells. Patient 1 was a 5-month-old girl with Janus kinase 3-deficient SCID who had 4% circulating CD3(+) T cells but no lymphocyte proliferative response to mitogens. Although the number of T cells increased after 2 nonchemoablated, T cell-depleted, haploidentical, paternal bone marrow transplantations, T-cell function failed to develop, and she became pancytopenic. Restriction fragment length polymorphism studies of flow cytometry-sorted blood T cells revealed all to be of maternal origin. A subsequent nonchemoablated, T cell-depleted maternal transplantation resulted in normal T-cell function and marrow recovery. Patient 2 was a 9-month-old girl with IL-7Ralpha-deficient SCID who presented with autoimmune pancytopenia. She had 8% blood T cells (all CD45RO(+)) but no response to mitogens. High-resolution HLA sequence-specific priming typing detected both maternal haplotypes, indicating the presence of maternal cells. Her pancytopenia resolved after treatment with rituximab and was thought to be due to host B-cell activation by transplacentally acquired maternal T cells. Persistent transplacentally acquired maternal T cells in infants with SCID can mediate immunologic functions despite failing to respond to mitogens in vitro. We present evidence that these cells can cause allograft rejection and immune cytopenias.

Genetic and Morphological Diversity of Temperate and Tropical Isolates of Phytophthora capsici

ABSTRACT Phytophthora capsici is a diverse species causing disease on a broad range of both temperate and tropical plants. In this study, we used cultural characteristics, amplified fragment length polymorphism (AFLP), and DNA sequence analyses of the ribosomal internal transcribed spacer (ITS) region and mitochondrial cytochrome oxidase II (cox II) genes to characterize temperate and tropical isolates from a wide range of host species. All but one temperate isolate grew at 35 degrees C, while all tropical isolates did not. All but two tropical isolates formed chlamydospores, while temperate isolates did not. There was strong bootstrap support for separation of temperate and tropical isolates using AFLP analysis; however, the temperate isolates appeared as a subgroup within the observed variation of the tropical isolates. The majority of temperate isolates clustered within a single clade with low variation regardless of host or geographical origin, while the tropical isolates were more variable and grouped into three distinct clades. Two clades of tropical isolates grouped together and were affiliated closely with the temperate isolates, while the third tropical clade was more distantly related. Phylogenetic analysis of the ITS regions resulted in similar groupings and variation within and between the temperate and tropical isolates as with the AFLP results. Sequence divergence among isolates and clades was low, with more variation within the tropical isolates than within the temperate isolates. Analysis of other species revealed shorter branch lengths separating temperate and tropical isolates than were observed in comparisons among other phylogenetically closely related species in the genus. Analysis of cox II sequence data was less clear. Although the temperate and tropical isolates grouped together apart from other species, there was no bootstrap support for separating these isolates. Restriction fragment length polymorphism (RFLP) analysis of the ITS regions separated the temperate and tropical isolates, as in the AFLP and ITS phylogenetic analyses. However, RFLP analysis of the cox I and II gene cluster did not distinguish between temperate and tropical isolates. The differences in grouping of isolates in these two RFLP studies should be helpful in identifying isolate subgroups. Our data do not fully clarify whether or not temperate and tropical isolates should be separated into different species. The available worldwide data are incomplete and the full range of variation in the species is not yet known. We suggest refraining from using the epithet P. tropicalis until more data are available.

UP-02.27: The association between genetic variant of luteinizing hormone and the incidence and prognosis of prostate cancer in groups of multi-ethnic origin

Luteinizing hormone (LH) secreted from the anterior pituitary, signals Leydig cells of the testes to produce testosterone. A common genetic variant of LH (vLH) has been identified. The prevalence of vLH allele has been estimated worldwide as a carrier frequency of 0% to 53% of Caucasians and is known to be more active than the wild-type protein. Screening for vLH has primarily relied on relative immunoreactoivity in variant-sensitive and insensitive assays. In addition, restriction fragment length polymorphism (RFLP) studies are largely been used to confirm immunoassay results.

Bacterial community structure of a pesticide-contaminated site and assessment of changes induced in community structure during bioremediation

The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.

The factor V Glu1608Lys mutation is recurrent in familial thrombophilia

Co-inheritance of heterozygous factor V deficiency with FV Leiden enhances the activated protein C resistance (APCR) associated with this mutation, resulting in pseudo-homozygous APCR. The role of FV deficiency in modulating thrombotic risk in this rare condition is poorly understood. We have identified in thrombophilic patients with FV deficiency a novel FV gene mutation (c. 4996G>A), predicting the Glu1608Lys substitution in the A3 domain. The heterozygous mutation was detected in three unrelated patients, two carriers of the FV Leiden mutation, and one of the FVHR2 haplotype. The Glu1608Lys change was also present in two subjects with mild FV deficiency, and absent in 200 controls. The FV1608Lys carriers showed reduced mean FV activity (42% +/- 12%) and antigen (53% +/- 18%) levels and, in Western blot analysis, reduced amounts of intact platelet FV. The restriction fragment length polymorphism (RFLP) study identified two haplotypes underlying the mutation, which suggests that it is recurrent. In heterozygous subjects the amount of FV1608Lys mRNA in white blood cells was similar to that produced by the counterpart alleles (FVWt or FVHR2). Recombinant FV1608Lys (rFV1608Lys), detected by Western blot in the conditioned medium, was indistinguishable from rFVWt and FV antigen and activity were found to be respectively 44% +/- 20% and 13% +/- 4% of rFVWt. Our data indicate that FVGlu1608Lys predicts a CRM (plasma)/CRMred (cell culture) FV deficiency, and may contribute to thrombophilia in carriers of FV Leiden and FVHR2 haplotype via a pseudo-homozygosity mechanism. Our findings help to define the molecular bases of FV deficiency and thrombophilia.

The immunoglobulin IGHD gene locus in C57BL/6 mice.

Four immunoglobulin heavy chain diversity (IGHD) gene subgroups (DFL16, DSP2, DQ52, and DST4) have been identified previously in BALB/c mice. Although the locations of most IGHD genes have been established based on restriction map and Southern blot analysis, a complete mouse IGHD gene locus map at the sequence level is still not available. In addition, a previous restriction fragment length polymorphism study suggested that significant difference in the IGHD gene locus exists between C57BL/6 and BALB/c mice. The author has now analyzed the C57BL/6 mouse genomic data and established a complete map of the IGHD gene locus. All four IGHD subgroups previously identified in BALB/c mice were found to be present in C57BL/6 mice. However, unlike the BALB/c mice, which have at least 13 IGHD genes, the C57BL/6 genome contains only ten IGHD genes, which include one DFL16, six DSP2, one DQ52, and two DST4 genes. There are also differences in the coding regions of the DST4 and DQ52 genes between the two mouse strains.

Systematics of Ipomoea subgenus Quamoclit (Convolvulaceae) based on ITS sequence data and a Bayesian phylogenetic analysis

A Bayesian phylogenetic analysis of 36 Ipomoea species using sequence data from the internal transcribed spacer region was compared with classification schemes based on traditional methods and a previously published cpDNA restriction fragment length polymorphism (RFLP) study. These molecular studies support a diversity of groups that were circumscribed on the basis of phenetic principles and agree generally with the results from cpDNA RFLP analyses. The congruence between the phylogenetic hypotheses based on new molecular data and the understanding of relationships developed in earlier studies indicate that these classifications may reflect evolutionary history. Two large clades of species, with one including sections Tricolores, Calonyction, and Pharbitis and the other including sections Mina and Leptocallis, were identified. Furthermore, morphologically distinct groups of Ipomoea species received support from the DNA sequence data. Indices of convergence for the Markov chain Monte Carlo (MCMC) in the Bayesian phylogenetic analysis were evaluated. A limited range of posterior probabilities for each node in the trees from a set of five MCMC samples provides a useful index of convergence. Bayesian node support values were generally higher than bootstrap values from a maximum parsimony analysis. This is consistent with the notion that these measures of support estimate different qualities of the data.

Cell-mediated immune responses of healthy laboratory volunteers to sonicate antigens prepared from the most prevalent strains of Mycobacterium tuberculosis from South India harboring a single copy of IS6110.

Our restriction fragment length polymorphism (RFLP) studies have shown that the most prevalent (40%) strains of Mycobacterium tuberculosis from South India contain a single copy of the IS6110 insertion sequence and are of importance in studying virulence and immunity. Sonicate antigens from seven such strains were used to study in vitro T-cell proliferation and gamma interferon (IFN-gamma) and interleukin-12 (IL-12) secretion as markers of protective immunity in 25 healthy subjects positive for purified protein derivative (PPD). The standard PPD and heat-killed H37Rv antigens induced the maximum levels of T-cell proliferation and IFN-gamma secretion but low levels of IL-12. All sonicate antigens induced T-cell proliferation and IFN-gamma secretion with strong positive correlation. Our results suggest that sonicate antigens from the most prevalent and recent strains of M. tuberculosis from clinical isolates have the potential to induce T-cell activation and may allow newer and specific antigens to be further characterized for diagnosis and vaccine development.