Chromosome engineering in Saccharomyces cerevisiae by using a site-specific recombination system of a yeast plasmid.

We have developed an effective method to delete or invert a chromosomal segment and to create reciprocal recombination between two nonhomologous chromosomes in Saccharomyces cerevisiae, using the site-specific recombination system of pSR1, a circular cryptic DNA plasmid resembling 2 microns DNA of S. cerevisiae but originating from another yeast, Zygosaccharomyces rouxii. A 2.1-kilobase-pair DNA fragment bearing the specific recombination site on the inverted repeats of pSR1 was inserted at target sites on a single or two different chromosomes of S. cerevisiae by using integrative vectors. The cells were then transformed with a plasmid bearing the R gene of pSR1, which encodes the site-specific recombination enzyme and is placed downstream of the GAL1 promoter. When the transformants were cultivated in galactose medium, the recombination enzyme produced by expression of the R gene created the modified chromosome(s) by recombination between two specific recombination sites inserted on the chromosome(s).     

Occurrence of D-rhamnan as the common antigen reactive against monoclonal antibody E87 in Pseudomonas aeruginosa IFO 3080 and other strains.

S. Sawada and co-workers reported that a monoclonal antibody (MAb), E87, interacted with about 80% of Pseudomonas aeruginosa isolates, and they separated a rhamnose-rich polysaccharide as the probable antigen for MAb E87 from P. aeruginosa IFO 3080 (S. Sawada, T. Kawamura, Y. Masuho, and K. Tomibe, J. Infec. Dis. 152:1290-1299, 1985). In the present study, the rhamnose-rich polysaccharide was shown to be structurally and immunologically identical to the D-rhamnan of P. aeruginosa IID 1008 (S. Yokota, S. Kaya, S. Sawada, T. Kawamura, Y. Araki, and E. Ito, Eur. J. Biochem. 167:203-209, 1987). Furthermore, a set of enzymes responsible for the formation of GDP-rhamnose (probably in a D-form) from GDP-D-mannose was found in the 100,000 x g supernatant fractions obtained from all of nine P. aeruginosa strains reactive against MAb E87. The result strongly supports a possibility that lipopolysaccharides having a D-rhamnan chain widely occur as the common antigen among various P. aeruginosa isolates.     

Microquantification of cholesterol and cholesteryl esters in rat peritoneal macrophages by reverse-phase high-performance liquid chromatography

A simple and rapid method for the microquantification of cholesterol and cholesteryl esters by reverse-phase high performance liquid chromatography has been established. Comparison of elution patterns of authentic cholesterol and cholesteryl esters revealed that a mu Bondasphere reverse-phase C8 (300-A) column was more suitable than a corresponding reverse-phase C4 or C18 column in terms of rapidity and sensitivity. Recovery of cholesterol and cholesteryl esters from a C8 column was greater than 98% when determined either by radioactive cholesterol and cholesteryl oleate or by cholesteryl heptadecanoate. The sensitivity of the quantification ranged from 5 ng to 50 micrograms for both cholesterol and cholesteryl esters. This method was applied to determination of cellular cholesterol and cholesteryl esters of rat peritoneal macrophages. Lipid extracts of these cells were found to contain 38.01 +/- 2.60 micrograms of cholesterol and 3.18 +/- 0.36 micrograms of cholesteryl esters per milligram of cell protein. When the cells were loaded with cholesteryl esters by incubation for 24 h with various concentrations of acetylated low-density lipoprotein, a cellular level of cholesteryl esters showed a dose-dependent increase and reached a maximal level of 106.60 +/- 3.05 micrograms/mg cell protein. Thus, the present method is useful for the microquantification of cholesterol and cholesteryl esters from lipid extracts of biological samples.     

Biosynthesis of glycerol teichoic acid in Bacillus cereus: formation of linkage unit disaccharide on a lipid intermediate

A tunicamycin-like antibiotic 24010 at a concentration of 1 μg/ml selectively inhibited the $\text{in vivo}$ synthesis of glycerol teichoic acid of cell walls in $\text{Bacillus cereus}$ AHU 1030. Incubation of membranes of this strain with $\text{N}$-acetylglucosaminyl pyrophosphorylundecaprenol and UDP-$\text{N}$-acetylmannosamine led to formation of a glycolipid having a saccharide moiety identical with the cell wall teichoic acid linkage unit, $\text{N}$-acetylmannosaminylβ(1→4)-$\text{N}$-acetylglucosamine. The membranes also catalyzed transfer of glycerol phosphate units from CDP-glycerol to this disaccharide-linked lipid. Thus the biosynthesis of the cell wall glycerol teichoic acid in this strain seems to involve the disaccharide-linked lipid as an intermediate.     

Bacillus cereus autolytic endoglucosaminidase active on cell wall peptidoglycan with N-unsubstituted glucosamine residues

An autolytic glycosidase from a lysozyme-resistant strain of Bacillus cereus capable of cleaving the glycosidic linkages of N-unsubstituted glucosamine in the cell wall peptidoglycan was studied. This glycosidase activity, together with N-acetylmuramyl-L-alanine amidase activity, was found in an autolytic enzyme preparation obtained from the 20,000 x g precipitate fraction by means of autolysis followed by ammonium sulfate fractionation. The major saccharide fragments resulting from digestion of the untreated, non-N-acetylated, cell wall peptidoglycan of B. cereus with the autolytic enzyme preparation were identified as N-acetylmuramyl-glucosamine and its dimer. The peptidoglycan N-acetylated with acetic anhydride could also be digested with the same enzyme preparation, giving N-acetylmuramyl-N-acetylglucosamine and its dimer as the major saccharide fragments.     

Possible demonstration of multipotential nature of embryonic neural retina by clonal cell culture.

The possible multipotential nature of the neural retina of early chick embryos was examined by the technique of clonal cell culture. Cultures were prepared from cells dissociated from freshly excised neural retinas of 3.5-day-old chick embryos or from cells harvested from primary highdensity cultures. The following four colony types were obtained: colonies differentiating into “lentoid bodies”; colonies with pigment cells; colonies with both “lentoid bodies” and pigment cells; and colonies comprised entirely of unidentifiable cells. Neuronal differentiation occurred frequently in the early stages of culture (up to about 10 days). In some of these neuronal colonies, “lentoid bodies” and, rarely, both “lentoid bodies” and pigment cells differentiated after a further culture period of up to 30 days. Secondary colonies established from primary colonies after 9–10 days demonstrated that these original colonies fell into four different categories: those giving rise to secondary colonies containing only “lentoid bodies,” those giving rise to pigmented colonies only, those developing both lentoid and pigmented colonies, and finally those which gave rise to secondary colonies of all three types, lentoid, pigmented, and mixed colonies. When primary pigmented colonies were recloned at about 30 days after inoculation, the differentiated pigment cells transdifferentiated into lens. Whether multispecific colonies were really of clonal origin or not is discussed. The possible presence of a multipotent progenitor cell able to give rise to multispecific clones in the neural retina of 3.5-day-old chick embryos is suggested. A sequence of differentiation starting from multipotent neural retinal cells to be terminated with lens through the differentiation of neuronal and pigment cells is hypothetically proposed.     

Qualitative abnormality of insulin secretion in a case with insulinoma.

We have presented here a case of atypical insulinoma. Despite the recurrent episodes of hypoglycemic symptoms, the plasma level of insulin has never been excessive at fasting or by regular provocative tests. Detailed examination had demonstrated qualitative abnormality of insulin secretion. Hyposuppressibility of insulin secretion by hypoglycemia, borderline diabetic curve of glucose tolerance test, blunted response ot insulin to glucagon and leucine were the principle characteristics of these abnormalities. After removal of adenoma, insulin response to glucose, glucagon and leucine was improved. Only secretion provoked a high level of insulin and this abnormal elevation was no longer seen after the removal of adenoma. A removed elevation was no longer seen after the removal of adenoma. A removed insulinoma contained 25 U of immunoreactive insulin per gram tissue, but was negative for aldehyde-fuchsin staining. On electromicroscopy only atypical beta-cell granules were seen.     

A soluble enzyme activity that attaches free diaminopimelic acid to bdelloplast peptidoglycan

An enzyme activity, responsible for the attachment of diaminopimelic acid (DAP) to bdelloplast wall peptidoglycan, was studied in an in vitro, cell-free system. Most of the activity was found in the high-speed (20000g) supernatant fraction of homogenates of bdelloplasts prepared from a culture of the intracellular bacterium Bdellovibrio bacteriovorus 109J, growing synchronously within cells of Escherichia coli. Peptidoglycan preparations obtained either from E. coli ML35 or from the walls of bdelloplasts synchronously cultured for 40 or 90 min served as the acceptors in this reaction, whereas cell wall or peptidoglycan preparations obtained from Gram-positive bacteria could not function as acceptors of DAP. The attachment activity had an apparent Km value for DAP of 10 microM; for bdelloplast peptidoglycan, it was approximately 0.43 mg/mL, which is 13 microM with respect to peptidoglycan disaccharide peptide units. DAP attachment was partially inhibited by the structural analogues lanthionine, L-ornithine, beta-aminobutyric acid, and D-serine, as well as the cell wall synthesis inhibitors penicillin G, ampicillin, and cephalexin. This enzyme activity is present only during the intracellular stage of the bdellovibrio's developmental growth cycle and may serve a stage-specific function of biochemically modifying the cell in which it grows.