Construction and characterization of Escherichia coli disruptants defective in the yaeM gene.

Escherichia coli disruptants defective in the yaeM gene, which is located at 4.2 min on the chromosome map, were constructed and characterized. The disruptants showed auxotrophy for 2-C-methylerythritol, a free alcohol of 2-C-methyl-D-erythritol 4-phosphate that is a biosynthetic precursor in the nonmevalonate pathway. This result clearly shows that the yaeM gene is indeed involved in this pathway in E. coli.     

The Gene Encoding Flavanone 3-Hydroxylase is Expressed Normally in the Pale Yellow Flowers of the Japanese Morning Glory Carrying the speckled Mutation Which Produce Neither Flavonol nor Anthocyanin but Accumulate Chalcone, Aurone and Flavanone

The Japanese morning glory carrying the recessive mutable speckledallele with the dominant speckled-activator bears colorlessflowers with fine and round colored spots distributed over thecorolla whereas the plant without the speckled-activator producespale yellow flowers. Previous chemical analysis has indicatedthat a mutation in the gene for flavanone 3-hydroxylase (F3H)is a likely candidate for the speckled allele. However, theF3HmRNA without sequence alteration accumulates normally inthe pale yellow flowers, indicating that the speckled alleleis neither the F3H gene nor a regulatory gene acting on theF3H gene expression. (Received April 4, 1997; Accepted June 2, 1997)     

Murine C4-binding protein: a rapid purification method by affinity chromatography

A simple, 3-step method was described for purification of murine C4 binding protein (C4-bp), a recently recognized serum protein that functions as one of the regulatory proteins of the complement system. The method consists of 1) affinity chromatography using TNBS-BGG-conjugated Sepharose beads, 2) gel filtration on a Sepharose 6B column, and 3) heparin-Sepharose chromatography. By this method, milligram quantities of C4-bp can be easily purified by more than 500-fold from EDTA-serum of various mouse strains, and the whole purification process can be completed within 1 wk. The overall yield of C4-bp is about 15%. The C4-bp thus prepared is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunelectrophoresis. The purified mouse C4-bp showed physicochemical properties very similar to those described for human C4-bp. Like human C4-bp, mouse C4-bp is composed of several apparently identical subunits of the m.w. of 80,000. However unlike the human counterpart, the subunits of mouse C4-bp are not linked by disulfide bonds but are connected by non-covalent forces that can be disrupted by SDS. The purified mouse C4-bp retained binding affinity for C4 and showed unaltered antigenicity. Immunization of rabbits with the purified mouse C4-bp resulted in the production of potent and monospecific antisera.     

Isolation of lactoperoxidase, lactoferrin, α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. β-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, α-lactalbumin could not be adsorbed onto the resin. α-Lactalbumin and β-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. α-Lactalbumin and β-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. α-Lactalbumin was eluted using a linear (0–0.15 M) concentration gradient of NaCl in 0.05 M Tris–HCl buffer (pH 8.5). Subsequently, β-lactoglobulin B and β-lactoglobulin A were eluted from the column with 0.05 M Tris–HCl (pH 6.8), using a linear (0.1–0.25 M) concentration gradient of NaCl. The yields were 1260 mg α-lactalbumin, 1290 mg β-lactoglobulin B and 2280 mg β-lactoglobulin A from 1 l rennet whey.