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PubMed1996

Genetic characterization of five powdery mildew disease resistance loci in Arabidopsis thaliana.

This paper reports on six Arabidopsis accessions that show resistance to a wild isolate of the powdery mildew pathogen, Erysiphe cichoracearum. Resistance at 7 days post-inoculation in these accessions was characterized by limited fungal growth and sporadic development of chlorotic or necrotic lesions at inoculation sites. Three accessions, Wa-1, Kas-1 and SI-0, were highly resistant, while the other accessions permitted some fungal growth and conidiation. Papilla formation was a frequent host response; however, cell death appeared to be neither a rapid nor a common response to infection. To determine the genetic basis of resistance, segregation analyses of progeny from crosses between each of the resistant accessions and Columbia (gl1), which is susceptible to the powdery mildew pathogen, were performed. For all accessions except Sl-0, resistance was conferred by a single locus. Sl-0 was unique in that two unlinked loci controlled the disease reaction phenotype. In accessions Wa-1, Kas-1, Stw-0 and Su-0, powdery mildew resistance was encoded by a semi-dominant allele. However, susceptibility was dominant to resistance in accessions Te-0 and Sl-0. Mapping studies revealed that powdery mildew resistances in Kas-1, Wa-1, Te-0, Su-0 and Stw-0 were controlled by five independent loci. This study suggests that the Arabidopsis powdery mildew disease will be a suitable model system in which to investigate powdery mildew diseases.

Arabidopsis↗

PubMed1999

The structural, biochemical, and genetic characterization of a new radiation-induced, variegated leaf mutant of soybean [Glycine max (L.) Merr].

A variegated leaf mutant in soybean [Glycine max (L.) Merr.] has been identified and characterized. E25-10 was derived by exposure of seeds of the "Williams' 82" cultivar to gamma-radiation. In this mutant, yellow leaf sectors contain defective chloroplasts, in which the thylakoid membranes are presented as long, parallel structures with little or no overlap. No starch grains have been detected in the mutant chloroplasts. Small vesicles and plastoglobuli can be found within the defective chloroplasts. Genetic studies revealed that a single nuclear-encoded gene is responsible for the mutation in E25-10. The total chlorophyll content is reduced in yellow leaf tissue by 70-80%. However, the chlorophyll a/b ratio is not altered. The absorbance spectrum of pigments in the mutant leaf tissue differed from that of the green extracts in the range of 400-500 nm. This reduction in total chlorophyll and the change in the absorbance spectrum pattern in the yellow tissue is related to a loss of certain photosynthetic complexes. Green gel analysis revealed that four major pigment-protein complexes (CP1, LHCP1, LHCP2, and CPa) of the thylakoid membranes were absent in the E25-10 mutant. Lithium dodecyl sulphate polyacrylamide gel analysis showed that at least 5-6 polypeptides (51, 44, 25, 15, 13, and 12 kDa) were missing in the thylakoid membranes of chloroplasts from the yellow tissue. Changes in chloroplast- and nuclear-encoded gene message levels were detected. The psaA transcripts which code for the P700 apoprotein in PSI were reduced in chloroplasts from the E25-10 mutant yellow tissue. The levels of the large subunit of ribulose bisphosphate carboxylase (rbcL) and light harvest complex protein (LHCP) of PSII mRNA appeared to be reduced slightly in the mutant plants. However, a much more significant reduction in the 16S rRNA and the small subunit of ribulose bisphosphate carboxylase (rbcS) expression was detected in the yellow leaf sectors. Our results suggest that the possible lesion in E25-10 is located in the photosystem I even though fewer grana were observed in the defective chloroplasts.

Chlorophyll↗

PubMed2003

Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis.

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of either an O-linked trisaccharide, Gal (beta1-4) Gal (alpha1-3) 2,4-diacetamido-2,4,6-trideoxyhexose or an O-linked disaccharide Gal (alpha1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pglB/B2, pglF, pglG and pglH. A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pglB2 polymorphisms were not found in strain C311 musical sharp 3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311 musical sharp 3, a strain with well-defined lipopolysaccharide (LPS) and pilin-linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating truncation of the trisaccharide structure. Antisera specific for the C311 musical sharp 3 trisaccharide failed to react with pilin from these pglE and pglF mutants. GC-MS analysis of the sugar composition of the pglE mutant showed a reduction in galactose compared with C311 musical sharp 3 wild type. Analysis of amino acid sequence homologies has suggested specific roles for pglE and pglF in the biosynthesis of the trisaccharide structure. Further, we present evidence that pglE, which contains heptanucleotide repeats, is responsible for the phase variation between trisaccharide and disaccharide structures in strain C311 musical sharp 3 and other strains. We also present evidence that pglG, pglH and pglB2 are potentially phase variable.

Amino Acid Sequence↗

PubMed1999

Genetic characterization of multifocal tumor growth in renal cell carcinoma.

The primary cancer found in the kidney is renal cell carcinoma (RCC), which does not as yet have a delineation of the types of lesions that progress to tumor malignancy. We studied tissues from primary tumors and secondary lesions, and compared it with the surrounding normal kidney tissue using comparative genomic hybridization to determine their potential for malignancy. Although our sample size was small, we found that small multifocal tumors have the same potential as primary carcinoma to lead to RCC malignancy. Further genetic studies in a larger sample of patients is necessary before the risk of different lesions to remain benign or progress to RCC can be defined absolutely.

Carcinoma, Renal Cell↗

PubMed1998

Genetic characterization of canine distemper virus in Serengeti carnivores.

The lion (Panthera leo) population in the Serengeti ecosystem was recently afflicted by a fatal epidemic involving neurological disease, encephalitis and pneumonia. The cause was identified as canine distemper virus (CDV). Several other species in the Serengeti were also affected. This report presents CDV H and P gene sequences isolated from Serengeti lions (Panthera leo), spotted hyenas (Crocuta crocuta), bat-eared fox (Otocyon megalotis) and domestic dog (Canis familiaris). Sequence analyses demonstrated that the four Serengeti species carry closely related CDV isolates which are genetically distinct from other CDV isolates from various species and locations. The results are consistent with the conclusions that: (1) a particularly virulent strain of CDV emerged among Serengeti carnivores within the last few years; (2) that strain has recognizable shared-derived (synapomorphic) genetic differences in both H and P genes when compared to CDV from other parts of the world; and (3) that the CDV strain has frequently crossed host species among Serengeti carnivores.

Africa↗

PubMed1984

Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene.

Two temperature-sensitive mutations have been constructed in the single actin gene (ACT1) of the yeast Saccharomyces cerevisiae by in vitro mutagenesis of the cloned gene followed by integrative transformation of mutagenized DNA into yeast cells. A strategy of allele replacement was used that allowed recessive mutations to be phenotypically expressed in the initial transformants, thus simplifying the screening of large numbers of independently transformed cells. After confirming that several ts mutations were located within the actin structural gene by genetic methods, these mutant alleles were cloned, and the altered amino acid residues were defined by DNA sequence analysis. The two unique mutations resulted in substitution of proline-32 with leucine and alanine-58 with threonine. In the course of isolating these mutations, the observation was made that a high proportion of yeast cells transformed with exogenous DNA by the spheroplast method are temperature sensitive for growth because of genetic changes unrelated to the transforming DNA.

Actins↗

PubMed1999

RAPD profile based genetic characterization of chemotypic variants of Artemisia annua L.

The annual herbaceous plant, Artemisia annua L., belonging to family Asteraceae, is the natural source of the highly potent antimalarial compound, artemisinin, besides producing valuable essential oil. The plant is at present the sole commercial source for artemisinin production since all the chemical syntheses are non-viable. Therefore, economic and practical considerations dictate that plants with maximum content of artemisinin be found and/or ways to increase their artemisinin content be sought. The key to this selection and breeding is a comprehension of chemical and genetic variability and suitable selection(s) of elites from within the available population. In the present study, RAPD analyses of selected chemotypes from a decade old introduced population in India were carried out using arbitrary primers. The RAPD data clearly indicate the distinction amongst these plants. Further, the detection of highly polymorphic profiles (97 polymorphic markers out of a total of 101 markers) suggests the existence of very high levels of genetic variation in the Indian population despite geographical isolation and opens out a strong possibility of further genetic improvement for superior artemisinin content. UPGMA analyses of RAPD and phytochemical trait data indicate that the wide phytochemical diversity is included within the genetic diversity. These results further support the prospects for selection and breeding of superior artemisinin containing lines.

Antimalarials↗

PubMed2004

Structural and genetic characterization of the Shigella boydii type 13 O antigen.

Shigella is an important human pathogen. It is generally agreed that Shigella and Escherichia coli constitute a single species; the only exception is Shigella boydii type 13, which is more distantly related to E. coli and other Shigella forms and seems to represent another species. This gives S. boydii type 13 an important status in evolution. O antigen is the polysaccharide part of the lipopolysaccharide in the outer membrane of gram-negative bacteria and plays an important role in pathogenicity. The chemical structure and genetic organization of the S. boydii type 13 O antigen were investigated. The O polysaccharide was found to be acid labile owing to the presence of a glycosyl phosphate linkage in the main chain. The structure of the linear pentasaccharide phosphate repeating unit (O unit) was established by nuclear magnetic resonance spectroscopy, including two-dimensional COSY, TOCSY, ROESY, and H-detected 1H, 13C and 1H, 31P HMQC experiments, along with chemical methods. The O antigen gene cluster of S. boydii type 13 was located and sequenced. Genes for synthesis of UDP-2-acetamido-2,6-dideoxy-L-glucose and genes that encode putative sugar transferases, O unit flippase, and O antigen polymerase were identified. Seven genes were found to be specific to S. boydii type 13. The S. boydii type 13 O antigen gene cluster has higher levels of sequence similarity with Vibrio cholerae gene clusters and may be evolutionarily related to these gene clusters.

Carbohydrates↗

PubMed1999

Familial Mediterranean fever: clinical and genetic characterization in a mixed pediatric population of Jewish and Arab patients.

OBJECTIVE: Familial Mediterranean fever (FMF) is an autosomal recessive hereditary disease which primarily affects non-Ashkenazi Jews, Armenians, Arabs, and Turks. The gene responsible for the disease (MEFV/FMF) has been recently identified. Four common mutations in exon 10 of the MEFV gene seem to account for 86% of the DNA variations identified in patients with FMF. We conducted a phenotype/genotype correlation study in a mixed population of Jewish and Arab children with FMF. STUDY DESIGN: Seventy patients clinically diagnosed as having FMF underwent molecular genetic studies using polymerase chain reaction and restriction endonuclease digestion methods to detect the presence of the four mutations (M694V, M680I, V726A, M694I). We then correlated the presence of each mutation with ethnic origin, age of onset, clinical manifestations, disease severity, and occurrence of amyloidosis. RESULTS: The M694V mutation, which is predominant in non-Ashkenazi Jews, was found in 92% of our Jewish patients and in only 30% of the Arab patients. All four mutations were identified among 94% of the Arab patients, but with no particular prevalence for any one of them. The presence of a homozygous M694V mutation was significantly associated with a more severe form of the disease: the clinical onset of the disease manifested at an earlier age; the number of attacks per month was higher; the global assessment by the treating physician and the severity of pain scored higher; and arthritis was more frequent. Only patients with the M694V mutation had a family history of amyloidosis. No association was found between the type of mutation and the predominance of fever, abdominal pain, pleuritis, skin eruption, or response to colchicine in the clinical picture. CONCLUSIONS: Homozygosity for the M694V mutation, predominant among North African Jews, is associated with a severe course and prognosis for FMF. This mutation is less common among Arabs and, when present, occurs almost only in heterozygous form. In Arab patients, the disease tends to run a milder course and seems to bear a better prognosis. The phenotype/genotype patterns that are evident from our study of a mixed series of Jewish and Arab children with FMF might provide a rational basis for counseling about the natural history of the disease and for clinical treatment of FMF patients and their families.

Adolescent↗

PubMed1981

Genetic characterization of aggregation-defective developmental mutants of Myxococcus xanthus.

The transposon Tn5 was used to map temperature-sensitive mutants of Myxococcus xanthus defective in aggregation (C. E. Morrison and D. R. Zusman, J. Bacteriol. 140:1036-1042, 1979). Seven of the eight mutants showing a similar terminal phenotype (rough) were found to be tightly linked. These mapped in a group of loci which we have designated aggR1, aggR2, aggR3, and aggR4. Temperature-sensitive mutants having a different terminal phenotype were not liked to aggR. A search through a group of nonconditional rough mutants indicated that a much lower proportion of these (1 of 35) mapped in aggR. Thus, aggR is probably only one of many sites which can lead to the rough phenotype when mutated. Localized mutagenesis was used to isolate nine additional aggR mutants. All mapped within aggR1, aggR2, or aggR3, and none was found outside this region. Thus, we have characterized a cluster of developmental genes which are needed for aggregation in M. xanthus. The localization of a Tn5 insert adjacent to this region makes possible further manipulation of these genes.

Chromosome Mapping↗

PubMed2001

Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene.

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.

Adenosine↗

PubMed2026

Endemic Circulation and Genetic Characterization of Foot-and-Mouth Disease Virus in Buffalo Populations of Bangladesh.

Foot-and-mouth disease (FMD) virus (FMDV) is endemic in Bangladesh, causing severe economic losses in the livestock sector. While it primarily affects cattle, buffaloes (Bubalus bubalis) remain highly susceptible. Therefore, this study aimed to determine the prevalence and molecular characteristics of FMDV in buffaloes across three districts (Sylhet, Rajshahi, and Noakhali) of Bangladesh from January to June 2024. In a cross-sectional study, a total of 622 nasal swabs from 67 herds were collected and tested for FMDV RNA using reverse transcription polymerase chain reaction (RT-PCR). Overall, 255 samples were positive, resulting in an individual-level prevalence of 41.0%(255/622), while 88.1% (59/67) of herds were FMDV-positive. Animal-level prevalence was highest in Sylhet (47.1%), followed by Noakhali (38.9%) and Rajshahi (37.1%). To further characterize circulating strains, eight representative RT-PCR-positive samples were sequenced, revealing the co-circulation of serotypes O (n = 5) and Asia-1 (n = 3). Phylogenetic analysis showed that the isolates belonged to the ME-SA/Ind2001e lineage of the serotype O and the Asia-1 Group V lineage, clustering with contemporary strains from Bangladesh and neighboring countries, suggesting possible intra- and transboundary transmission. Pairwise genetic distance evaluation revealed high regional similarity, while Mantel tests indicated significant associations between genetic, geographic, and temporal distances. Comparative genomic analysis revealed largely conserved genomic regions, whereas VP1 analysis indicated that purifying selection predominated across both serotypes, with serotype O exhibiting greater genetic diversity (π = 0.11642) than Asia-1 (π = 0.05258), suggesting localized antigenic variability and possible immune-mediated viral evolution. These findings highlight the need for strengthened surveillance, improved biosecurity, and integrated vaccination strategies to enhance FMD control and reduce economic losses in Bangladesh.

Animals↗

PubMed2004

Genetic characterization of pcpS, encoding the multifunctional phosphopantetheinyl transferase of Pseudomonas aeruginosa.

Fatty acid synthases (primary metabolism), non-ribosomal peptide synthases and polyketide synthases (secondary metabolism) contain phosphopantetheinyl (Ppant)-dependent carrier proteins that must be made functionally active by transfer of the 4'-Ppant moiety from coenzyme A. These reactions are usually catalysed by dedicated Ppant transferases. Although rich in Ppant-dependent carrier proteins, it was previously shown that Pseudomonas aeruginosa possesses only one Ppant transferase, encoded by pcpS, which functions in both primary and secondary metabolism. Consistent with this notion are our findings that pcpS can genetically complement mutations in the Escherichia coli acpS and entD genes, encoding the apo-acyl carrier protein (ACP) synthase of fatty acid synthesis and a Ppant transferase of enterobactin synthesis, respectively. It also complements a Bacillus subtilis sfp mutation affecting a gene encoding a Ppant transferase essential for surfactin synthesis. A pcpS insertion mutant could only be constructed in a strain carrying the E. coli acpS gene on a chromosomally integrated element in trans, implying that the in vitro essentiality of pcpS is due to its requirement for activation of apo-ACP of fatty acid synthesis. The conditional pcpS mutant is non-fluorescent, does not produce pyoverdine and pyochelin, and does not grow in the presence of iron chelators. The data presented here for the first time confirm that PcpS plays an essential role in both fatty acid and siderophore metabolism.

Bacterial Proteins↗

PubMed2003

Genetic characterization of the three medicinal Echinacea species using RAPD analysis.

The three medicinal species of the Echinacea genus, E. angustifolia DC., E. pallida (Nutt.) Nutt. and E. purpurea (L.) Moench were distinguished using the RAPD (random amplified polymorphic DNA) technique. Species-specific markers were identified from amplicons obtained with four of the twenty 10-mer primers contained in the Operon RAPD kit A. In particular, one marker was identified for E. angustifolia (OPA 20, 1800 pb) and E. pallida (OPA 10, 600 pb) and three markers for E. purpurea (OPA 11 : 1250 pb; OPA 17 : 750, 1800 pb). Genetic distance analysis indicated a high degree of difference among the three species with a relative lower difference between E. angustifolia and E. pallida.

Echinacea↗

PubMed2004

Identification and genetic characterization of an Aegilops tauschii ortholog of the wheat leaf rust disease resistance gene Lr1.

Aegilops tauschii (goat grass) is the progenitor of the D genome in hexaploid bread wheat. We have screened more than 200 Ae. tauschii accessions for resistance against leaf rust (Puccinia triticina) isolates,which are avirulent on the leaf rust resistance gene Lrl. Approximately 3.5% of the Ae. tauschii accessions displayed the same low infection type as the tester line Thatcher Lrl. The accession Tr.t. 213, which showed resistance after artificial infection with Lrl isolates both in Mexico and in Switzerland, was chosen for further analysis. Genetic analysis showed that the resistance in this accession is controlled by a single dominant gene,which mapped at the same chromosomal position as Lrl in wheat. It was delimited in a 1.3-cM region between the restriction fragment length polymorphism (RFLP) markers ABC718 and PSR567 on chromosome 5DL of Ae.tauschii. The gene was more tightly linked to PSR567(0.47 cM) than to ABC718 (0.79 cM). These results indicate that the resistance gene in Ae. tauschii accession Tr.t. 213 is an ortholog of the leaf rust resistance gene Lrlof bread wheat, suggesting that Lrl originally evolved in diploid goat grass and was introgressed into the wheat D genome during or after domestication of hexaploidwheat. Compared to hexaploid wheat, higher marker polymorphism and recombination frequencies were ob-served in the region of the Lrl ortholog in Ae. tauschii. The identification of LrlAe, the orthologous gene of wheatLrl, in Ae. tauschii will allow map-based cloning of Lrlfrom this genetically simpler, diploid genome.

Basidiomycota↗

PubMed2001

Genetic characterization of Pseudomonas 'NZI7'--a novel pathogen that results in a brown blotch disease of Agaricus bisporus.

AIMS: To characterize a novel pseudomonad isolate capable of causing brown blotch disease of Agaricus bisporus. METHODS AND RESULTS: Using the white-line-in-agar (WLA) assay, fluorescent pseudomonads isolated from a New Zealand mushroom farm were screened for the lipodepsipeptide tolaasin, a characteristic marker of Pseudomonas tolaasii. One isolate, NZI7, produced a positive WLA assay and caused brown lesions of A. bisporus comparable with those produced by Ps. tolaasii. However, genetic analysis suggested that Ps. tolaasii and NZI7 were genetically dissimilar, and that NZI7 is closely related to Pseudomonas syringae. Nucleotide sequence analyses of a gene involved in tolaasin production indicated that similar genes are present in both NZI7 and Ps. tolaasii. CONCLUSION: NZI7 represents a novel Pseudomonas species capable of causing brown blotch disease of A. bisporus. SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic identification of Ps. tolaasii based on A. bisporus browning and positive WLA may have limited specificity.

Agaricales↗

PubMed1989

Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.

The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). Mutations in two genes, hemA and hemL, result in auxotrophy for ALA in Salmonella typhimurium, but the roles played by these genes and the mechanism of ALA synthesis are not understood. I have cloned and sequenced the S. typhimurium hemA gene. The predicted polypeptide sequence for the HemA protein shows no similarity to known ALA synthases, and no ALA synthase activity was detected in extracts prepared from strains carrying the cloned hemA gene. Genetic analysis, DNA sequencing of amber mutations, and maxicell studies proved that the open reading frame identified in the DNA sequence encodes HemA. Another surprising finding of this study is that hemA lies directly upstream of prfA, which encodes peptide chain release factor 1 (RF-1). A hemA::Kan insertion mutation, constructed in vitro, was transferred to the chromosome and used to show that these two genes form an operon. The hemA gene ends with an amber codon, recognized by RF-1. I suggest a model for autogenous control of prfA expression by translation reinitiation.

5-Aminolevulinate Synthetase↗

PubMed1975

Genetic analysis of adenovirus type 2. I. Isolation and genetic characterization of temperature-sensitive mutants.

Temperature-sensitive mutants which replicate normally at 33 C but poorly at 39 C were isolated from nitrosoguanidine- or nitrous acid-mutagenized adenovirus 2 by (i) testing the cytopathic effect or inclusion body-forming capacity of random plaque isolates, or (ii) reduced plaque enlargement upon shifting from 33 to 39 C. Thirty-six mutants were isolated with 33 C/39 C plaque ratios varying from 20 to 10-5. Some of these mutants could be arranged into 13 groups by the complementation test. By means of recombination analysis a provisional linear genetic map was constructed.

Adenoviridae↗