Rapid increases in cytokinin concentration in lateral buds of chickpea (Cicer arietinum L) during release of apical dominance

We examined the role of cytokinins (CKs) in release of apical dominance in lateral buds of chickpea (Cicer arietinum L.). Shoot decapitation or application of CKs (benzyladenine, zeatin or dihydrozeatin) stimulated rapid bud growth. Time-lapse video recording revealed growth initiation within 2 h of application of 200 pmol benzyladenine or within 3 h of decapitation. Endogenous CK content in buds changed little in the first 2 h after shoot decapitation, but significantly increased by 6 h, somewhat later than the initiation of bud growth. The main elevated CK was zeatin riboside, whose content per bud increased 7-fold by 6 h and 25-fold by 24 h. Lesser changes were found in amounts of zeatin and isopentenyl adenine CKs. We have yet to distinguish whether these CKs are imported from the roots via the xylem stream or are synthesised in situ in the buds, but CKs may be part of an endogenous signal involved in lateral bud growth stimulation following shoot decapitation. To our knowledge, this is the first detailed report of CK levels in buds themselves during release of apical dominance.

Differential display and suppression subtractive hybridization analysis of the pulp of ripening banana

The fruit of banana undergoes several important physico-chemical changes during ripening. Analysis of gene expression would permit identification of important genes and regulatory elements involved in this process. Therefore, transcript profiling of preclimacteric and climacteric fruit was performed using differential display and Suppression subtractive hybridization. Our analyses resulted in the isolation of 12 differentially expressed cDNAs, which were confirmed by dot-blots and northern blots. Among the sequences identified were sequences homologous to plant aquaporins, adenine nucleotide translocator, immunophilin, legumin-like proteins, deoxyguanosine kinase and omega-3 fatty acid desaturase. Some of these cDNAs correspond to newly isolated genes involved in changes related to the respiratory climacteric, or stress-defense responses. Functional characterization of ripening-associated genes could provide information useful in controlling biochemical pathways that would have an impact on banana quality and shelf life. (C) 2009 Elsevier B.V. All rights reserved.

Human leukocyte antigen (HLA) and single nucleotide polymorphisms (SNPs) tumor necrosis factor (TNF)-alpha-238 and-308 as genetic markers of susceptibility to psoriasis and severity of the disease in a long-term follow-up Brazilian study

Background The strongest genetic marker for psoriasis is Cw*06. Polymorphisms in the tumor necrosis factor (TNF)-alpha promoter region, especially replacement of guanine with adenine in positions -238 and -308 are related to higher TNF-alpha production and higher risk for psoriasis in Caucasoid populations, not found in Asians. We performed a case-control study of 69 patients with psoriasis type I and 70 controls, characterized clinical progression along 10-years of follow-up in mild or severe disease and determined HLA class I, II, and TNF single nucleotide polymorphisms (SNPs) -238 and -308 polymorphisms to demonstrate whether these polymorphisms may be genetic risk for susceptibility to psoriasis or severity of the disease in Brazilians. Methods Polymorphisms were identified using PCR/SSP. Alleles, genotypes, and haplotypes frequencies were compared using Fisher`s test. Results More severe disease was found in male patients. It may be suggested that alleles B*37, Cw*06, Cw*12, and DRB1*07 were associated with severe disease course, while B*57 with mild disease. No statistical difference was found between the patients and controls regarding polymorphisms frequencies in TNF SNPs. This study pointed to a higher TNF-238 G/G genotype frequency (OR: 3.21; CI: 1.06-9.71; P = 0.04) in the group with severe disease. Conclusions Polymorphisms in the TNF-alpha SNPs do not seem to be a more important genetic risk factor for psoriasis than the already known Cw*06 in Brazilian patients, but these markers may be related to clinical manifestations.

Analysis of five polymorphic DNA markers for indirect genetic diagnosis of haemophilia A in the Brazilian population

Hemophilia A is an X-linked, inherited, bleeding disorder caused by the partial or total inactivity of the coagulation factor VIII (FVIII). Due to difficulties in the direct recognition of the disease-associated mutation in the F8 gene, indirect diagnosis using polymorphic markers located inside or close to the gene is used as an alternative for determining the segregation of the mutant gene within families and thus for detecting carrier individuals and/or assisting in prenatal diagnosis. This study characterizes the allelic and haplotype frequencies, genetic diversity, population differentiation and linkage disequilibrium of five microsatellites (F8Int1, F8Int13, F8Int22, F8Int25.3 and IKBKG) in samples of healthy individuals from Sao Paulo, Rio Grande do Sul and Pernambuco and of patients from Sao Paulo with haemophilia A to determine the degree of informativeness of these microsatellites for diagnostic purposes. The interpopulational diversity parameters highlight the differences among the analyzed population samples. Regional differences in allelic frequencies must be taken into account when conducting indirect diagnosis of haemophilia A. With the exception of IKBKG, all of the microsatellites presented high heterozygosity levels. Using the markers described, diagnosis was possible in 10 of 11 families. The F8Int22, F8Int1, F8Int13, F8Int25.3 and IKBKG microsatellites were informative in seven, six, five and two of the cases, respectively, demonstrating the effectiveness of using these microsatellites in prenatal diagnosis and in carrier identification in the Brazilian population.

Sequence variation of the alpha-lactalbumin gene in Holstein and Nellore cows

The alpha-lactalbumin is a subunit of lactose-synthase, an enzyme responsible for lactose production, a disaccharide that influences milk production. Sequence variations of bovine alpha-lactalbumin have been associated with differences in milk yield. This study aimed to analyze allelic frequency differences at position-1689 (g. AG) and+15 (g. AG) of the alpha-lactalbumin gene in Holstein (Bos taurus) and Nellore (Bos indicus) cows. Blood samples were analyzed from 34 Holstein, 104 Nellore, and 99 Dairy Nellore cows using PCR-RFLP. The different RFLP patterns were sequenced and a novel sequence variation on nucleotide-46 was identified. An adenine at this position was designated as the A allele and a guanine was designated B allele. The frequencies of alleles A-1689, A-46, and A+15 differed between Holstein and both Nellore breeds. The results show that differences in alpha-lactalbumin allelic variants in the 5`-flanking and the 5`-UTR region might be associated with differences in milk production between Holstein cows and cows from Nellore breeds. However, the lack of difference between Nellore and Dairy Nellore suggests that other sequence variantions that regulate milk production might be responsible for the selection of Dairy Nellore cows with superior milk production.

GABAA receptor β3 subunit gene and psychiatric morbidity in a post-traumatic stress disorder population

GABAergic systems have been implicated in the pathogenesis of anxiety, depression and insomnia. These symptoms are part of the core and comorbid psychiatric disturbances in post-traumatic stress disorder (PTSD) In a sample of Caucasian male PTSD patients, dinucleotide repeat polymorphisms of the GABAA receptor beta3 subunit gene were compared to scores on the General Health Questionnaire-28 (GHQ). As the major allele at this gene locus (GABRB3) was GI, the alleles were divided into GI and non-GI groups. On the total score of the GHQ, which comprises the somatic symptoms, anxiety/insomnia, social dysfunction and depression subscales, patients with the GI non-GI genotype had a significantly higher score when compared to either the G1G1 genotype (alpha = 0.01) or the non-GI non-GI genotype (alpha = 0.05). No significant difference was found between the G1G1 and non-Gl non-G1 genotypes. When the GI non-G1 heterozygotes were compared to the combined G1G1 and non-GI non-GI homozygotes, a significantly higher total GHQ score was found in the heterozygotes (P = 0.002). These observations suggest a heterosis effect. Further analysis of GHQ subscale scores showed that heterozygotes compared to the combined homozygotes had higher scores on the somatic symptoms (P = 0.006), anxiety/insomnia (P = 0.003), social dysfunction (P = 0.054) and depression (P = 0.004) subscales. In conclusion, the present study indicates that in a population of PTSD patients, heterozygosity of the GABRB3 major (GI) allele confers higher levels of somatic symptoms, anxiety/insomnia, social dysfunction and depression than found in homozygosity. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Low intraspecific variation in the rRNA internal transcribed spacer 2 (ITS2) of the Australian paralysis tick, Ixodes holocyclus

Ixodes holocyclus has a narrow, discontinuous distribution along the east coast of Australia. We studied ticks from 17 localities throughout the geographic range of this tick. The ITS2 of I. holocyclus is 793 bp long. We found nucleotide variation at eight of the 588 nucleotide positions (1.4%) that were compared for all ticks. There were eight different nucleotide sequences. Most sequences were not restricted to a particular geographic region. However, sequences F, G and H, which had an adenine at position 197, were found only in the far north of Queensland - all other ticks had a guanine at this position. The low level of intraspecific variation in this tick (0.7%) contrasts with the sequence divergence between L holocyclus and its close relative, I. cornuatus (13.1 %). These data indicate that L holocyclus does not contain cryptic species despite possible geographic isolation of some populations. We conclude that variation in the ITS2 is likely to be informative about the phylogeny of the group.

Molecular analysis of dimethyl sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide reductase family of microbial molybdopterin-containing enzymes

Dimethyl sulphide dehydrogenase catalyses the oxidation of dimethyl sulphide to dimethyl sulphoxide (DMSO) during photoautotrophic growth of Rhodovulum sulfidophilum . Dimethyl sulphide dehydrogenase was shown to contain bis (molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor unique to enzymes of the DMSO reductase family. Sequence analysis of the ddh gene cluster showed that the ddhA gene encodes a polypeptide with highest sequence similarity to the molybdop-terin-containing subunits of selenate reductase, ethylbenzene dehydrogenase. These polypeptides form a distinct clade within the DMSO reductase family. Further sequence analysis of the ddh gene cluster identified three genes, ddhB , ddhD and ddhC . DdhB showed sequence homology to NarH, suggesting that it contains multiple iron-sulphur clusters. Analysis of the N-terminal signal sequence of DdhA suggests that it is secreted via the Tat secretory system in complex with DdhB, whereas DdhC is probably secreted via a Sec-dependent mechanism. Analysis of a ddhA mutant showed that dimethyl sulphide dehydrogenase was essential for photolithotrophic growth of Rv. sulfidophilum on dimethyl sulphide but not for chemo-trophic growth on the same substrate. Mutational analysis showed that cytochrome c (2) mediated photosynthetic electron transfer from dimethyl sulphide dehydrogenase to the photochemical reaction centre, although this cytochrome was not essential for photoheterotrophic growth of the bacterium.

Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture

Rapid accumulation of few polyhedra (FP) mutants was detected during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in cell culture. 100% FP infected cells were observed by passage 6. The specific yield decreased from 178 polyhedra per cell at passage 2 to two polyhedra per cell at passage 6. The polyhedra at passage 6 were not biologically active, with a 28-fold reduction in potency compared to passage 3. Electron microscopy studies revealed that very few polyhedra were produced in an FP infected cell (< 10 polyhedra per section) and in most cases these polyhedra contained no virions. A specific failure in the intranuclear nucleocapsid envelopment process in the FP infected cells, leading to the accumulation of naked nucleocapsids, was observed. Genomic restriction endonuclease digestion profiles of budded virus DNA from all passages did not indicate any large DNA insertions or deletions that are often associated with such FP phenotypes for the extensively studied Autographa californica nucleopolyhedrovirus and Gaileria mellonella nucleopolyhedrovirus. Within an HaSNPV 25K FP gene homologue, a single base-pair insertion (an adenine residue) within a region of repetitive sequences (seven adenine residues) was identified in one plaque-purified HaSNPV FP mutant. Furthermore, the sequences obtained from individual clones of the 25KFP gene PCR products of a late passage revealed point mutations or single base-pair insertions occurring throughout the gene. The mechanism of FP mutation in HaSNPV is likely similar to that seen for Lymantria dispar nucleopolyhedrovirus, involving point mutations or small insertions/deletions of the 25K FP gene.

The DMSO reductase family of microbial molybdenum enzymes; Molecular properties and role in the dissimilatory reduction of toxic elements

The dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes is a large and diverse group that is found in bacteria and archaea. These enzymes are characterised by a bis(molybdopterin guanine dinucleotide)Mo form of the molybdenum cofactor, and they are particularly important in anaerobic respiration including the dissimilatory reduction of certain toxic oxoanions. The structural and phylogenetic relationship between the proteins of this family is discussed. High-resolution crystal structures of enzymes of the DMSO reductase family have revealed a high degree of similarity in tertiary structure. However, there is considerable variation in the structure of the molybdenum active site and it seems likely that these subtle but important differences lead to the great diversity of function seen in this family of enzymes. This diversity of catalytic capability is associated with several distinct pathways of electron transport.

Development and characterization of polymorphic microsatellite markers in taro (Colocasia esculenta)

Microsatellite-containing sequences were isolated from enriched genomic libraries of taro (Colocasia esculenta (L.) Schott). The sequencing of 269 clones yielded 77 inserts containing repeat motifs. The majority of these (81.7%) were dinucleotide or trinucleotide repeats. The GT/CA repeat motif was the most common, accounting for 42% of all repeat types. From a total of 43 primer pairs designed, 41 produced markers within the expected size range. Sixteen (39%) were polymorphic when screened against a restricted set of taro genotypes from Southeast Asia and Oceania, with an average of 3.2 alleles detected on each locus. These markers represent a useful resource for taro germplasm management, genome mapping, and marker-assisted selection.

Characterization of microsatellite loci in Anopheles flavirostris, the principal malaria vector in the Philippines

Microsatellites were isolated and characterized from Anopheles flavirostris, the principal malaria vector in the Philippines. Fifty of the 150 positive clones sequenced contained mostly dinucleotide microsatellites and only 16 had trinucleotide repeats. We designed primers from the unique sequences flanking 18 microsatellite loci. Of these, 11 loci produced successful amplification and revealed high levels of polymorphism; 86 alleles were detected with allele number ranging from 2 to 16 at each locus. The high allelic variability will make these microsatellite loci very useful for taxonomic and population genetic studies.

Isolation and characterization of nine microsatellite loci from the Hawaiian grouper Epinephelus quernus (Serranidae) for population genetic analyses

The availability of variable genetic markers for groupers (Serranidae) has generally been limited to mitochondrial DNA. For studies of population genetic structure, more loci are usually required; particularly useful are those that are nuclear in origin such as microsatellites. Here, we isolated and characterized 9 microsatellite loci from the endemic Hawaiian grouper Epinephelus quernus using a biotin-labeled oligonucleotide-streptavidin-coated magnetic bead approach. Of the 20 repeat-containing fragments isolated, 15 had sufficient flanking region in which to design primers. Among these, 9 produced consistent polymerase chain reaction product, and 6 were highly variable. These 6 loci were all composed of dinucleotide repeats, with the number of alleles ranging from 6 to 18, and heterozygosities from 33.3% to 91.7%. The high levels of variability observed should make these markers useful for population genetic studies of E. quernus, and potentially other epinephelines.

Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [H-3]zeatin riboside ([H-3]ZR), a water-soluble blue dye or [H-3]H2O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within I h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [H-3]ZR and of blue dye in the same bud position was increased 3- to 10-fold relative to intact plants, whereas content of [H-3]H2O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [H-3]H2O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [H-3]ZR injected at physiological concentrations. Within 1 h, 80-95% of [H-3]ZR was converted to other active CKs (mainly zeatin riboside-5'phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O-acetylZR, O-acetylZRMP and a compound correlated with sites of high CK-concentrations) and inactive catabolites (adenosine, adenine, 5'AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.