Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman-Birk protease inhibitor and suppresses the accumulation of the ß-subunit of ß-conglycinin in soybean seeds.

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5'-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the ß-subunit of ß-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman-Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC-MS and GC-MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37-52% and 15-19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.

Candida albicans biofilms: a developmental state associated with specific and stable gene expression patterns.

Like many bacteria, yeast species can form biofilms on several surfaces. Candida albicans colonizes the surfaces of catheters, prostheses, and epithelia, forming biofilms that are extremely resistant to antifungal drugs. We have used transcript profiling to investigate the specific properties of C. albicans biofilms. Biofilm and planktonic cultures produced under different conditions of nutrient flow, aerobiosis, or glucose concentration were compared by overall gene expression correlation. Correlation was much higher between biofilms than planktonic populations irrespective of the growth conditions, indicating that biofilm populations formed in different environments display very similar and specific transcript profiles. A first cluster of 325 differentially expressed genes was identified. In agreement with the overrepresentation of amino acid biosynthesis genes in this cluster, Gcn4p, a regulator of amino acid metabolism, was shown to be required for normal biofilm growth. To identify biofilm-related genes that are independent of mycelial development, we studied the transcriptome of biofilms produced by a wild-type, hypha-producing strain and a cph1/cph1 efg1/efg1 strain defective for hypha production. This analysis identified a cluster of 317 genes expressed independently of hypha formation, whereas 86 genes were dependent on mycelial development. Both sets revealed the activation of the sulfur-amino acid biosynthesis pathway as a feature of C. albicans biofilms.

Decreased expression of genes involved in sulfur amino acid metabolism in frataxin-deficient cells.

Inherited deficiency of the mitochondrial protein frataxin causes neural and cardiac cell degeneration, and Friedreich's ataxia. Five hypotheses for frataxin's mitochondrial function have been generated, largely from work in non-human cells: iron transporter, iron-sulfur cluster assembler, iron-storage protein, antioxidant and stimulator of oxidative phosphorylation. We analyzed gene expression in three human cell types using microarrays, and identified just 48 transcripts whose expression was significantly frataxin-dependent in at least two cell types. Significant decreases in seven transcripts occurred in the sulfur amino acid (SAA) biosynthetic pathway and the iron-sulfur cluster (ISC) biosynthetic pathway to which it is connected. By contrast, we did not observe a single frataxin-dependent transcript that fits with the other four current hypotheses. Quantitative reverse-transcriptase PCR analysis of ISC-S and rhodanese transcripts confirmed that the expression of these genes involved in ISC metabolism was lower in mutants. Amino acid analysis confirmed the defect in SAA metabolism: homocystine, cysteine, cystathionine and serine were significantly decreased in frataxin-deficient cell extracts and mitochondria. An ISC defect was further confirmed by observing decreases in succinate dehydrogenase and aconitase activities, whose activities require ISCs. The ISC-U scaffold protein was specifically decreased in frataxin-deficient cells, suggesting a role for frataxin in its expression or maintenance, and sodium sulfide partially rescued the oxidant-sensitivity of the FRDA cells. Also, multiple transcripts involved in the Fas/TNF/INF apoptosis pathway were up-regulated in frataxin-deficient cells, consistent with a multi-step mechanism of Friedreich's ataxia pathophysiology, and suggesting alternative possibilities for therapeutic intervention.

Limits to sulfur accumulation in transgenic lupin seeds expressing a foreign sulfur-rich protein.

The low sulfur amino acid content of legume seeds restricts their nutritive value for animals. We have investigated the limitations to the accumulation of sulfur amino acids in the storage proteins of narrow leaf lupin (Lupinus angustifolius) seeds. Variation in sulfur supply to lupin plants affected the sulfur amino acid accumulation in the mature seed. However, when sulfur was in abundant supply, it accumulated to a large extent in oxidized form, rather than reduced form, in the seeds. At all but severely limiting sulfur supply, addition of a transgenic (Tg) sink for organic sulfur resulted in an increase in seed sulfur amino acid content. We hypothesize that demand, or sink strength for organic sulfur, which is itself responsive to environmental sulfur supply, was the first limit to the methionine (Met) and cysteine (Cys) content of wild-type lupin seed protein under most growing conditions. In Tg, soil-grown seeds expressing a foreign Met- and Cys-rich protein, decreased pools of free Met, free Cys, and glutathione indicated that the rate of synthesis of sulfur amino acids in the cotyledon had become limiting. Homeostatic mechanisms similar to those mediating the responses of plants to environmental sulfur stress resulted in an adjustment of endogenous protein composition in Tg seeds, even when grown at adequate sulfur supply. Uptake of sulfur by lupin cotyledons, as indicated by total seed sulfur at maturity, responded positively to increased sulfur supply, but not to increased demand in the Tg seeds.

[Regeneration of HNP transgenic alfalfa plants by Agrobacterium mediated gene transfer].

Transgenic plants regenerated from cotyledons of M. sativa L. infected using Agrobacterium tumefaciens A281 with plasmid pBF649 containing a gene encoding protein of high sulfur-amino acid content (HNP) were obtained successfully. The plants grew and fertiled well in field. Cotyledon explants were better recipient for transformation of M. sativa L. Environment of suitable temperature (15 degrees C) and high humidity on high viability of the plants transplanted into soil were essential conditions.

Statistical approaches for evaluation of genetic risk factor of peripheral arterial occlusive disease.

In the paper we show results of medical study from statistical point of view. The medical study was aimed to study genetic risk factors of peripheral arterial occlusive diseases in Czech population. Two genes, CBS and MTHFR were examined, as various genotypes of these genes are thought to have impact on amino thiols, who participate in variety of reactions in vasculature. Statistical part of the study was responsible for analysis and interpretation of collected data.

Metabolism of sulfur amino acids in Saccharomyces cerevisiae.

Sulfur amino acid biosynthesis in Saccharomyces cerevisiae involves a large number of enzymes required for the de novo biosynthesis of methionine and cysteine and the recycling of organic sulfur metabolites. This review summarizes the details of these processes and analyzes the molecular data which have been acquired in this metabolic area. Sulfur biochemistry appears not to be unique through terrestrial life, and S. cerevisiae is one of the species of sulfate-assimilatory organisms possessing a larger set of enzymes for sulfur metabolism. The review also deals with several enzyme deficiencies that lead to a nutritional requirement for organic sulfur, although they do not correspond to defects within the biosynthetic pathway. In S. cerevisiae, the sulfur amino acid biosynthetic pathway is tightly controlled: in response to an increase in the amount of intracellular S-adenosylmethionine (AdoMet), transcription of the coregulated genes is turned off. The second part of the review is devoted to the molecular mechanisms underlying this regulation. The coordinated response to AdoMet requires two cis-acting promoter elements. One centers on the sequence TCACGTG, which also constitutes a component of all S. cerevisiae centromeres. Situated upstream of the sulfur genes, this element is the binding site of a transcription activation complex consisting of a basic helix-loop-helix factor, Cbf1p, and two basic leucine zipper factors, Met4p and Met28p. Molecular studies have unraveled the specific functions for each subunit of the Cbf1p-Met4p-Met28p complex as well as the modalities of its assembly on the DNA. The Cbf1p-Met4p-Met28p complex contains only one transcription activation module, the Met4p subunit. Detailed mutational analysis of Met4p has elucidated its functional organization. In addition to its activation and bZIP domains, Met4p contains two regulatory domains, called the inhibitory region and the auxiliary domain. When the level of intracellular AdoMet increases, the transcription activation function of Met4 is prevented by Met30p, which binds to the Met4 inhibitory region. In addition to the Cbf1p-Met4p-Met28p complex, transcriptional regulation involves two zinc finger-containing proteins, Met31p and Met32p. The AdoMet-mediated control of the sulfur amino acid pathway illustrates the molecular strategies used by eucaryotic cells to couple gene expression to metabolic changes.

Cloning and characterization of the Aspergillus nidulans cysB gene encoding cysteine synthase.

The cysB gene of A. nidulans was cloned by complementation of a cysB mutation. This is the first cloned eukaryotic genomic sequence coding for cysteine synthase. The gene contains one 71-bp intron and codes for a protein of 370 amino acids. Its N-terminal region has characteristic features of transit peptides, suggesting mitochondrial localisation of the enzyme. The protein shows homology with bacterial and plant cysteine synthases among which it occupies a remote phylogenetic position and apparently represents a distinct subfamily. Transcription of the cysB gene is not appreciably regulated by the concentration of methionine in the growth medium.

The vacuolar compartment is required for sulfur amino acid homeostasis in Saccharomyces cerevisiae.

In order to isolate new mutations impairing transcriptional regulation of sulfur metabolism in Saccharomyces cerevisiae, we used a potent genetic screen based on a gene fusion expressing XylE (from Pseudomonas putida) under the control of the promoter region of MET25. This selection yielded strains mutated in various different genes. We describe in this paper the properties of one of them, MET27. Mutation or disruption of MET27 leads to a methionine requirement and affects S-adenosylmethionine (AdoMet)-mediated transcriptional control of genes involved in sulfur metabolism. The cloning and sequencing of MET27 showed that it is identical to VPS33. Disruptions or mutations of gene VPS33 are well known to impair the biogenesis and inheritance of the vacuolar compartment. However, the methionine requirement of vps33 mutants has not been reported previously. We show here, moreover, that other vps mutants of class C (no apparent vacuoles) also require methionine for growth. Northern blotting experiments revealed that the met27-1 mutation delayed derepression of the transcription of genes involved in sulfur metabolism. By contrast, this delay was not observed in a met27 disrupted strain. Physiological and morphological analyses of met27-1 and met27 disrupted strains showed that these results could be explained by alterations in the ability of the vacuole to transport or store AdoMet, the physiological effector of the transcriptional regulation of sulfur metabolism.

Deficiency of a mouse kidney metalloendopeptidase activity: immunological demonstration of an altered gene product.

Meprin, an 85,000 molecular weight metalloendopeptidase is a major component of the kidney brush border membrane in mice. Some inbred mouse strains exhibit low levels of meprin activity. These strains were characterized by little, if any, protein in brush border preparations corresponding to the native enzyme. However, material exhibiting partial identity to meprin was identified by Ouchterlony immunodiffusion. Immunoblots of brush border proteins confirmed that this immunoreactive material was present but of higher molecular weight than the native enzyme. The implication of these data is that the structural gene for meprin is expressed, albeit incorrectly, in the low-meprin strains.

Proximity of the Mep-1 gene to H-2D on chromosome 17 in mice.

The Mep-1 gene on chromosome 17 in mice controls the activity of meprin, a kidney brush border metalloendopeptidase. Most inbred mouse strains of the k haplotype (e.g., CBA, C3H, AKR) are markedly deficient in meprin activity; these mice carry the Mep-1b allele. Mouse strains in which meprin activity levels are normal are designated Mep-1a. Studies using congenic and recombinant strains mapped the Mep-1 gene telomeric to H-2D near the Tla gene. To further study the relationship between the major histocompatibility complex and Mep-1, a linkage study was conducted. Mep-1a F1 hybrids [C3H.A (KkDd) X C3H.OH (KdDk)] were backcrossed with Mep-1b C3H.OH (KdDk) parents. The progeny were assayed for H-2D markers, Pgk-2 isozymes, and meprin activity. Recombination between H-2D and Mep-1 occurred in 6 out of 284 mice, a crossover frequency of 2.1%. Mep-1 is therefore 2.1 crossover units telomeric to H-2D and approximately 0.6 crossover units from Tla. The Mep-1 locus provides a new genetic marker for the future mapping of this important area of the mouse genome.