Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation.     

Studies on the Glucanase of Sclerotinia Libertiana

Glucanase-treatment of yeast cells was shown to increase the glucose fermenting activity, and decrease the sucrose and maltose fermenting activity. Also, lipase–and phospholipase–treatment decreased the fermenting activity on these sugars. However, the effects on the disaccharide fermenting activity could be reversed under various growth conditions of the yeast cells.

From these results, structural factors envolved in the transport of fermentable sugars into yeast cells are discussed.     

Photoaffinity labeling identifies the substrate-binding site of mammalian squalene epoxidase

Squalene epoxidase (SE) catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) in order to identify the location of the substrate-binding site and the roles of key residues in catalysis. Truncated 50-kDa rrSE was purified and photoaffinity labeled by competitive SE inhibitor (Ki=18.4 microM), [(3)H]TNSA-Dza. An 8-kDa CNBr/BNPS-skatole peptide was purified and the first 24 amino acids were sequenced by Edman degradation. The sequence PASFLPPSSVNKRGVLLLGDAYNL corresponded to residues 388-411 of the full-length rat SE. Three nucleophilic residues (Lys-399, Arg-400, and Asp-407) were labeled by [(3)H]TNSA-Dza. Triple mutants were prepared in which bulky groups were used to replace the labeled charged residues. Purified mutant enzymes showed lower enzymatic activity and reduced photoaffinity labeling by [(3)H]TNSA-Dza. This constitutes the first evidence as to the identity of the substrate-binding site of SE.     

A novel and simple method for quantification of small,dense LDL

A preponderance of small, dense (sd) LDL is strongly associated with the development of coronary heart disease, but the method for the measurement of sd LDL is too laborious for clinical use. We report a simple method for the quantification of sd LDL that is applicable to an autoanalyzer. This method consists of two steps: first, to precipitate the lipoprotein of density (d) <1.044 g/ml using heparin-magnesium; and second, to measure LDL-cholesterol in the supernatant by the homogeneous method or apolipoprotein B (apoB) by an immunoturbidometric assay. The cholesterol and apoB values obtained by the precipitation method (45 +/- 26 and 33 +/- 20 mg/dl, respectively) were similar to those obtained in the lipoprotein (d = 1.044-1.063) separated by ultracentrifugation (42 +/- 22 and 31 +/- 17 mg/dl, respectively), and there was an excellent correlation between the two methods for sd LDL-cholesterol (y = 1.05X + 1, r = 0.88, n = 69) and apoB (y = 1.07X, r = 0.90). Sd LDL values had a significant inverse correlation with LDL size. A high correlation was found between sd LDL-cholesterol and apoB values (r = 0.94). Sd LDL value was related to triglyceride, apoB, and LDL-cholesterol, but not to the buoyant LDL level. These results suggest that this precipitation method is a simple and rapid method for the measurement of sd LDL concentration.     

A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.     

Group X secretory phospholipase A2 can induce arachidonic acid release and eicosanoid production without activation of cytosolic phospholipase A2 alpha

Group X secretory phospholipase A2 (sPLA2-X) and cytosolic phospholipase A2 alpha (cPLA2alpha) are involved in the release of arachidonic acid (AA) from membrane phospholipids linked to the eicosanoid production in various pathological states. Recent studies have indicated the presence of various types of cross-talk between sPLA2s and cPLA2alpha resulting in effective AA release. Here we examined the dependence of sPLA2-X-induced potent AA release on the cPLA2alpha activation by using specific cPLA2alpha or sPLA2 inhibitors as well as cPLA2alpha-deficient mice. We found that Pyrrophenone, a cPLA2alpha-specific inhibitor, did not suppress the sPLA2-X-induced potent AA release and prostaglandin E2 formation in mouse spleen cells. Furthermore, the amount of AA released by sPLA2-X from spleen cells was not significantly altered by cPLA2alpha deficiency. These results suggest that sPLA2-X induces potent AA release without activation of cPLA2a, which might be relevant to eicosanoid production in some pathological states where cPLA2a is not activated.     

Plasmid genes increase membrane permeability in Escherichia coli

The membrane permeability to o-nitrophenyl beta-D-galactoside is increased in the presence of rifampicin in Escherichia coli cells carrying srnB+ or pnd+ plasmids, but not in the cells carrying srnB- or pnd- mutant plasmids. The same permeability alteration was also observed at 42 degrees C when a rpoC4- mutant strain was used as a host strain in the absence of rifampicin. These results and the blockage of the effects by action of chloramphenicol suggest that the increase of permeability to o-nitrophenyl galactoside was caused by the expression of srnB+ or pnd+ gene, respectively. srnB+ gene expression leads to massive RNA degradation, probably through the activation of the rna+ gene product. In an rna- strain carrying the srnB+ plasmid, the extent of RNA degradation was reduced, whereas the permeability to o-nitrophenyl galactoside was increased to the same level as in the rna+ strain. Also, the increase in permeability to o-nitrophenyl galactoside was observed at 30 degrees C, although high-temperature incubation (42 degrees C) was necessary for the induction of RNA degradation. These results suggest that the alteration in permeability is a more direct effect of the expression of srnB+ or pnd+ gene and that the RNA degradation is a secondary phenomenon caused by the alteration in the membrane.     

Recent developments in yeast cell surface display toward extended applications in biotechnology

Yeasts are promising hosts for industrial bio-refinery applications. In yeast cell surface displays, functional proteins, such as cellulases or lipases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae is the most commonly used yeast for cell surface display. Engineered yeasts have been utilized for a variety of applications, such as bioethanol production, chemicals synthesis, adsorption of environmental pollutants, and protein evolution. Here, we summarize recent developments in yeast cell surface display techniques for bio-refinery applications, including methods using hosts such as Pichia pastoris, Yarrowia lipolytica, and S. cerevisiae, focusing on the characteristics of anchor proteins and applications.     

Studies on the ɛ-Lysine Acylase

?-Lysine acylase of Achromobacter pestifer EA was purified by fractionations with ammonium sulfate and acetone, and by vertical zone electrophoresis. As a result, this bacterial ?-lysine acylase was obtained as an electrophoretically homogeneous protein, specific activity of which is the highest among ?-lysine acylases ever reported.