MIPS Gene Research Articles

Sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of D-glucose 6-phosphate to L-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989-998, 2004). One of the forms with an approximately 50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as an approximately 65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified approximately 1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive approximately 65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.

GENE FUNCTION PREDICTION BY A COMBINED ANALYSIS OF GENE EXPRESSION DATA AND PROTEIN-PROTEIN INTERACTION DATA

Prediction of biological functions of genes is an important issue in basic biology research and has applications in drug discoveries and gene therapies. Previous studies have shown either gene expression data or protein-protein interaction data alone can be used for predicting gene functions. In particular, clustering gene expression profiles has been widely used for gene function prediction. In this paper, we first propose a new method for gene function prediction using protein-protein interaction data, which will facilitate combining prediction results based on clustering gene expression profiles. We then propose a new method to combine the prediction results based on either source of data by weighting on the evidence provided by each. Using protein-protein interaction data downloaded from the GRID database, published gene expression profiles from 300 microarray experiments for the yeast S. cerevisiae, we show that this new combined analysis provides improved predictive performance over that of using either data source alone in a cross-validated analysis of the MIPS gene annotations. Finally, we propose a logistic regression method that is flexible enough to combine information from any number of data sources while maintaining computational feasibility.

Molecular Cloning, Bacterial Overexpression and Characterization of L-myo- inositol 1- Phosphate Synthase from a Monocotyledonous Resurrection Plant, Xerophyta viscosa Baker

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS), an evolutionarily conserved enzyme-protein, catalyses the first and rate limiting step of inositol biosynthesis. Inositol and its derivatives play important roles in biological kingdom like growth regulation, membrane biogenesis, signal transduction and also acts as an osmolyte or osmoprotectant in abiotic stress tolerance. Here we report the cloning, sequencing and the characterization of the INO1 gene from Xerophyta viscosa (XINO1), a monocotyledonous resurrection plant. Nucleotide sequences of XINO1 show striking homology (70–99%) with a number of INO1 genes from plant sources particularly with the monocots. The gene is functionally identified through genetic complementation using a yeast inositol auxotrophic strain FY250. The gene is expressed in E. coli BL21, recombinant protein purified to homogeneity, biochemically characterized and compared with Oryza INO1 (RINO1) gene product. The XINO1 gene product is catalytically active in a broader range of lower temperature (between 10–40 °C) than the RINO1 gene- product. This is the first report of MIPS gene from any resurrection plant.

Expression and nucleotide sequence of an INS (3) P1 synthase gene associated with low-phytate kernels in maize (Zea mays L.).

Most of the phosphorus (P) in maize (Zea mays L.) kernels is in the form of phytic acid. A low phytic acid (lpa) maize mutant, lpa1-1, displays levels reduced by 66%. A goal of genetic breeding is development of low phytic acid feedstocks to improve P nutrition of nonruminant animals and reduce the adverse environmental impacts of excess P in manure. The genetic basis of the lpa1-1 mutation is not known, but previous genetic mapping has shown both the mutant phenotype and the Ins (3) P(1) synthase (MIPS) gene, which encodes the first enzyme, myo-inositol phosphate synthase, in the phytic acid biosynthetic pathway, map to the same chromosomal region in maize. Research was conducted to determine the expression of the MIPS gene in lpa1-1 and wild-type kernels with similar genetic backgrounds and to ascertain if variation in the MIPS coding sequence could be inferred to be the basis of the mutation. MIPS enzyme activity determined by TLC was reduced 2-3-fold in mutant kernels. RT-PCR-based experiments using primers specific for the 1S-MIPS sequence indicated gene expression was reduced 50-60% in the mutant. Sequence analysis of the MIPS genomic sequence revealed 10 exons and 9 introns that are identical in both mutant and wild-type developing kernels. These findings support an association between reduced MIPS gene activity and low phytic acid content, but additional research should examine the promoter, the 5'UTR, or transcriptional controlling elements of the MIPS gene to ascertain the possible presence of a genetic lesion in those regions.

Sll1722, an unassigned open reading frame of Synechocystis PCC 6803, codes for L-myo-inositol 1-phosphate synthase.

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes conversion of glucose 6-phosphate to L-myo-inositol 1-phosphate, the first and the rate-limiting step in the production of inositol, and has been reported from evolutionarily diverse organisms. Two forms of the enzyme have been characterized from higher plants, viz. cytosolic and chloroplastic, and the presence of MIPS has been earlier reported from the cyanobacteria (e.g. Spirulina sp.), the presumed chloroplast progenitors. The present study demonstrates possible multiple forms of MIPS and identifies the gene for one of them in the cyanobacterium Synechocystis sp. PCC 6803. Following detection of at least two immunologically cross-reactive MIPS forms, we have been able to identify from the fully sequenced Synechocystis genome an as yet unassigned open reading frame (ORF), sll1722, coding for the approx. 50-kDa MIPS protein, by using biochemical, molecular and bioinformatics tools. The DNA fragment corresponding to sll1722 was PCR-amplified and functional identity of the gene was confirmed by a complementation assay in Saccharomyces cerevisiae mutants containing a disrupted INO1 gene for the yeast MIPS. The sll1722 PCR product was cloned in Escherichia coli expression vector pET20b and the isopropyl beta-D-thiogalactopyranoside (IPTG)-induced overexpressed protein product was characterized following complete purification. Comparison of the sll1722 sequences with other MIPS sequences and its phylogenetic analysis revealed that the Synechocystis MIPS gene is quite divergent from the others.

Expression of 1L-myoinositol-1-phosphate synthase in organelles.

We have studied the expression of 1L-myoinositol-1-phosphate synthase (MIPS; EC 5.5.1.4) in developing organs of Phaseolus vulgaris to define genetic controls that spatially regulate inositol phosphate biosynthesis. MIPS, the pivotal biosynthetic enzyme in inositol metabolism, is the only enzyme known to catalyze the conversion of glucose 6-phosphate to inositol phosphate. It is found in unicellular and multicellular eukaryotes and has been isolated as a soluble enzyme from both. Thus, it is widely accepted that inositol phosphate biosynthesis is largely restricted to the cytosol. Here, we report findings that suggest the enzyme is also expressed in membrane-bound organelles. Microscopic and biochemical analyses detected MIPS expression in plasma membranes, plastids, mitochondria, endoplasmic reticula, nuclei, and cell walls of bean. To address mechanisms by which the enzyme could be targeted to or through membranes, MIPS genes were analyzed for sorting signals within primary structures and upstream open reading frames that we discovered through our sequence analyses. Comprehensive computer analyses revealed putative transit peptides that are predicted to target the enzyme to different cellular compartments. Reverse transcriptase PCR experiments suggest that these putative targeting peptides are expressed in bean roots and leaves.

Expression of D-myo-inositol-3-phosphate synthase in soybean. Implications for phytic acid biosynthesis.

Phytic acid, a phosphorylated derivative of myo-inositol, functions as the major storage form of phosphorus in plant seeds. Myo-inositol phosphates, including phytic acid, play diverse roles in plants as signal transduction molecules, osmoprotectants, and cell wall constituents. D-myo-inositol-3-phosphate synthase (MIPS EC 5.5.1.4) catalyzes the first step in de novo synthesis of myo-inositol. A soybean (Glycine max) MIPS cDNA (GmMIPS1) was isolated by reverse transcriptase-PCR using consensus primers designed from highly conserved regions in other plant MIPS sequences. Southern-blot analysis and database searches indicated the presence of at least four MIPS genes in the soybean genome. Northern-blot and immunoblot analyses indicated higher MIPS expression and accumulation in immature seeds than in other soybean tissues. MIPS was expressed early in the cotyledonary stage of seed development. The GmMIPS1 expression pattern suggested that it encodes a MIPS isoform that functions in seeds to generate D-myo-inositol-3-phosphate as a substrate for phytic acid biosynthesis.