Identification of a new human mtDNA polymorphism (A14290G) in the NADH dehydrogenase subunit 6 gene

Leber's hereditary optic neuropathy (LHON) is a maternally inherited form of retinal ganglion cell degeneration leading to optic atrophy in young adults. Several mutations in different genes can cause LHON (heterogeneity). The ND6 gene is one of the mitochondrial genes that encodes subunit 6 of complex I of the respiratory chain. This gene is a hot spot gene. Fourteen Persian LHON patients were analyzed with single-strand conformational polymorphism and DNA sequencing techniques. None of these patients had four primary mutations, G3460A, G11788A, T14484C, and G14459A, related to this disease. We identified twelve nucleotide substitutions, G13702C, T13879C, T14110C, C14167T, G14199T, A14233G, G14272C, A14290G, G14365C, G14368C, T14766C, and T14798C. Eleven of twelve nucleotide substitutions had already been reported as polymorphism. One of the nucleotide substitutions (A14290G) has not been reported. The A14290G nucleotide substitution does not change its amino acid (glutamic acid). We looked for base conservation using DNA star software (MEGALIGN program) as a criterion for pathogenic or nonpathogenic nucleotide substitution in A14290G. The results of ND6 gene alignment in humans and in other species (mouse, cow, elegans worm, and Neurospora crassa mold) revealed that the 14290th base was not conserved. Fifty normal controls were also investigated for this polymorphism in the Iranian population and two had A14290G polymorphism (4%). This study provides evidence that the mtDNA A14290G allele is a new nonpathogenic polymorphism. We suggest follow-up studies regarding this polymorphism in different populations.

Strain variation in ppGpp concentration and RpoS levels in laboratory strains of Escherichia coli K-12

Laboratory strains and natural isolates of Escherichia coli differ in their level of stress resistance due to strain variation in the level of the sigma factor sigma(S) (or RpoS), the transcriptional master controller of the general stress response. We found that the high level of RpoS in one laboratory strain (MC4100) was partially dependent on an elevated basal level of ppGpp, an alarmone responding to stress and starvation. The elevated ppGpp was caused by two mutations in spoT, a gene associated with ppGpp synthesis and degradation. The nature of the spoT allele influenced the level of ppGpp in both MC4100 and another commonly used K-12 strain, MG1655. Introduction of the spoT mutation into MG1655 also resulted in an increased level of RpoS, but the amount of RpoS was lower in MG1655 than in MC4100 with either the wild-type or mutant spoT allele. In both MC4100 and MG1655, high ppGpp concentration increased RpoS levels, which in turn reduced growth with poor carbon sources like acetate. The growth inhibition resulting from elevated ppGpp was relieved by rpoS mutations. The extent of the growth inhibition by ppGpp, as well as the magnitude of the relief by rpoS mutations, differed between MG1655 and MC4100. These results together suggest that spoT mutations represent one of several polymorphisms influencing the strain variation of RpoS levels. Stress resistance was higher in strains with the spoT mutation, which is consistent with the conclusion that microevolution affecting either or both ppGpp and RpoS can reset the balance between self-protection and nutritional capability, the SPANC balance, in individual strains of E coli.

Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1 ), tryptose phosphate broth (2.95 g l 1), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 lgml 1 chloramphenicol, 100 lgml 1 streptomycin and 100 IU ml 1 penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-20-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT50/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC50. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals

Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression

Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immunerelated genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus–cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture

Deletion Analysis of 15 exons located outside and inside a “Hot Spot” in the Duchenne Muscular Dystrophy gene in Duchenne’s Muscular dystrophy patients

Introduction. Duchenne and Becker Muscular Dystrophies (DMD/DMB) are X-linked recessive diseases characterized by progressive muscle weakness and wasting, loss of motor skills and death after the second decade of life. Deletions are the most prevalent mutations that affect the dystrophin gene, which spans 79 exons.Objective: Identify deletions on the dystrophin gene in 58 patients affected with DMD.Methods: Through multiplex PCR identify deletions on the dystrophin gene in 58 patients with DMD and observe the frequency of this mutation in our population.Results: We found deletions in 1.72% of patients (1 of 58 persons). Deletions were not the principal cause of disease in our population. It is possible that duplications and point mutations caused this illness in our patients.Conclusions: The frequency of deletions in the 15 exons analyzed from the dystrophin gene was low. The predominant types of mutation in our patients` samples were not deletions as has been observed in the literature worldwide, therefore, it is important to determine other types of mutations as are duplications and point mutations.

Gene Mapping of 18S and 5S rDNA Genes in the Karyotype of the Three-Spot Gourami Trichogaster trichopterus (Perciformes, Osphronemidae)

Ornamental fish culture is important as an economic activity and for biodiversity conservation as well. The species of the genus Trichogaster (Perciformes, Osphronemidae), popularly known as three-spot gourami, are among the several commercial species raised around the world. In the present work, eight specimens of Thrichogaster trichopterus from aquarium trade facilities were analyzed. The karyotype was composed of 23 pairs of subtelo/acrocentric chromosomes. Fluorescent in situ hybridization allowed identifying the 18S ribosomal gene at telomeric region on long arms of the largest acrocentric pair. On the other hand, the 5S rRNA gene is located at a proximal region on a pair of medium-sized chromosomes. Such information is extremely useful in face of the risks of introduction and the development of ornamental fish trade, once many fish species can be identified only by genetic studies.

"Hot spot" in the PROP1 gene responsible for combined pituitary hormone deficiency.

As pituitary function depends on the integrity of the hypothalamic-pituitary axis, any defect in the development and organogenesis of this gland may account for a form of combined pituitary hormone deficiency (CPHD). Although pit-1 was 1 of the first factors identified as a cause of CPHD in mice, many other homeodomain and transcription factors have been characterized as being involved in different developmental stages of pituitary gland development, such as prophet of pit-1 (prop-1), P-Lim, ETS-1, and Brn 4. The aims of the present study were first to screen families and patients suffering from different forms of CPHD for PROP1 gene alterations, and second to define possible hot spots and the frequency of the different gene alterations found. Of 73 subjects (36 families) analyzed, we found 35 patients, belonging to 18 unrelated families, with CPHD caused by a PROP1 gene defect. The PROP1 gene alterations included 3 missense mutations, 2 frameshift mutations, and 1 splice site mutation. The 2 reported frameshift mutations could be caused by any 2-bp GA or AG deletion at either the 148-GGA-GGG-153 or 295-CGA-GAG-AGT-303 position. As any combination of a GA or AG deletion yields the same sequencing data, the frameshift mutations were called 149delGA and 296delGA, respectively. All but 1 mutation were located in the PROP1 gene encoding the homeodomain. Importantly, 3 tandem repeats of the dinucleotides GA at location 296-302 in the PROP1 gene represent a hot spot for CPHD. In conclusion, the PROP1 gene seems to be a major candidate gene for CPHD; however, further studies are needed to evaluate other genetic defects involved in pituitary development.

The “Spot 14” gene resides on the telomeric end of the 11q13 amplicon and is expressed in lipogenic breast cancers: Implications for control of tumor metabolism

Enhanced long chain fatty acid synthesis may occur in breast cancer, where it is necessary for tumor growth and predicts a poor prognosis. “Spot 14” (S14) is a carbohydrate- and thyroid hormone-inducible nuclear protein specific to liver, adipose, and lactating mammary tissues that functions to activate genes encoding the enzymes of fatty acid synthesis. Amplification of chromosome region 11q13, where the S14 gene (THRSP) resides, also predicts a poor prognosis in breast tumors. We localized the S14 gene between markers D11S906 and D11S937, at the telomeric end of the amplified region at 11q13, and found that it was amplified and expressed in breast cancer-derived cell lines. Moreover, concordant expression of S14 and a key lipogenic enzyme (acetyl-CoA carboxylase) in a panel of primary breast cancer specimens strongly supported a role for S14 as a determinant of tumor lipid metabolism. S14 expression provides a pathophysiological link between two prognostic indicators in breast cancer: enhanced lipogenesis and 11q13 amplification.

Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato

The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site–leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.

Heat and chemical stress modulate the expression of the alpha-RYR gene in broiler chickens

The biological cause of Pork Stress syndrome, which leads to PSE (pale, soft, exudative) meat, is excessive release of Ca(2+) ions, which is promoted by a genetic mutation in the ryanodine receptors (RyR) located in the sarcoplasmic reticulum of the skeletal muscle cells. We examined the relationship between the formation of PSE meat under halothane treatment and heat stress exposure in chicken alpha RYR hot spot fragments. Four test groups were compared: 1) birds slaughtered without any treatment, i.e., the control group (C); 2) birds slaughtered immediately after halothane treatment (H); 3) birds slaughtered immediately after heat stress treatment (HS), and 4) birds exposed to halothane and to heat stress (H+HS), before slaughtering. Breast muscle mRNA was extracted, amplified by RT-PCR, and sequenced. PSE meat was evaluated using color determination (L*value). The most common alteration was deletion of a single nucleotide, which generated a premature stop codon, resulting in the production of truncated proteins. The highest incidence of nonsense transcripts came with exposure to halothane; 80% of these abnormal transcripts were detected in H and H+HS groups. As a consequence, the incidence of abnormal meat was highest in the H+HS group (66%). In HS, H, and C groups, PSE meat developed in 60, 50, and 33% of the samples, respectively. Thus, halothane apparently modulates alpha RYR gene expression in this region, and synergically with exposure to heat stress, causes Avian Stress syndrome, resulting in PSE meat in broiler chickens.

QTL mapping for reaction to Phaeosphaeria leaf spot in a tropical maize population

Phaeosphaeria leaf spot (PLS) is an important disease in tropical and subtropical maize (Zea mays, L.) growing areas, but there is limited information on its inheritance. Thus, this research was conducted to study the inheritance of the PLS disease in tropical maize by using QTL mapping and to assess the feasibility of using marker-assisted selection aimed to develop genotypes resistance to this disease. Highly susceptible L14-04B and highly resistant L08-05F inbred lines were crossed to develop an F(2) population. Two-hundred and fifty six F(2) plants were genotyped with 143 microsatellite markers and their F(2:3) progenies were evaluated at seven environments. Ten plants per plot were evaluated 30 days after silk emergence following a rating scale, and the plot means were used for analyses. The heritability coefficient on a progeny mean basis was high (91.37%), and six QTL were mapped, with one QTL on chromosomes 1, 3, 4, and 6, and two QTL on chromosome 8. The gene action of the QTL ranged from additive to partial dominance, and the average level of dominance was partial dominance; also a dominance x dominance epistatic effect was detected between the QTL mapped on chromosome 8. The phenotypic variance explained by each QTL ranged from 2.91 to 11.86%, and the joint QTL effects explained 41.62% of the phenotypic variance. The alleles conditioning resistance to PLS disease of all mapped QTL were in the resistant parental inbred L08-05F. Thus, these alleles could be transferred to other elite maize inbreds by marker-assisted backcross selection to develop hybrids resistant to PLS disease.

Development and agronomic performance of common bean lines simultaneously resistant to anthracnose, angular leaf spot and rust

The common bean is affected by several pathogens that can cause severe yield losses. Here we report the introgression of resistance genes to anthracnose, angular leaf spot and rust in the `carioca-type` bean cultivar `Ruda`. Initially, four backcross (BC) lines were obtained using `TO`, `AB 136`, `Ouro Negro` and `AND 277` as donor parents. Molecular fingerprinting was used to select the lines genetically closer to the recurrent parent. The relative genetic distances between `Ruda` and the BC lines varied between 0.0% and 1.99%. The BC lines were intercrossed and molecular markers linked to the resistance genes were used to identify the plants containing the genes of interest. These plants were selfed to obtain the F(2), F(3) and F(4) plants which were selected based on the presence of the molecular markers mentioned and resistance was confirmed in the F(4) generation by inoculation. Four F(4:7) pyramid lines with all the resistance genes showed resistance spectra equivalent to those of their respective donor parents. Yield tests showed that these lines are as productive as the best `carioca-type` cultivars.

Patterns of resistance to angular leaf spot, anthracnose and common bacterial blight in common bean germplasm

Diseases and insect pests are major causes of low yields of common bean (Phaseolus vulgaris L.) in Latin America and Africa. Anthracnose, angular leaf spot and common bacterial blight are widespread foliar diseases of common bean that also infect pods and seeds. One thousand and eighty-two accessions from a common bean core collection from the primary centres of origin were investigated for reaction to these three diseases. Angular leaf spot and common bacterial blight were evaluated in the field at Santander de Quilichao, Colombia, and anthracnose was evaluated in a screenhouse in Popayan, Colombia. By using the 15-group level from a hierarchical clustering procedure, it was found that 7 groups were formed with mainly Andean common bean accessions (Andean gene pool), 7 groups with mainly Middle American accessions (Middle American gene pool), while 1 group contained mixed accessions. Consistent with the theory of co-evolution, it was generally observed that accessions from the Andean gene pool were resistant to Middle American pathogen isolates causing anthracnoxe, while the Middle American accessions were resistant to pathogen isolates from the Andes. Different combinations of resistance patterns were found, and breeders can use this information to select a specific group of accessions on the basis of their need.

Adaptive sequence evolution in a color gene involved in the formation of the characteristic egg-dummies of male haplochromine cichlid fishes.

BACKGROUND: The exceptionally diverse species flocks of cichlid fishes in East Africa are prime examples of parallel adaptive radiations. About 80% of East Africa's more than 1 800 endemic cichlid species, and all species of the flocks of Lakes Victoria and Malawi, belong to a particularly rapidly evolving lineage, the haplochromines. One characteristic feature of the haplochromines is their possession of egg-dummies on the males' anal fins. These egg-spots mimic real eggs and play an important role in the mating system of these maternal mouthbrooding fish. RESULTS: Here, we show that the egg-spots of haplochromines are made up of yellow pigment cells, xanthophores, and that a gene coding for a type III receptor tyrosine kinase, colony-stimulating factor 1 receptor a (csf1ra), is expressed in egg-spot tissue. Molecular evolutionary analyses reveal that the extracellular ligand-binding and receptor-interacting domain of csf1ra underwent adaptive sequence evolution in the ancestral lineage of the haplochromines, coinciding with the emergence of egg-dummies. We also find that csf1ra is expressed in the egg-dummies of a distantly related cichlid species, the ectodine cichlid Ophthalmotilapia ventralis, in which markings with similar functions evolved on the pelvic fin in convergence to those of the haplochromines. CONCLUSION: We conclude that modifications of existing signal transduction mechanisms might have evolved in the haplochromine lineage in association with the origination of anal fin egg-dummies. That positive selection has acted during the evolution of a color gene that seems to be involved in the morphogenesis of a sexually selected trait, the egg-dummies, highlights the importance of further investigations of the comparative genomic basis of the phenotypic diversification of cichlid fishes.

Caracterização do Tomato chlorotic spot virus isolado de jiló no Vale do Paraíba, Estado de São Paulo

Os tospovírus são responsáveis por perdas significativas em diversas culturas, principalmente solanáceas. No município de São José dos Campos (SP), plantas de jiló (Solanum gilo) apresentando sintomas de mosaico, bolhosidades, nanismo e queda acentuada da produção foram coletadas para análise. Visando a caracterização do agente causador dos sintomas, testes biológicos, elétrono microscópicos, sorológicos e moleculares foram realizados. Através de inoculação mecânica em plantas indicadoras das famílias Amaranthaceae, Chenopodiaceae e Solanaceae obtiveram-se resultados típicos aos esperados para tospovírus. Ao microscópio eletrônico de transmissão, observaram-se, em contrastação negativa, partículas pleomórficas com diâmetro entre 80 e 110 nm e em cortes ultra-finos partículas presentes em vesículas do retículo endoplasmático. Através de DAS-ELISA, identificou-se o Tomato chlorotic spot virus (TCSV). A partir de RNA total extraído de folhas infetadas, amplificaram-se, via RT-PCR, fragmentos correspondentes ao gene da proteína do capsídeo (cp) os quais foram seqüenciados e comparados com outros depositados no "GenBank". A homologia de nucleotídeos e aminoácidos deduzidos foi respectivamente de 99 e 95% quando comparada com seqüências de isolados de TCSV. A comparação com as outras espécies do gênero Tospovirus apresentou valores de homologia entre 72 e 84%. Estes resultados confirmam a identidade deste vírus como pertencente à espécie TCSV, que é predominante no Estado de São Paulo e importante patógeno de outras plantas cultivadas. Além disso, variedades de jiló quando inoculadas foram susceptíveis tanto ao TCSV como às espécies Tomato spotted wilt virus (TSWV) e Groundnut ringspot virus (GRSV).