A Novel Method for Generating Nested Deletions Using the in Vitro Bacteriophage T3 DNA Packaging System

To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained.     

Phylogenetic Position of the Mitochondrion-Lacking Protozoan Trichomonas tenax,Based on Amino Acid Sequences of Elongation Factors 1α and 2

Major parts of amino-acid-coding regions of elongation factor (EF)-1α and EF-2 in Trichomonas tenax were amplified by PCR from total genomic DNA and the products were cloned into a plasmid vector, pGEM-T. The three clones from each of the products of the EF-1α and EF-2 were isolated and sequenced. The insert DNAs of the clones containing EF-1α coding regions were each 1,185 bp long with the same nucleotide sequence and contained 53.1% of G + C nucleotides. Those of the clones containing EF-2 coding regions had two different sequences; one was 2,283 bp long and the other was 2,286 bp long, and their G + C contents were 52.5 and 52.9%, respectively. The copy numbers of the EF-1α and EF-2 gene per chromosome were estimated as four and two, respectively. The deduced amino acid sequences obtained by the conceptual translation were 395 residues from EF-1α and 761 and 762 residues from the EF-2s. The sequences were aligned with the other eukaryotic and archaebacterial EF-1αs and EF-2s, respectively. The phylogenetic position of T. tenax was inferred by the maximum likelihood (ML) method using the EF-1α and EF-2 data sets. The EF-1α analysis suggested that three mitochondrion-lacking protozoa, Glugea plecoglossi, Giardia lamblia, and T. tenax, respectively, diverge in this order in the very early phase of eukaryotic evolution. The EF-2 analysis also supported the divergence of T. tenax to be immediately next to G. lamblia. Received: 15 February 1996 / Accepted: 28 June 1996     

Concanavalin A can trap insulin and increase insulin internalization into cells cultured in monolayer

When rat hepatoma cells (R-Y121B) were incubated with insulin at 37 degrees C, concanavalin A increased insulin internalization into cells. When R-Y121B cells were first incubated with labeled insulin at 4 degrees C then with concanavalin A at various concentrations at 37 degrees C, the total cellular radioactivity was much higher at high lectin concentrations than at low lectin concentrations. This increase was not only due to an increase in insulin internalization into cells but also to an increase in insulin binding to cell surfaces. Concanavalin A can trap insulin on the insulin receptors - a "trapping" effect. It has been concluded that insulin and concanavalin A binding sites are very close to each other on the insulin receptors.     

Continuous culture of reuber hepatoma cells in serum-free,arginine-, glutamine-and tyrosine-deprived chemically defined medium

Summary The rat hepatoma cell line, H4-II-E, was grown serially over a I-year period and about 30 passages in arginine-, glutamine-, and tyrosine-deprived and ornithine-supplemented Eagle's mininum essential medium with no supplements other than biotin. The adapted cel line, R-Y121B, proliferates in the above mentioned medium with a doubling time of about 4 days and maintains hepatic “marker” enzymes such as tyrosine aminotransferase, phenylalanine hydroxylase, and all the enzymes of the urea cycle. This work was supported in part by Grant-in-Aid for Cancer Research 301050 and Science Research Grant 337013 from the Ministry of Education, Science and Culture, Japan.     

Biosynthesis of membrane proteins of Pseudomonas aeruginosa: effects of various antibiotics

The biosynthesis of membrane proteins of Pseudomonas aeruginosa was examined using various antibiotics (puromycin, streptomycin, chloramphenicol, tetracycline, and rifampin). Among six major membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the biosynthesis of two membrane proteins (proteins I and II) was found to be unusually resistant to these antibiotics. The biosynthesis of protein I (apparent molecular weight of 6,500) was completely resistant to puromycin, streptomycin, chloramphenicol, and tetracycline at conditions which severely inhibited the biosynthesis of all the other membrane proteins except for protein II. Under the same conditions, the biosynthesis of protein II (apparent molecular weight of 9,000) was also resistant to puromycin, streptomycin, and tetracycline, but was sensitive to chloramphenicol. The effect of rifampin on the biosynthesis of proteins I and II indicated that their messenger RNAs are extremely stable; their functional half-lives are 16 and 8 min for proteins I and II, respectively, in contrast with 2.0 and 3.5 min for the average half-lives of the cytoplasmic and membrane proteins, respectively. Protein II was identified as the lipoprotein of the outer membrane from its amino acid composition and mobility in gel electrophoresis. Protein I is a cytoplasmic membrane protein lacking histidine. From the content of arginine residues, the number of protein I molecules per cell was estimated to be as many as, and most likely more than, that of the lipoprotein (protein II). Therefore, protein I is the most abundant protein in P. aeruginosa.     

The effect of dexamethasone on the functioning of C3 receptor sites on the surfaces of human fibroblasts

The presence of C₃ receptor sites on the cell surfaces of WI-38 fibroblasts was reported in a previous paper. Here the effect of dexamethasone sodium sulfate of C₃ receptor site function was studied. The incubation of WI-38 fibroblasts with dexamethasone sodium sulfate produces the biphasic mode of action, i.e., the growth-stimulating phase with low doses (90–230 μg/ml) and the growth-inhibiting phase with high doses (450–900 μg/ml). The function of C₃ receptor sites on WI-38 fibroblasts seems to be very stable and cannot be suppressed by the pretreatment of WI-38 fibroblasts with dexamethasone in high concentrations, where the cell growth is inhibited.     

Effects of relaxation of gluten network on rehydration kinetics of pasta

The aim of this study was to investigate the effects of the relaxation of the gluten network on pasta rehydration kinetics. The moisture content of pasta, under conditions where the effects of the diffusion of water on the moisture content were negligible, was estimated by extrapolating the average moisture content of pasta of various diameters to 0?mm. The moisture content of imaginary, infinitely thin pasta did not reach equilibrium even after 1?h of rehydration. The rehydration of pasta made of only gluten was also measured. The rate constants estimated by the Long and Richman equation for both the pasta indicated that the rehydration kinetics of infinitely thin pasta were similar to those of gluten pasta. These results suggest that the swelling of starch by fast gelatinization was restricted by the honeycomb structural network of gluten and the relaxation of the gluten network controlled pasta rehydration kinetics.