Biodiesel production from crude Jatropha curcas L. seed oil with a high content of free fatty acids

A technique to produce biodiesel from crude Jatropha curcas seed oil (CJCO) having high free fatty acids (15%FFA) has been developed. The high FFA level of JCJO was reduced to less than 1% by a two-step pretreatment process. The first step was carried out with 0.60 w/w methanol-to-oil ratio in the presence of 1% w/w H(2)SO(4) as an acid catalyst in 1-h reaction at 50 degrees C. After the reaction, the mixture was allowed to settle for 2h and the methanol-water mixture separated at the top layer was removed. The second step was transesterified using 0.24 w/w methanol to oil and 1.4% w/w NaOH to oil as alkaline catalyst to produce biodiesel at 65 degrees C. The final yield for methyl esters of fatty acids was achieved ca. 90% in 2 h.     

A specific beta-glucosidase-aggregating factor is responsible for the beta-glucosidase null phenotype in maize

Maize (Zea mays L.) beta-glucosidase was extracted from shoots of a wild-type (K55) and a "null" (H95) maize genotype. Enzyme activity assays and electrophoretic data showed that extracts from the null genotype had about 10% of the activity present in the normal genotype. Zymograms of the null genotype were devoid of any activity bands in the resolving gel, but had a smeared zone of activity in the stacking gel after native polyacrylamide gel electrophoresis. When extracts were made with buffers containing 0.5% to 2% sodium dodecyl sulfate, the smeared activity zone entered the resolving gel as a distinct band. These data indicated that the null genotypes have beta-glucosidase activity, but the enzyme occurs as insoluble or poorly soluble large quaternary complexes mediated by a beta-glucosidase-aggregating factor (BGAF). BGAF is a 35-kD protein and binds specifically to beta-glucosidase and renders it insoluble during extraction. BGAF also precipitates beta-glucosidase that is added exogenously to supernatant fluids of the null tissue extracts. The specific beta-glucosidase-aggregating activity of BGAF is unequivocally demonstrated. These data clearly show that the monogenic inheritance reported for the null alleles at the beta-glucosidase gene is actually for the BGAF protein, and BGAF is solely responsible for beta-glucosidase aggregation and insolubility and, thus, the apparent null phenotype.     

Compelling evidence that a single nucleotide substitution in TYRP1 is responsible for coat-colour polymorphism in a free-living population of Soay sheep

Identifying the genes that underlie phenotypic variation in natural populations is a central objective of evolutionary genetics. Here, we report the identification of the gene and causal mutation underlying coat colour variation in a free-living population of Soay sheep (Ovis aries). We targeted tyrosinase-related protein 1 (TYRP1), a positional candidate gene based on previous work that mapped the Coat colour locus to an approximately 15cM window on chromosome 2. We identified a non-synonymous substitution in exon IV that was perfectly associated with coat colour. This polymorphism is predicted to cause the loss of a cysteine residue that is highly evolutionarily conserved and likely to be of functional significance. We eliminated the possibility that this association is due to the presence of strong linkage disequilibrium with an unknown regulatory mutation by demonstrating that there is no difference in relative TYRP1 expression between colour morphs. Analysis of this putative causal mutation in a complex pedigree of more than 500 sheep revealed almost perfect co-segregation with coat colour (chi2-test, p<0.0001, LOD=110.20), and very tight linkage between Coat colour and TYRP1 (LOD=29.50).     

A dual-probe hybridization method for reducing variability in single nucleotide polymorphism analysis with oligonucleotide microarrays

DNA microarray technology has become powerful and popular in mutation/single nucleotide polymorphism (SNP) discovery and genotyping. However, this method is often associated with considerable signal noise of nonbiological origin that may compromise the data quality and interpretation. To achieve a high degree of reliability, accuracy, and sensitivity in data analysis, an effective normalization method to minimize the technical variability is highly desired. In the current study, a simple and robust normalization method is described. The method is based on introduction of a reference probe coimmobilized with SNP probes on the microarray for a dual-probe hybridization (DPH) reaction. The reference probe is used as an intraspot control for the customized microarrays. Using this method, the interassay coefficient of variation (CV) was reduced significantly by approximately 10%. After DPH normalization, the CVs and ranges of the ratios were reduced by two to five times. The relative magnitudes of variation of different sources were also analyzed by analysis of variance. Glass slides were shown to contribute the most to the variance, whereas sampling and residual errors had relatively modest contribution. The results showed that this DPH-based spot-dependent normalization method is an effective solution for reducing experimental variation associated with microarray genotyping data.     

A single nucleotide polymorphism in the DNA polymerase gamma gene of Saccharomyces cerevisiae laboratory strains is responsible for increased mitochondrial DNA mutability

In the Saccharomyces cerevisiae strains used for genome sequencing and functional analysis, the mitochondrial DNA replicase Mip1p contains a single nucleotide polymorphism changing the strictly conserved threonine 661 to alanine. This substitution is responsible for the increased rate of mitochondrial DNA point mutations and deletions in these strains.     

Increased quality of in natura and cryopreserved semen of water buffaloes supplemented with saturated and unsaturated fatty acids from the palm oil industry

Ruminant energy supplementation with vegetable oils or fats has been standing out worldwide and oil palm processing has been receiving growing interest. This study assessed the effect of supplementation with saturated and unsaturated fatty acids from the palm oil industry on the lipid profile of seminal plasma and of the sperm membrane, as well as on the morphological and functional characteristics of raw and cryopreserved buffalo semen. Twelve purebred Murrah bulls (Bubalus bubalis) were assigned to the experimental groups and fed diets for 120 days with no added lipids (CONT, four bulls), or with an extra amount of 3% lipids from crude palm oil (PALM, four bulls), or from palm oil deodorizer distillate (PODD, four bulls). Semen was collected and cryopreserved every 15 days. The lipid composition of membranes and semen quality were determined after collections. Lipid supplementation did not impact feed intake (P>0.05). Diet enrichment with PALM increased the linoleic acid (C18:2,ω6) in seminal plasma. Lipid supplementation did not increase the polyunsaturated fatty acids in the sperm membrane composition, but significantly increased the lignoceric acid (C24:0). Cryopreserved semen of the supplemented bulls presented higher progressive motility (60.2 vs. 67.9 vs. 65.2%; P<0.05) and sperm viability detected by eosin-nigrosin staining (61.1 vs. 69.4 vs. 67.8%; P<0.05). Palm oil reduced major sperm defects in both raw (12.2 vs. 9.3 vs. 13.2%; P<0.0001) and cryopreserved semen (12.4 vs. 9.4 vs. 11.2%; P<0.0001). The lipids added to the diet did not impact the population of spermatozoa with intact plasma and acrosomal membranes (PI-/PSA-), but significantly increased the percentage of spermatozoa with high mitochondrial potential (25.6 vs. 31.5 vs. 32.0%; P=0.008). The results suggest that lipid supplementation based on crude palm oil or palm oil deodorizer distillate can be safely used to feed buffalo bulls and may increase sperm attributes related to male fertility.     

A porphyrin pathway impairment is responsible for the phenotype of a dominant disease lesion mimic mutant of maize.

The maize lesion mimic gene Les22 is defined by dominant mutations and characterized by the production of minute necrotic spots on leaves in a developmentally specified and light-dependent manner. Phenotypically, Les22 lesions resemble those that are triggered during a hypersensitive disease resistance response of plants to pathogens. We have cloned Les22 by using a Mutator-tagging technique. It encodes uroporphyrinogen decarboxylase (UROD), a key enzyme in the biosynthetic pathway of chlorophyll and heme in plants. Urod mutations in humans are also dominant and cause the metabolic disorder porphyria, which manifests itself as light-induced skin morbidity resulting from an excessive accumulation of photoexcitable uroporphyrin. The phenotypic and genetic similarities between porphyria and Les22 along with our observation that Les22 is also associated with an accumulation of uroporphyrin revealed what appears to be a case of natural porphyria in plants.     

A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.     

Activation by saturated and monounsaturated fatty acids of the O2- -generating system in a cell-free preparation from neutrophils

Saturated and monounsaturated fatty acids with appropriate chain length such as laurate and oleate activated an O2- -generating enzyme system in a cell-free preparation from porcine neutrophils. The activated preparation catalyzed a stoichiometric conversion of O2 to O2- by utilizing NADPH as the electron donor. The preparation contained both membrane and soluble fractions and, upon separation into subfractions, the O2- -generating activity resided exclusively in the membrane fraction. Polyunsaturated fatty acids including arachidonate also activated the system, but they concurrently stimulated NADPH-independent O2 consuming reactions which yield neither O2- nor H2O2. The amount of such a non-O2- -producing O2 consumption often reached twice as much as that of O2- production. For the activation of the O2- -generating system in the membrane, the presence of the soluble fraction was essential. However, the soluble fraction was no longer effective when once used for the activation, suggesting that the effective component(s) in the fraction was consumed or translocated to the membrane during the activation. When the activated membrane was incubated with delipidated albumin, the activity was lost with concomitant decreases in the amount of membrane-associated fatty acids. The lost activity was restored by the replenishment of the fatty acid in the presence of a fresh soluble fraction. We also found that Ca2+ augmented a non-O2- -producing O2 consumption in the cell-free preparation by unsaturated fatty acids and interfered with the activation of the O2- -generating system, especially that by saturated fatty acids.     

A single amino acid change is responsible for evolution of acyltransferase specificity in bacterial methionine biosynthesis

Bacteria and yeast rely on either homoserine transsuccinylase (HTS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 A resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bona fide HTS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites.     

A major locus expressed in the male gametophyte with incomplete penetrance is responsible for in situ gynogenesis in maize

In flowering plants, double fertilization occurs when the egg cell and the central cell are each fertilized by one sperm cell. In maize, some lines produce pollen capable of inducing in situ gynogenesis thereby leading to maternal haploids that originate exclusively from the female plant. In this paper, we present a genetic analysis of in situ gynogenesis in maize. Using a cross between non-inducing and inducing lines, we identified a major locus on maize chromosome 1 controlling in situ gynogenesis (ggi1, for gynogenesis inducer 1). Fine mapping of this locus was performed, and BAC physical contigs spanning the locus were identified using the rice genome as anchor. Genetic component analysis showed that (a) a segregation distortion against the inducer parent was present at this locus, (b) segregation resulted only from male deficiency and (c) there was a correlation between the rate of segregation distortion and the level of gynogenetic induction. In addition, our results showed that the genotype of the pollen determined its capacity to induce the formation of a haploid female embryo, indicating gametophytic expression of the character with incomplete penetrance. We propose the occurrence of a gametophytic-specific process which leads to segregation distortion at the ggi1 locus associated with gynogenetic induction with incomplete penetrance.