Journal articles on the topic 'Wzb gene'

ABSTRACT Group 1 capsular polysaccharides (CPSs) of Escherichia coli and some loosely cell-associated exopolysaccharides (EPSs), such as colanic acid, are assembled by a Wzy-dependent polymerization system. In this biosynthesis pathway, Wza, Wzb, and Wzc homologues are required for surface expression of wild-type CPS or EPS. Multimeric complexes of Wza in the outer membrane are believed to provide a channel for polymer export; Wzc is an inner membrane tyrosine autokinase and Wzb is its cognate phosphatase. This study was performed to determine whether the Wza, Wzb, and Wzc proteins for colanic acid expression in E. coli K-12 could function in the E. coli K30 prototype group 1 capsule system. When expressed together, colanic acid Wza, Wzb, and Wzc could complement a wza-wzb-wzc defect in E. coli K30, suggesting conservation in their collective function in Wzy-dependent CPS and EPS systems. Expressed individually, colanic acid Wza and Wzb could also function in K30 CPS expression. In contrast, the structural requirements for Wzc function were more stringent because colanic acid Wzc could restore translocation of K30 CPS to the cell surface only when expressed with its cognate Wza protein. Chimeric colanic acid-K30 Wzc proteins were constructed to further study this interaction. These proteins could restore K30 biosynthesis but were unable to couple synthesis to export. The chimeric protein comprising the periplasmic domain of colanic acid Wzc was functional for effective K30 CPS surface expression only when coexpressed with colanic acid Wza. These data highlight the importance of Wza-Wzc interactions in group 1 CPS assembly.

ABSTRACT Escherichia coli group 1 K antigens form a tightly associated capsule structure on the cell surface. Although the general features of the early steps in capsular polysaccharide biosynthesis have been described, little is known about the later stages that culminate in assembly of a capsular structure on the cell surface. Group 1 capsule biosynthesis gene clusters (cps) in E. coli and Klebsiella pneumoniae include a conserved open reading frame, wzi. The wzi gene is the first of a block of four conserved genes (wzi-wza-wzb-wzc) found in all group 1 K-antigen serotypes. Unlike wza, wzb, and wzc homologs that are found in gene clusters responsible for production of exopolysaccharides (i.e., predominantly cell-free polymer) in a range of bacteria, wzi is found only in systems that assemble capsular polysaccharides. The predicted Wzi protein shows no similarity to any other known proteins in the databases, but computer analysis of Wzi predicted a cleavable signal sequence. Wzi was expressed with a C-terminal hexahistidine tag, purified, and used for the production of specific antibodies that facilitated localization of Wzi to the outer membrane. Circular dichroism spectroscopy indicates that Wzi consists primarily of a β-barrel structure, and dynamic light scattering studies established that the protein behaves as a monomer in solution. A nonpolar wzi chromosomal mutant retained a mucoid phenotype and remained sensitive to lysis by a K30-specific bacteriophage. However, the mutant showed a significant reduction in cell-bound polymer, with a corresponding increase in cell-free material. Furthermore, examination of the mutant by electron microscopy showed that it lacked a coherent capsule structure. It is proposed that the Wzi protein plays a late role in capsule assembly, perhaps in the process that links high-molecular-weight capsule to the cell surface.

ABSTRACT Strains of Vibrio vulnificus incubated in situ in natural estuarine waters during warm months continued to express katG (periplasmic catalase), rpoS (stress sigma factor), tufA (elongation factor), wza, and wzb (capsule synthesis). vvhA (hemolysin) was differentially expressed between environmental and clinical isolates. These results paralleled our in vitro findings.

ABSTRACT The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1 are clustered within a 27-kbp region termed the wee cluster. This report demonstrates the involvement of two genes of the wee cluster of RAG-1, wzb and wzc, in emulsan biosynthesis. The two gene products, Wzc and Wzb were overexpressed and purified. Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity. Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc. Using the synthetic substrate p-nitrophenyl phosphate (PNPP) Wzb exhibited a V max of 12 μmol of PNPP min−1 mg−1 and a Km of 8 mM PNPP at 30°C. The emulsifying activity of mutants lacking either wzb or wzc was 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production. Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus. Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency. Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively. Neither of these mutants contained cell-associated emulsan. However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide. A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation.

ABSTRACT Vibrio vulnificus causes both food-borne disease and wound infections. Most V. vulnificus strains express capsular polysaccharide (CPS), which is required for the virulence of this organism. Under standard growth conditions, CPS expression is lost at a relatively high frequency (10−3 to 10−4), resulting in a switch from an opaque (Op, CPS+) colony morphology to a translucent (Tr, CPS−) colony morphology. The wzb gene, which encodes a phosphatase required for CPS expression, has been proposed to be involved in this switch through a site-specific deletion of the entire gene. In an examination of five strains, we found that the frequency of wzb deletion in Tr colonies varies by strain and therefore does not account for all the Tr colonies that are seen. In addition, we have identified a third, intermediate (Int) colony morphotype, in which the colonies appear less opaque but are not fully translucent. PCR studies have demonstrated that Int colonies still contain the wzb gene, while reverse transcriptase PCR studies have shown that although Int strains retain expression of wzb, in some cases the transcription of wzb is reduced. Int strains switch to a true Tr (wzb negative) morphotype at a very high frequency (nearly 100%) under certain conditions. Finally, Int colonies, which in some cases can easily be mistaken for Tr colonies, have been observed to occasionally revert to Op, while Tr colonies containing a wzb deletion presumably are unable to revert to the encapsulated form.

ABSTRACT Two proteins of Escherichia coli, termed Wzc and Wzb, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, Wzc was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive ATP, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, Wzb was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate intop-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Wzb was able to dephosphorylate previously autophosphorylated Wzc. Together, these data demonstrate, for the first time, that E. coli cells contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. From comparative analysis of amino acid sequences, Wzc was found to be similar to a number of proteins present in other bacterial species which are all involved in the synthesis or export of exopolysaccharides. Since these polymers are considered important virulence factors, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.

The exopolysaccharide (EPS) biosynthesis gene clusters of four Lactobacillus rhamnosus strains consist of chromosomal DNA regions of 18·5 kb encoding 17 ORFs that are highly similar among the strains. However, under identical conditions, EPS production varies considerably among these strains, from 61 to 1611 mg l−1. Fifteen genes are co-transcribed starting from the first promoter upstream of wzd. Nevertheless, five transcription start sites were identified by 5′-RACE PCR analysis, and these were associated with promoter sequences upstream of wzd, rmlA, welE, wzr and wzb. Six potential glycosyltransferase genes were identified that account for the assembly of the heptasaccharide repeat unit containing an unusually high proportion of rhamnose. Four genes involved in the biosynthesis of the sugar nucleotide precursor dTDP-l-rhamnose were identified in the EPS biosynthesis locus, which is unusual for lactic acid bacteria. These four genes are expressed from their own promoter (P2), as well as co-transcribed with the upstream EPS genes, resulting in coordinated production of the rhamnose precursor with the enzymes involved in EPS biosynthesis. This is believed to be the first report demonstrating that the sequence, original organization and transcription of genes encoding EPS production are highly similar among four strains of Lb. rhamnosus, and do not vary with the amount of EPS produced.

ABSTRACT Group 1 capsules of Escherichia coli are similar to the capsules produced by strains of Klebsiella spp. in terms of structure, genetics, and patterns of expression. The striking similarities between the capsules of these organisms prompted a more detailed investigation of the cps loci encoding group 1 capsule synthesis. Six strains of K. pneumoniae and 12 strains of E. coli were examined. PCR analysis showed that the clusters in these strains are conserved in their chromosomal locations. A highly conserved block of four genes,orfX-wza-wzb-wzc, was identified in all of the strains. Thewza and wzc genes are required for translocation and surface assembly of E. coli K30 antigen. The conservation of these genes points to a common pathway for capsule translocation. A characteristic JUMPstart sequence was identified upstream of each cluster which may function in conjunction with RfaH to inhibit transcriptional termination at a stem-loop structure found immediately downstream of the “translocation-surface assembly” region of the cluster. Interestingly, the sequence upstream of thecps clusters in five E. coli strains and oneKlebsiella strain indicated the presence of IS elements. We propose that the IS elements were responsible for the transfer of thecps locus between organisms and that they may continue to mediate recombination between strains.

Vibrio vulnificus populates coastal waters around the world, where it exists freely or becomes concentrated in filter feeding mollusks. It also causes rapid and life-threatening sepsis and wound infections in humans. Of its many virulence factors, it is the V. vulnificus capsule, composed of capsular polysaccharide (CPS), that plays a critical role in evasion of the host innate immune system by conferring antiphagocytic ability and resistance to complement-mediated killing. CPS may also provoke a portion of the host inflammatory cytokine response to this bacterium. CPS production is biochemically and genetically diverse among strains of V. vulnificus, and the carbohydrate diversity of CPS is likely affected by horizontal gene transfer events that result in new combinations of biosynthetic genes. Phase variation between virulent encapsulated opaque colonial variants and attenuated translucent colonial variants, which have little or no CPS, is a common phenotype among strains of this species. One mechanism for generating acapsular variants likely involves homologous recombination between repeat sequences flanking the wzb phosphatase gene within the Group 1 CPS biosynthetic and transport operon. A considerable number of environmental, genetic, and regulatory factors have now been identified that affect CPS gene expression and CPS production in this pathogen.

ABSTRACT Isolation of Vibrio vulnificus during winter months is difficult due to the entrance of these cells into the viable but nonculturable (VBNC) state. While several studies have investigated in vitro gene expression upon entrance into and persistence within the VBNC state, to our knowledge, no in situ studies have been reported. We incubated clinical and environmental isolates of V. vulnificus in estuarine waters during winter months to monitor the expression of several genes during the VBNC state and compared these to results from in vitro studies. katG (periplasmic catalase) was down-regulated during the VBNC state in vitro and in situ compared to the constitutively expressed gene tufA. Our results indicate that the loss of catalase activity we previously reported is a direct result of katG repression, which likely accounts for the VBNC response of this pathogen. While expression of vvhA (hemolysin) was detectable in environmental strains during in situ incubation, it ceased in all cases by ca. 1 h. These results suggest that the natural role of hemolysin in V. vulnificus may be in osmoprotection and/or the cold shock response. Differences in expression of the capsular genes wza and wzb were observed in the two recently reported genotypes of this species. Expression of rpoS, encoding the stress sigma factor RpoS, was continuous upon entry into the VBNC state during both in situ and in vitro studies. We found the half-life of mRNA to be less than 60 minutes, confirming that mRNA detection in these VBNC cells is a result of de novo RNA synthesis.

Secoisolariciresinol (SECO) is a lignan of potential therapeutic value for diseases such as cancer, but its use has been limited by the lack of ideal production methods, even though its precursors are abundant in plants, such as flaxseeds. Here, we report the characterization of a bacterial strain, S1, isolated from the human intestinal flora, which could produce secoisolariciresinol by biotransformation of precursors in defatted flaxseeds. This bacterium was a Gram-negative and facultatively anaerobic straight rod without capsules. Biochemical assays showed that it was negative for production of oxidase, lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, and β-glucolase. The G + C content of genomic DNA was 57.37 mol%. Phylogenetic analysis by 16S rRNA and rpoB gene sequences demonstrated S1’s close relatedness to Klebsiella. No homologues were found for wzb or wzc (capsular genes), which may explain why Klebsiella sp. strain S1 does not have the capsule and was isolated from a healthy human individual. Based on the percentages of homologous genes with identical nucleotide sequences between the bacteria in comparison, we found that clear-cut genetic boundaries had been formed between S1 and any other Klebsiella strains compared, dividing them into distinct phylogenetic lineages. This work demonstrates that the intestinal Klebsiella, well known as important opportunistic pathogens prevalent in potentially fatal nosocomial infections, may contain lineages that are particularly beneficial to the human health.

Data is considered to be the important element in the field of Data Science and Machine Learning. Performance of Machine Learning and Data Mining algorithms greatly influenced by the characteristics of data and data with missing values. Performance of all these Machine Learning algorithms greatly improved and they can give accurate results when the data is in full without missing values. So before applying these algorithms; dataset and its missing values are completely filled. To impute these missing values in the dataset there are numerous methods were proposed. In this paper we used micro array gene expression dataset; by introducing various percentages of missing values a new methodology is proposed to impute these missing values in the data set. The nature of micro array gene expression dataset is huge in dimensionality, so at first, we used recursive feature elimination method to select the best features which contributes much for model was selected then we apply the Ridge Regression for imputation. Imputations with other methods are compared. We evaluate the performance of all models by using the metrics like MSE, MAE, R-square. To select the best model in the set of models we used Normalized Criteria Distance (NCD) to rank the models under proposed metrics. The model with least NCD rank selected as the best model among other models, in our paper proposed model has got the lowest value among other models and considered to be the best model among other models.

Huntington gene is located on chromosome 4p16.3 IT15 locus considered a major causative gene of Huntington disorder. HTT is a neurodegenerative disorder mutation in gene cause abnormal movements and psychiatric disturbances. HTT is inherited in an autosomal dominant manner with almost complete penetrance and till now, no research studies provide insight into HTT gene. Bioinformatics analysis includes transcription factors binding sites, phylogenetic studies with reference to various selected orthologs and syntenic relationship of HTT gene. Our study showed that in HTT gene majority of the portion is conserved among two orthologs chimpanzee and mouse in significance to human. These studies also revealed information about conservation of genes among different ortholog species and their evolutionary relationship.

Ribonucleic acid (RNA) silencing, RNA interference (RNAi) or post-transcriptional gene silencing takes place in a variety of eukaryotes and it was discovered firstly in the plants. The RNA silencing process is activated by a trigger from dsRNA predecessor. A very important step in the silencing pathways the conversion of dsRNA into small duplexes of RNA of the representative length and arrangement. Then these small dsRNA monitor RNA silencing by different mechanisms. Post transcriptional gene silencing mechanisms were initially identified as an anti-viral process that give protection to the organisms from the viruses or which inhibit the unsystematic incorporation of transposable components. The basic aim of this review article is to study the mechanism of gene silencing by dsRNA and the roles of certain proteins in cellular post transcriptional RNA silencing machinery and finally we also discuss the RNA silencing as an anti-viral defense mechanism in the plants.

Noninvasive in vivo molecular imaging has developed over the past decade and involves nuclear (positron emission tomography [PET], gamma camera), magnetic resonance, and in vivo optical imaging systems. Most current in vivo molecular imaging strategies are “indirect” and involve the coupling of a “reporter gene” with a complementary “reporter probe.” Imaging the level of probe accumulation provides indirect information related to the level of reporter gene expression. Reporter gene constructs are driven by upstream promoter/enhancer elements; reporter gene expression can be constitutive, leading to continuous transcription and used to identify the site of transduction and to monitor the level and duration of gene (vector) activity. Alternatively, reporter gene expression can be inducible, leading to controlled gene expression, or reporter genes can function as a “sensor” to monitor the level of endogenous promoters and transcription factors. The development of versatile and sensitive assays that do not require tissue sampling will be of considerable value for monitoring molecular-genetic and cellular processes in animal models of human disease, as well as for studies in human subjects in the future. Noninvasive imaging of molecular-genetic and cellular processes will complement established ex vivo molecular-biologic assays that require tissue sampling, and will provide a spatial as well as a temporal dimension to our understanding of various diseases. Several examples of imaging endogenous biologic processes in animals using reporter constructs, radiolabeled probes, and PET imaging are reviewed (e.g., p53-dependent gene expression, T-cell receptor-dependent activation of T-lymphocytes, and preliminary studies of endogenous HIF-1α expression). Issues related to the translation of noninvasive molecular imaging technology into the clinic are also discussed.

Citrus greening caused by (Candidatus liberibacter spp.) is one of the most destructive bacterial disease of citrus all over the world and it is transmitted by grafting and psyllid (Diaphorina citri) is responsible for its spread. This study was aimed to detect the prevalence of CLas in different varieties of citrus in Pakistan and molecular characterization of mtco1 gene in Diaphorina citri. DNA was extracted from fourteen different citrus cultivars infected with citrus greening from different orchards of Sargodha Punjab, Pakistan. Specific primers were used to amplify (Mtco1 gene) in Diaphorina citri and also Las specific primers were also used to detect HLB pathogen. Amplified fragments of 507bp, 1500bp and 2500bp were observed in CLas and Mtco1 gene of Asian isolates. The resulted fragments were TA cloned and sequenced from both strands. The infectious bacterium was found in 3 samples out of 14 (21.5 %) while from psyllid it was found in 5 samples (37.5%). The weather changes effect the prevalence of CLas in citrus but usually it decrease during autumn and found Las positive vector in spring or summer season up to 42oC. So there is a need to research on management of citrus greening disease in Pakistan as it caused major yield loses

Gene transfer involves the use of an engineered biologic vehicle known as a vector to introduce a gene encoding a protein of interest into a particular tissue. In diseases with known defects at a genetic level, gene transfer offers a potential means of restoring a normal molecular environment via vector-mediated entry (transduction) and expression of genes encoding potentially therapeutic proteins selectively in diseased tissues. The technology of gene transfer therefore underlies the concept of gene therapy and falls under the umbrella of the current genomics revolution. Particularly since 1995, numerous attempts have been made to introduce genes into intracranial blood vessels to demonstrate and characterize viable transduction. More recently, in attempting to translate cerebrovascular gene transfer technology closer to the clinical arena, successful transductions of normal human cerebral arteries ex vivo and diseased animal cerebral arteries in vivo have been reported using vasomodulatory vectors. Considering the emerging importance of gene-based strategies for the treatment of the spectrum of human disease, the goals of the present report are to overview the fundamentals of gene transfer and review experimental studies germane to the clinical translation of a technology that can facilitate genetic modification of cerebral blood vessels.

Ischemia induces a complex response of differentially expressed genes in the brain. In order to understand the specific mechanisms of injury in the developing brain, it is important to obtain information on global changes in the transcriptome after neonatal hypoxia-ischemia. In this study, oligonucleotide arrays were used to investigate genomic changes at 2, 8, 24, and 72 hours after neonatal hypoxia-ischemia, which was induced in 9-day-old mice by left carotid artery ligation followed by hypoxia (10% O2). In total, 343 genes were differentially expressed in cortex, hippocampus, thalamus, and striatum 2 to 72 hours after hypoxia-ischemia, when comparing ipsilateral with contralateral hemispheres and with controls, using the significance analysis for microarrays. A total of 283 genes were upregulated and 60 were downregulated, and 94% of the genes had not previously been shown after neonatal hypoxia-ischemia. Genes related to transcription factors and metabolism had mostly upregulated transcripts, whereas most downregulated genes belonged to the categories of ion and vesicular transport and signal transduction. Genes involved in transcription, stress, and apoptosis were induced early after the insult, and many new genes that may play important roles in the pathophysiology of neonatal hypoxiaischemia were identified.

Fibroblast growth factor (FGF)-2 is a potent neurotrophic and angiogenic peptide. To examine possible protective effects of FGF-2 gene expression against transient focal cerebral ischemia in rats, a replication defective, recombinant adenovirus vector expressing FGF-2, was injected intraventricularly 2 hours after middle cerebral artery occlusion (MCAO). The treatment group showed significant recovery compared with the vehicle-treated groups in terms of serial neurologic severity scores over the 35 days after MCAO. Further, 2,3,5-triphenyltetrazolium chloride staining showed that FGF-2 gene transfer decreased infarct volume by 44% as compared with that in the vehicle-treated groups at 2 days after MCAO. The same tendency of gene transfer effects on infarct volume was confirmed at 35 days after MCAO with hematoxylin/eosin staining. Enzyme-linked immunosorbent assay revealed that FGF-2 concentration was increased significantly at 2 days after MCAO, not only in cerebrospinal fluid but also in cerebral substance in the lesioned and treated animals. These results suggested that FGF-2 gene transfer using these adenoviral vectors might be a useful modality for the treatment of occlusive cerebrovascular disease even after the onset of stroke.

Brain ischemia triggers an inflammatory reaction that progresses for days to weeks and seems to have a role in secondary progression of injury. Inflammation induces a complex pattern of signaling molecules with partly contradictory actions, and the responses may be different in the immature and adult brain. The authors characterized the global inflammatory gene expression in the developing brain as a first step toward understanding the protective and deleterious effects of inflammation after hypoxia-ischemia. Oligonucleotide arrays were used to investigate inflammatory genes in cortex, hippocampus, thalamus, and striatum at 2, 8, 24, and 72 hours after hypoxia-ischemia, which was induced in 9-day-old mice by left carotid artery ligation followed by hypoxia. After hypoxia-ischemia, 148 inflammatory genes were differentially expressed. More than 97% of the genes were upregulated and 93% had not previously been reported after hypoxia-ischemia in the immature brain. The results indicate that microglia/macrophages, T-and B-cells, NK-cells, mast cells, dendritic cells, and polymorphonuclear leukocytes may participate in the response to hypoxia-ischemia. In addition, novel cytokines/chemokines, complement-related, interferon-regulated, components of the TIR/nuclear factor-κB pathway, and a number of immunomodulatory genes were induced. Several of these genes may be of pathophysiologic significance after neonatal hypoxia-ischemia.

Monocyte chemoattractant protein-1 (MCP-1) is expressed in the ischemic cortex after focal brain ischemia and appears to exacerbate ischemic damage. The authors examined the effect of gene transfer of dominant negative MCP-1, called 7ND, 90 minutes after induction of focal brain ischemia in hypertensive rats. Adenoviral vectors encoding mutant MCP-1 (Ad7ND; n = 11), or Escherichia coli β-galactosidase (AdlacZ; n = 17) as control were injected into the lateral ventricle of male spontaneously hypertensive rats. Both AdlacZ (n = 12) and Ad7ND (n = 6) administration provided transgene expression as early as 6 hours after injection and the expression further increased on day 1, followed by a sustained detection on day 5. Five days after ischemia, infarct volume (75 ± 13 mm3, n = 5, mean ± SD) significantly reduced to 72% of control (104 ± 22 mm3, n = 5, P < 0.05) by 7ND gene transfer. Numbers of leukocytes in the vessels (48.3 ± 32.9/cm2) and macrophage/monocyte infiltration (475.2 ± 125.5 /mm2) of the infarct area in the Ad7ND group were significantly less than those measured in the AdlacZ group (143.8 ± 72.1/cm2 and 671.8 ± 125.5/mm2, P < 0.05, respectively). In summary, the postischemic gene transfer of dominant negative MCP-1 attenuated the infarct volume and infiltration of inflammatory cells, suggesting potential usefulness of the anti–MCP-1 gene therapy.

Starch quality is one of the most important agronomic traits in rice (Oryza sativa L). In this study, we identified single nucleotide polymorphisms (SNPs) in the Waxy and Alk genes of eight rice varieties and their associations with starch physicochemical properties.vi.e.vamylose content (AC) and gelatinization temperature (GT). Seven Sri Lankan rice varieties, Pachchaperumal, Herathbanda, At 354, Bg 352, Balasuriya, H 6 and Bw 295-5 were detected as high amylose varieties while Nipponbare exhibited low amylose content. In silico analysis of the Waxy gene revealed that all tested Sri Lankan varieties possessed ‘G’ (Wxa allele) instead of ‘T’ in the first intron which could explain varieties with high and intermediate amylose content. All Sri Lankan varieties had ‘A’ instead of ‘C’ in exon 6 of the Waxy gene and this fact was tally with the varieties showing high amylose content. Therefore, possessing the Wxa allele in the first intron and ‘A’ in exon 6 could be used as a molecular marker for the selection of high amylose varieties as validated using several Sri Lankan varieties. All Sri Lankan varieties except, Bw 295-5 exhibited the intermediate type of GT which could not be explained using the so far reported allelic differences in the Alk gene. However, Bw 295-5 which is a low GT variety had two nucleotide polymorphisms in the last exon of the Alk gene, i.e. ‘G’ and ‘TT’ that represent low GT class. Therefore, it can be concluded that sequence variations of Waxy and Alk genes reported in this study are useful in breeding local rice varieties with preferential amylose content and GT class.Key word Alk gene, amylose content, single nucleotide polymorphism, Waxy gene.INTRODUCTIONRice (Oryza sativa L.) is one of the leading food crops of the world. More than half of the world’s population relies on rice as the major daily source of calories and protein (Sartaj and Suraweera, 2005). After grain yield, quality is the most important aspect of rice breeding. Grain size and shape largely determine the market acceptability of rice, while cooking quality is influenced by the properties of starch. In rice grains starch is the major component that primarily controls rice quality. Starch consists of two forms of glucose polymers, relatively unbranched amylose and a highly branched amylopectin. Starch-synthesizing genes may contribute to variation in starch physicochemical properties because they affect the amount and structure of amylose and amylopectin in rice grain (Kharabian-Masouleh et al., 2012). Amylose content (AC), gelatinization temperature (GT) and gel consistency (GC) is the three most important determinants of eating and cooking quality. Amylose content is the ratio of amylose amount present in endosperm to total starch content. Rice varieties are grouped based on their amylose content into waxy (0-2%), very low (3-9%), low (10-19%), intermediate (20-25%), and high (> 25%) (Kongseree and Juliano, 1972). The most widely used method for amylose determination is a colorimetric assay where iodine binds with amylose to produce a blue-purple color, which is measured spectrophotometrically at a single wavelength (620nm). Low amylose content is usually associated with tender, cohesive and glossy cooked rice; while, high amylose content is associated with firm, fluffy and separate grains of cooked rice. The Waxy (Wx) gene, which encodes granule-bound starch synthase I (GBSSI), is the major gene controlling AC in rice (Nakamura, 2002). The Waxy gene is located on chromosome six and various single nucleotide polymorphisms (SNPs) of Wx were found, including a ‘G’ to ‘T’ SNP of the first intron, ‘A’ to ‘C’ SNP of the sixth exon and ‘C’ to ‘T’ SNP of the tenth exon (Larkin and Park, 2003). The ‘AGGTATA’ sequence at the 5’splice-junction coincides with the presence of the Wxa allele, while the ‘AGTTATA’ sequence coincides with the presence of the Wxb allele. Therefore, all intermediate and high amylose cultivars had ‘G’ nucleotide while low amylose cultivars had ‘T’ nucleotide at the putative leader intron 5′ splice site. The cytosine and thymidine (CT) dinucleotide repeats in the 5’- untranslated region (UTR) of the Waxy gene were reported to be a factor associated with AC. However, the relationship between these polymorphisms and amylose contents is not clear. Amylopectin chain length distribution plays a very important role to determine GT in cooked rice. The time required for cooking is determined by the gelatinization temperature of starch. It is important because it affects the texture of cooked rice and it is related to the cooking time of rice. The gelatinization temperature is estimated by the alkali digestibility test. It is measured by the alkali spreading value (ASV). The degree of spreading value of individual milled rice kernels in a weak alkali solution (1.7% KOH) is very closely correlated with gelatinized temperature. According to the ASV, rice varieties may be classified as low (55 to 69°C), intermediate (70 to 74°C) and high (> 74°C) GT classes. In a breeding program ASV is extensively used to estimate the gelatinization temperature. The synthesis of amylopectin is more complex than that of amylose. Polymorphisms in the starch synthase IIa (SSIIa) gene which is recognized as the Alk gene are responsible for the differences in GT in rice (Umemoto and Aoki, 2005; Waters et al., 2006). Two single nucleotide polymorphisms (SNPs) in the last exon of the Alk gene are responsible for the differences in GT in rice. The biochemical analysis clearly showed that the function of the amino acids caused by these two SNPs is essential for SSIIa enzyme activity (Nakamura et al., 2005) and those are ‘G’/‘A’ SNP at 4424 bp position and ‘GC’/‘TT’ SNPs at 4533/4534 bp position with reference to Nipponbare rice genomic sequence. Based on the SNPs, Low SSIIa enzyme activity results in S-type amylopectin, which is enriched in short chains whereas high SSIIa enzyme activity produces L-type amylopectin (Umemoto et al., 2004). Therefore, the combination of ‘G’ at SNP3 and ‘GC’ at SNP4 is required to produce L-type rice starch and this has a higher GT relative to S-type starch. GC is a standard assay that is used in rice improvement programs to determine the texture of softness and firmness in high amylose rice cultivars. Intermediate and low amylose rice usually has soft gel consistency. Sequence variation in exon 10 of the Waxy gene associates with GC (Tran et al., 2011).OBJECTIVES The objectives of this study were to detect polymorphisms in major starch synthesizing genes among several rice cultivars as models and to determine the relationship between their SNP variations and starch physicochemical properties. Also, we analyzed major starch synthesizing gene sequences of several Sri Lankan rice varieties in silico aiming at utilizing this information in rice breeding programs.MATERIALS AND METHODSPlant materials: Seeds of eight Oryza sativa L. accessions were obtained from the Rice Research and Development Institute (RRDI), Bathalagoda, Sri Lanka and Gene Bank of Plant Genetic Resource Center (PGRC), Gannoruwa.Characterization of grain physical parameters: Grain length and width were determined using a vernier caliper. Ten grains from each sample were collected randomly and measured to obtain the average length and width of the milled rice. The average length and width were recorded as their length and width. Based on the length and width of the grains, the milled rice grains were classified into four classes (table 1) according to the method accepted by RRDI Bathalagoda, Sri Lanka.According to the scale L/S – Long Slender, L/M – Long Medium, I/B – Intermediate Bold and S/R –Short RoundAnalysis of amylose content: Initially, rice samples were dehusked and polished prior to milling. Ten whole – milled rice kernels of eight rice samples were ground separately by using mortar and pestle. Amylose content per 100 mg was determined by measuring the blue value of rice varieties as described by Juliano (1971). About 100mg rice sample was shifted into a 100 mL volumetric flask and 1mL of 95% ethanol was added. Then 9mL of 1N NaOH was added and the content was boiled for 20min. at boiling temperature to gelatinize the starch. After cooling the content, the volume was made up to 100mL and 5mL of starch solution was pipetted out into a 100mL volumetric flask. The blue color was developed by adding 1mL of 1N acetic acid and 2 mL of iodine solution (0.2g iodine and 2.0g potassium iodine in 10 mL aqueous solution). Then volume was made up to 100mL with distilled water and the solution was kept for 20min. after shaking. Finally, the absorbance of the solution was measured at 620nm using Spectrophotometer T80 (PG Instruments Limited) as described by Juliano (1971). The standard curve was prepared using 40mg of potato-amylose to calculate the amylose content of rice varieties through absorbance values. Forty mg of potato amylose was put into a 100 mL of volumetric flask and 1ml of 95% ethanol and 9mL of NaOH were added and content was heated for 20min at boiling temperature. After cooling the content volume of the solution was made up to 100mL using distilled water. Then 1mL, 2mL, 3mL, 4mL and 5mL of amylose solution were pipetted out into 100mL flasks. Then 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1mL of 1N acetic acid were added to the flasks respectively. Finally, 2mL of iodine solution was added to each flask and volume was made up to 100mL with distilled water. Solutions were stood up for 20min. after shaking and absorbance values were measured at 620nm. Measured absorbance values were plotted at 620nm against the concentration of anhydrous amylose (mg).Analysis of gelatinization temperature: GT was indirectly measured on rice by the alkali spreading value. Husked and polished seeds per accession were used for the analysis. Selected duplicate sets of six milled grains without cracks of each sample were put into Petri dishes. About 10mL of 1.7% KOH was added and grains were spread in the petri dish to provide enough space. The constant temperature at 30°C was maintained to ensure better reproducibility. After 23hrs, the degree of disintegration was quantified by a standard protocol with a numerical scale of 1–7 (table 2) as reported by Cruz and Khush (2000). As reported by Juliano (2003), GT of rice was determined using the alkaline spreading scale, where 1.0-2.5: High (74-80 °C), 2.6-3.4: High-intermediate (70-74 °C), 3.5-5.4: Intermediate (70-74 °C) and 5.5-7.0 Low: (55-70 °C).Bioinformatics and statistical analysis: The available literature was used to identify the most likely candidate genes associated with rice starch quality and their SNPs of each gene (Hirose et al., 2006; Waters and Henry, 2007; Tran et al., 2011). In all the tested varieties except Bg 352 and At 354, the DNA sequence of each gene was retrieved from the Rice SNP Seek database (http://snp-seek.irri.org/). The gene sequences of At 354 and Bg 352 were obtained from the National Research Council 16-016 project, Wayamba University of Sri Lanka. Multiple sequence alignment was conducted for the DNA sequence using Clustal Omegavsoftware (https://www.ebi.ac.uk/Tools/msa/clustalo/). Starch physiochemical data obtained were subjected to a one-way analysis of variance (ANOVA) followed by Duncan’s New Multiple Range Test (DNMRT) to determine the statistical differences among varieties at the significance level of p ≤ 0.05. Statistical analysis was done using SAS version 9.1 (SAS, 2004).ESULTS AND DISCUSSION: Physical properties of rice grains: Physical properties such as length, width, size, shape and pericarp color of rice grains obtained from eight different rice varieties are given in table 3. Classification of rice grains was carried out, according to their sizes and shapes based on Juliano (1985). The size of the rice grains was determined as per grain length while grain shape was determined by means of length and width ratio of the rice kernel. In the local market, rice is classified as Samba (short grain), Nadu (intermediate grain) and Kora (long/medium) based on the size of the grain (Pathiraje et al., 2010). Lengths of rice kernels were varied from 5.58 to 6.725 mm for all varieties. The highest grain length and width were given by At 354 and Pachchaperumal respectively. The varieties, Bw 295-5 and H 6 showed a length: width ratio over 3 which is considered as slender in grain shape. Bw 295-5, H 6, At 354, Bg 352 and Nipponbare possessed white pericarp and others possessed red pericarp.Relationship between amylose content and SNPs variation of waxy loci in selected varieties: Amylose content was measured in seven Sri Lankan rice varieties and one exotic rice variety. Amylose content of the evaluated varieties varied significantly with p ≤ 0.05 with the lowest of 15.11% and highest of 28.63% which were found in Nipponbare and Bw 295-5, respectively (table 4). The majority of the evaluated varieties fell into the high AC category (between 25-28%). Only Nipponbare could be clearly categorized under the low amylose group (table 4). The amylose content of Bg 352, Pachchaperumal and Herathbanda have already been determined by early studies of Rebeira et al. (2014) and Fernando et al. (2015). Most of the data obtained in the present experiment has agreed with the results of previous studies. Major genes such as Waxy and their functional SNPs have a major influence on amylose in rice (Nakamura et al., 2005). Accordingly, single nucleotide polymorphism, ‘G’/‘T’, at the 5’ leader intron splice site of the GBSSI has explained the variation in amylose content of varieties. Accordingly, high and intermediate amylose varieties have ‘AGGTATA’ while low amylose varieties have the sequence ‘AGTTATA’, which might lead to a decrease in the splicing efficiency. Therefore, the GBSSI activity of Nipponbare might be considerably weak and resulted in starch with low amylose content. Hence, producing ‘G’/‘T’ polymorphism clearly differentiates low amylose rice varieties, as reported by Nakamura et al. (2005). In GBSSI, Larkin and Park (2003) identified an ‘A’/‘C’ polymorphism in exon 6 and a ‘C’/‘T’ polymorphism in exon 10 which resulted in non- synonymous amino acid change. Chen et al. (2008) reported that the non-synonymous ‘A’/‘C’ SNP at exon 6 had the highest possible impact on GBSSI. Accordingly, the ‘A’/‘C’ polymorphism in exon 6 causes a tyrosine/serine amino acid substitution while the ‘C’/‘T’ polymorphism in exon 10 causes a serine/proline amino acid substitution. In view of this information, there is a relationship between the polymorphism detected by in silico analysis and amylose content obtained from our experiment. Out of the eight tested rice varieties, only one variety, Nipponbare was categorized as low amylose variety (10-19%) and it exhibited ‘T’ nucleotide at the intron splice site (table 4; figure 1). Varieties such as Pachchaperumal, Balasuriya, Bw 295-5, H 6, Herathbanda, At 354 and Bg 352 which contained high amylose (> 25%), had ‘G’ and ‘A’ nucleotides at intron splice site and exon 6 respectively (table 4; figure 1). The predominant allelic pattern of intron splice site and exon 6 are different in varieties containing intermediate amylose content (20-25%) which showed ‘G’ and ‘C’ nucleotides respectively. Of these selected rice varieties, none of the intermediate type amylose variety was found.Relationship between gel consistency and SNPs variation in Waxy loci: In this study, GC data of Herathbanda, Hondarawalu, Kuruluthuda, Pachchaperumal and Bg 352 were obtained from Fernando et al. (2015). The results of Tran et al. (2011) showed that the exon 10 ‘C’/‘T’ SNP of Wx has mainly affected GC. Accordingly, rice with a ‘C’ at exon 10 had soft and viscous gels once cooked. However, a sample with a ‘T’ had short and firm gels. In this study, Herathbanda, Hondarawalu, Kuruluthuda and Pachchaperumal had ‘C’ nucleotide and Bg 352 had ‘T’ nucleotide in exon 10 (table 5; figure 2). However, ‘C’/‘T’ substitution analysis could not be used to explain the GC of tested varieties.Relationship between gelatinized temperature and SNPs variation of Alk loci in selected rice varieties: Although there were differences in the scores, the degree of disintegration of all samples was saturated at 23 hrs. Most of the selected rice varieties showed the intermediate disintegration score. Varieties, Pachchaperumal, Balasuriya, H 6, Herathbanda, At 354 and Bg 352 were categorized into intermediate GT class (70–74°C) as indicated by an alkali spreading (AS) value of 5 (table 6; figure 3). Nipponbare and Bw 295-5 showed the highest disintegration score indicating the dispersion of all grains. Hence these varieties were categorized into low GT class (55-69°C) as indicated by an AS value of 6 (table 6; figure 3). However, high GT class rice varieties (> 74°C) were not found in the tested samples. Chromosomal mutation within the Alk gene has led to a number of single nucleotide polymorphisms (SNPs). Umemoto et al. (2004) identified four SNPs in Alk gene. Thus, SNP3 and SNP4 may be important genetic polymorphisms that are associated with GT class. According to the SNP3 and SNP4, eight rice varieties could be classified into either high GT or low GT types. If there is ‘A’ instead of ‘G’ at 4424 bp position of Alk gene with reference to Nipponbare rice genomic sequence, it codes methionine instead of valine amino acid residue in SSIIa, whilst two adjacent SNPs at bases 4533 and 4534 code for either leucine (‘GC’) or phenylalanine (‘TT’). Rice varieties with high GT starch had a combination of valine and leucine at these residues. Rice varieties with low GT starch had a combination of either methionine and leucine or valine and phenylalanine at these same residues. Nipponbare carried the ‘A’ and ‘GC’ nucleotides, while Bw 295-5 carried the ‘G’ and ‘TT’ nucleotides. Hence these varieties were classified into low GT class. Varieties such as Pachchaperumal, Balasuriya, H 6, Herathbanda, At 354 and Bg 352 carried ‘G’ and ‘GC’ nucleotides and these varieties were classified into high GT rice varieties. However, intermediate GT status could not be determined by SNP3 and SNP4 mutation of Alk gene (table 6; figure 4).In silico analysis of the polymorphisms in GBSSI gene and Alk genes of rice varieties retrieved from Rice-SNP-database: In this study, GBSSI gene and Alk gene were compared with the sequences retrieved from the Rice-SNP-Seek database to validate the SNPs further. As previously reported by Ayres et al. (1997), all low amylose varieties had the sequence ‘AGTTATA’ in exon 1. In agreement with preliminary work done by Larkin and Park (2003), all of the intermediate amylose varieties have the allelic pattern of GCC. All of the high amylose varieties have either the GAC or GAT allele of GBSSI. Among 42 rice accessions with the Sri Lankan pedigree, four allelic patterns were found; TAC, GCC, GAC and GAT (table 7). In this allelic pattern, the first letter corresponds to the ‘G’/‘T’ polymorphism in 5’ leader intron splice-junction, the second letter corresponds to the ‘A’/‘C’ polymorphism in exon 6 and the third letter corresponds to the ‘C’/‘T’ polymorphism in exon10 of Waxy gene. Analysis of the ‘G’/‘T’ polymorphism in the Wx locus showed that 41 rice cultivars shared the same ‘AGGTATA’ sequence at the 5’ leader intron splice-junction. But only 1 rice cultivar, Puttu nellu was found with ‘T’ nucleotide in intron1/exon1 junction site, which could be categorized as a low amylose variety (table 7). As discussed above, varieties with an intermediate level of apparent amylose could be reliably distinguished from those with higher apparent amylose based on a SNP in exon 6. Hence, only three rice varieties Nalumoolai Karuppan, Pannithi and Godawel with ‘C’ nucleotide in exon 6 exhibited the possibility of containing intermediate amylose content (table 7). High activity of GBSSI produces high amylose content leading to a non-waxy, non-sticky or non-glutinous phenotype. Therefore, according to the in silico genotypic results, rest of the 38 rice varieties may produce high amylose content in the endosperm (table 7). Proving this phenomenon. Abeysekera et al. (2017) has reported that usually, most of Sri Lankan rice varieties contain high amylose content. Targeted sequence analysis of exon 8 of the Alk gene in 42 different rice cultivars were found with three SNP polymorphisms that resulted in a changed amino acid sequence and, of these three SNPs, two SNPs were reported to be correlated with possible GT differences. Accordingly, Puttu nellu and 3210 rice varieties carried the ‘G’ and ‘TT’ nucleotides in SNP3 and SNP4 respectively (table 7). Hence these varieties can be classified into low GT class and except these two; other rice varieties carried the ‘G’ and ‘GC’ nucleotides in SNP3 and SNP4 respectively. Therefore, those varieties can possibly be classified into high GT rice varieties (table 7). However, further experiments are necessary to check the phenotypic variations for grain amylose content and GT class of in silico analyzed rice varieties. CONCLUSION Present results revealed the relationship between SNPs variation at Waxy loci and the amylose content of selected rice varieties. Accordingly, Pachchaperumal, At 354, Bg 352, Herathbanda, H 6, Balasuriya and Bw 295-5 with high amylose content had ‘G’ instead of ‘T’ in the first intron exhibiting the presence of Wxa allele with reference to Nipponbare which had low amylose content. Also all tested varieties had ‘A’ in exon 6 of the Waxy gene. Thus present findings i.e. presence of Wxa allele and SNP ‘A’ in exon 6 could be used as a potential molecular marker for the selection of high amylose varieties. In addition, Bw 295-5 which is a low GT variety, had two SNPs variations in the last exon of the Alk gene i.e. ‘G’ and ‘TT’ which is likely to be used to represent low GT class. Accordingly, sequence variations identified in Waxy and Alk genes could be utilized in the future rice breeding programs for the development of varieties with preferential amylose content and GT class.ACKNOWLEDGMENTSDirector and staff of the Gene Bank, Plant Genetic Resources Center, Gannoruwa are acknowledged for giving rice accessions.CONFLICT OF INTERESTAuthors have no conflict of interest.REFERENCESAbeysekera, W., G. Premakumara, A. Bentota and D. S. Abeysiriwardena, 2017. Grain amylose content and its stability over seasons in a selected set of rice varieties grown in Sri Lanka. Journal of agricultural sciences Sri Lanka, 12(1): 43-50.Ayres, N., A. McClung, P. Larkin, H. Bligh, C. Jones and W. Park, 1997. Microsatellites and a single-nucleotide polymorphism differentiate apparentamylose classes in an extended pedigree of us rice germ plasm. Theoretical applied genetics, 94(6-7): 773-781.Chen, M.-H., C. Bergman, S. Pinson and R. Fjellstrom, 2008. Waxy gene haplotypes: Associations with apparent amylose content and the effect by the environment in an international rice germplasm collection. Journal of cereal science, 47(3): 536-545.Cruz, N. D. and G. Khush, 2000. Rice grain quality evaluation procedures. Aromatic rices, 3: 15-28.Fernando, H., T. Kajenthini, S. Rebeira, T. Bamunuarachchige and H. Wickramasinghe, 2015. Validation of molecular markers for the analysis of genetic diversity of amylase content and gel consistency among representative rice varieties in sri lanka. Tropical agricultural research, 26(2): 317-328.Hirose, T., T. Ohdan, Y. Nakamura and T. Terao, 2006. Expression profiling of genes related to starch synthesis in rice leaf sheaths during the heading period. Physiologia plantarum, 128(3): 425-435.Juliano, B., 1971. A simplified assay for milled rice amylose. Journal of cereal science today, 16: 334-360.Juliano, B. O., 1985. Rice: Chemistry and technology. The american association of cereal chemists. Inc. St. Paul, Minnesota, USA, 774.Juliano, B. O., 2003. Rice chemistry and quality. Island publishing house. Island publishing house, Manila: 1-7.Kharabian-Masouleh, A., D. L. Waters, R. F. Reinke, R. Ward and R. J. Henry, 2012. Snp in starch biosynthesis genes associated with nutritional and functional properties of rice. Scientific reports, 2(1): 1-9.Kongseree, N. and B. O. Juliano, 1972. Physicochemical properties of rice grain and starch from lines differing in amylose content and gelatinization temperature. Journal of agricultural food chemistry, 20(3): 714-718.Larkin, P. D. and W. D. Park, 2003. Association of waxy gene single nucleotide polymorphisms with starch characteristics in rice (Oryza sativa L.). Molecular Breeding, 12(4): 335-339.Nakamura, Y., 2002. Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: Rice endosperm as a model tissue. Plant cell physiology, 43(7): 718-725.Nakamura, Y., P. B. Francisco, Y. Hosaka, A. Sato, T. Sawada, A. Kubo and N. Fujita, 2005. Essential amino acids of starch synthase iia differentiate amylopectin structure and starch quality between Japonica and Indica rice varieties. Plant molecular biology, 58(2): 213-227.Pathiraje, P., W. Madhujith, A. Chandrasekara and S. Nissanka, 2010. The effect of rice variety and parboiling on in vivo glycemic response. Journal of tropical agricultural research, 22(1): 26-33.Rebeira, S., H. Wickramasinghe, W. Samarasinghe and B. Prashantha, 2014. Diversity of grain quality characteristics of traditional rice (Oryza sativa L.) varieties in sri lanka. Tropical agricultural research, 25(4): 470-478.Sartaj, I. Z. and S. A. E. R. Suraweera, 2005. Comparison of different parboiling methods on the quality characteristics of rice. Annals of the Sri Lankan Department of Agriculture, 7: 245-252.Tran, N., V. Daygon, A. Resurreccion, R. Cuevas, H. Corpuz and M. Fitzgerald, 2011. A single nucleotide polymorphism in the waxy gene explains a significant component of gel consistency. Theoretical applied genetics, 123(4): 519-525.Umemoto, T. and N. Aoki, 2005. Single-nucleotide polymorphisms in rice starch synthase iia that alter starch gelatinisation and starch association of the enzyme. Functional plant biology, 32(9): 763-768.Umemoto, T., N. Aoki, H. Lin, Y. Nakamura, N. Inouchi, Y. Sato, M. Yano, H. Hirabayashi and S. Maruyama, 2004. Natural variation in rice starch synthase iia affects enzyme and starch properties. Functional plant biology, 31(7): 671-684.Waters, D. L. and R. J. Henry, 2007. Genetic manipulation of starch properties in plants: Patents 2001-2006. Recent patents on biotechnology, 1(3): 252-259.Waters, D. L., R. J. Henry, R. F. Reinke and M. A. Fitzgerald, 2006. Gelatinization temperature of rice explained by polymorphisms in starch synthase. Plant biotechnology journal, 4(1): 115-122.

The tissue-specific gene expression at the brain microvasculature, which forms the blood–brain barrier (BBB) in vivo, can be elucidated with a brain vascular genomics program, which starts with the isolation of gene products derived from purified brain microvessels. Genes commonly expressed in peripheral organs are subtracted with the suppression subtractive hybridization method using driver cDNA produced from a pool of rat liver/kidney–derived poly A+RNA. From a screen of 480 clones in the subtracted tester cDNA library, 156 clones were sequenced. The clones fell into 3 groups: known genes (51%), rat expressed sequence tags (31%), and novel rat genes not found in databases (18%). The known genes could be categorized into families of common function including vascular remodeling, signal transduction, transcription factors, biologic transport, vascular amyloid, hemostasis, myelin, lipids, secretion, cytoskeleton, and junctional complexes. Brain vascular genomics, or BBB genomics, allows for an accelerated discovery of the gene families that are differentially expressed at the microvasculature in brain.

To correlate brain metabolic status with the molecular events during cerebral ischemia, a cDNA array was performed after positron emission tomography scanning in a model of focal cerebral ischemia in baboons. Cluster analysis for the expression of 74 genes allowed the identification of 4 groups of genes. In each of the distinct groups, the authors observed a marked inflection in the pattern of gene expression when the CMRo2 was reduced by 48% to 66%. These patterns of coordinated modifications in gene expression could define molecular checkpoints for the development of an ischemic infarct and a molecular definition of the penumbra.

Confounding any genome-scale analysis of gene expression after cerebral ischemia is massive suppression of protein synthesis. This inefficient translation questions the utility of examining profiles of total transcripts. Our approach to such postischemic gene profiling in the mouse by microarray analysis was to concentrate on those mRNAs bound to polyribosomes. In our proof-of-principle study, polysomally bound and unbound mRNAs were subjected to microarray analysis: of the 1,161 transcripts that we found to increase after ischemia, only 36% were bound to polyribosomes. In addition to the expected increases in heat-shock proteins and metallothioneins, increases in several other bound transcripts involved in the promotion of cell survival or antiinflammatory behavior were noted, such as CD63 (Lamp3), Lcn2 (lipocalin-2), Msn (moesin), and UCP2 (uncoupling protein 2), all of which showed increases in cognate protein by Western blotting. The list of heretofore nonfunctionally annotated transcripts (RIKEN clones/ESTs) that increased appeared to be novel. How some transcripts are selected in ischemic brain for translation into protein, while others are rejected, is not clear. The length of the 5′-UTR in the ischemically induced transcripts that occur in the NCBI RefSeq database did not indicate any general tendency to be more than 200 nt, nor to be longer than the 5′-UTRs of the unbound transcripts. Thus, the presence of a complex 5′-UTR region with internal ribosome entry sites (IRES) or polypyrimidine tracts (TOP) does not appear to be the basis of selection for translation in ischemic brain.

The present study determined whether gene transfer of human copper/zinc superoxide dismutase-1 (Cu/Zn SOD-1) prevented the autoregulatory impairment of CBF induced by subarachnoid hemorrhage (SAH). After application of recombinant adenovirus (100 μL of 1 × 1010 pfu/mL, intracisternally) encoding human Cu/Zn SOD-1 3 days before experiments, Cu/Zn SOD-1 activity significantly increased in association with increase in Cu/Zn SOD-1 mRNA and protein expression in the cerebral vasculature of both sham-operated and SAH rats as determined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry, and SAH-induced increase in superoxide anion was markedly reduced in accordance with increased nitric oxide production. In line with these findings, rats that received human Cu/Zn SOD-1 therapy showed the prominent restoration of blunted vasodilation of the pial artery in response to calcitonin gene-related peptide and levcromakalim, and the recovery of impaired autoregulatory vasodilation in response to acute hypotension, thereby leading to significant restoration of CBF autoregulation. These results provide a rational basis for application of Cu/Zn SOD-1 gene therapy for protection of the impairment of autoregulatory CBF during the acute stage of SAH.

Studies of gene expression changes after cerebral ischemia can provide novel insight into ischemic pathophysiology. Here we describe application of restriction-mediated differential display to screening for differentially expressed genes after focal cerebral ischemia. This method combines the nonredundant generation of biotin-labeled fragment sets with the excellent resolution of direct blotting electrophoresis, reliable fragment recovery, and a novel clone selection strategy. Using the filament model in mouse with 90 minutes MCA occlusion followed by 2, 6, and 20 hours reperfusion, we have compared gene expression in sham-operated animals to both the ipsi- and contralateral forebrain hemisphere of ischemic mice. Our screening method has resulted in the identification of 70 genes differentially regulated after transient middle cerebral artery occlusion (MCAO), several of which represent unknown clones. We have identified many of the previously published regulated genes, lending high credibility to our method. Surprisingly, we detected a high degree of correspondent regulation of genes in the nonischemic hemisphere. A high percentage of genes coding for proteins in the respiratory chain was found to be up-regulated after ischemia, potentially representing a new mechanism involved in counteracting energy failure or radical generation in cerebral ischemia. One particularly interesting gene, whose upregulation by ischemia has not been described before, is pip92; this gene shows a rapid and long-lasting induction after cerebral ischemia. Here we demonstrate that pip92 induces cell death in primary neurons and displays several hallmarks of pro-apoptotic activity upon overexpression, supporting the notion that we have identified a novel pathophysiological player in cerebral ischemia. In summary, restriction-mediated differential display has proven its suitability for screening complex samples such as brain to reliably identify regulated genes, which can uncover novel pathophysiological mechanisms.

Study was aimed at assessing the role of apoplastic invertase in synchronization of the microtuber formation in potato plants in vitro. A non-transgenic potato (Solanum tuberosum L.) plants (hereinafter nonTPs) and a line of potato plants, which expressed the SUC2 gene of Saccharomyces cerevisiae encoding invertase apoplastic localization under the control of the B33 promoter of the patatin gene (hereinafter TPs), was used to achieve this purpose. Cell divisions in the axillary meristems of single-node cuttings were synchronized by exposing to 7°C for 24 h in a MS medium (without agar). After the low-temperature, the synchronization (LTS) of proportion of simultaneously dividing cells in meristems of nonTPs was four-fold greater than without chilling. The LTS did not change the number of dividing cells in the axillary meristems of TPs. The LTS contributed to an increase in the mass and size of microtubers in both potato lines. All microtubers of nonTPs were of physiological maturity. At the same time, microtubers of TPs were physiologically immature, with high glucose content. During the tuber formation, the activity of acidic invertases in microtubers of TPs was higher than that of nonTPs. Microtubers of TPs were larger than the nonTPs, more hydrated with low starch amount. Propably glucose accumulated in the microtubers of the TPs may act as a signal to trigger cell division. It indicates the regulatory function of apoplastic invertase and sugars. The high activity of apoplastic invertase plays a negative role in the production of potato microtubers of identical physiological age.

Angiogenesis is regulated by concerted actions of angiogenic and angiostatic factors. Homeobox D3 gene ( HOXD3) is a potent proangiogenic transcription factor that promotes angiogenesis by modulating the expression of matrix-degrading proteinases, integrins, and extracellular matrix components. Application of HOXD3 can promote angiogenesis in the skin, but its role in other vascular beds has not been examined. The authors examined HOXD3 expression in human brain vessels by in situ hybridization. Although little or no HOXD3 mRNA was detected in normal brain vessels, increased levels of HOXD3 and its target gene, αVβ3, were found in angiogenic vessels in human brain arteriovenous malformations. The authors further investigated whether HOXD3 plays a role in cerebral angiogenesis in a murine model. Expression of HOXD3 in mouse brain was achieved through retroviral vector—mediated HOXD3 gene transfer. HOXD3 expression lead to a significant induction of cerebral angiogenesis as shown by quantitative microvessel counting (HOXD3: 241 ± 19 vessels/mm2 vs. saline: 150 ± 14 vessels/mm2, P < 0.05). The data also showed that focal cerebral blood flow was increased in the angiogenic region with less vascular leakage. Moreover, expression of HOXD3 led to an increase in the expression of a direct downstream target gene αVβ3 integrin. The data suggest that HOXD3 may play an important role in regulating cerebral angiogenesis, and that gene transfer of HOXD3 may provide a novel and potent means to stimulate angiogenesis.

Abstract Motivation Identifying the genes regulated by a given transcription factor (TF) (its ‘target genes’) is a key step in developing a comprehensive understanding of gene regulation. Previously, we developed a method (CisMapper) for predicting the target genes of a TF based solely on the correlation between a histone modification at the TF’s binding site and the expression of the gene across a set of tissues or cell lines. That approach is limited to organisms for which extensive histone and expression data are available, and does not explicitly incorporate the genomic distance between the TF and the gene. Results We present the T-Gene algorithm, which overcomes these limitations. It can be used to predict which genes are most likely to be regulated by a TF, and which of the TF’s binding sites are most likely involved in regulating particular genes. T-Gene calculates a novel score that combines distance and histone/expression correlation, and we show that this score accurately predicts when a regulatory element bound by a TF is in contact with a gene’s promoter, achieving median precision above 60%. T-Gene is easy to use via its web server or as a command-line tool, and can also make accurate predictions (median precision above 40%) based on distance alone when extensive histone/expression data is not available for the organism. T-Gene provides an estimate of the statistical significance of each of its predictions. Availability and implementation The T-Gene web server, source code, histone/expression data and genome annotation files are provided at http://meme-suite.org. Supplementary information Supplementary data are available at Bioinformatics online.

Rupture of an intracranial aneurysm (subarachnoid hemorrhage) is a potentially devastating condition frequently complicated by delayed cerebral ischemia from sustained contraction of intracranial arteries (cerebral vasospasm). There is mounting evidence linking the formation of intracranial aneurysms and the pathogenesis of post-subarachnoid hemorrhage vasospasm to aberrant bioavailability and action of the vasodilator molecule nitric oxide generated by isoforms of nitric oxide synthase. In humans, the gene encoding the endothelial isoform of nitric oxide synthase (eNOS) is known to be polymorphic, with certain polymorphisms associated with increased cardiovascular disease susceptibility. In this prospective clinical study involving 141 participants, we used gene microarray technology to demonstrate that the eNOS gene intron-4 27-base pair variable number tandem repeat polymorphism (eNOS 27 VNTR) predicts susceptibility to intracranial aneurysm rupture, while the eNOS gene promoter T-786C single nucleotide polymorphism (eNOS T-786C SNP) predicts susceptibility to post-subarachnoid hemorrhage vasospasm. We believe that genetic information such as this, which can be obtained expeditiously at the time of diagnosis, may be used as a helpful adjunct to other clinical information aimed at predicting and favorably modifying the clinical course of persons with intracranial aneurysms.