2-(2-Pyridyl)ethyl group: new type protecting group in the synthesis of DNA via phosphoramidite intermediates

2-(2-Pyridyl)ethyl group is a new type P-O protecting group for the synthesis of oligodeoxyribonucleotides by the phosphite triester method. This group is stable to alkali and acid conditions, and to be removed from internucleotidic bonds under mild conditions via two step procedures without any side reactions. Further we have found that bis(diisopropylamino)chlorophosphine is much more effective for the preparation of bis(diisopropylamino)alkoxyphosphines than various dichlorophosphines.     

Sequence and over-expression of subunits of adenosine triphosphate synthase in thermophilic bacterium PS3

The primary structures of all the subunits of thermophilic ATP synthase were determined, and its alpha, beta and gamma subunits could be over-expressed in Escherichia coli, because these subunits were stable and reconstitutable. DNA of 7500 base pairs in length was found to contain a cluster of nine genes for subunits of ATP synthase. The order of their reading frames (size in base pairs) was: I(381): a(630): c(216): b(489): delta(537): alpha(1507): gamma(858): beta(1419): epsilon(396), I being a gene for a small hydrophobic, basic protein expressed in vitro. All the termini of TF0F1 subunits were confirmed by peptide sequencing. Large quantities of the overexpressed thermophilic alpha, beta and gamma subunits were prepared from the extract of E. coli, by a few purification steps.     

Solubilization and reconstitution of voltage-dependent calcium channel from bovine cardiac muscle. Ca2+ influx assay using the fluorescent dye Quin2

Highly purified sarcolemmal membranes, prepared from fresh bovine heart left ventricle, were solubilized by n-octyl beta-D-glucopyranoside and reconstituted into proteoliposomes with soybean phospholipids by the detergent-dialysis method. Ca2+ flux into the proteoliposomes was determined using the fluorescent probe Quin2. A membrane potential (negative in the proteoliposome interior) that was created by K+ diffusion mediated by valinomycin accelerated the Ca2+ influx. The voltage-dependent Ca2+ influx was dependent on pretreatment of the sarcolemmal membranes with Bay K 8644 and was inhibited by various calcium antagonists including nicardipine (K0.5 = 4.5.10(-7) M), verapamil (K0.5 = 9.2.10(-9) M), diltiazem (K0.5 = 26.10(-8) M) and omega-conotoxin (K0.5 = 9.5.10(-9) M).     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.     

Response of end bud cells from immature rat mammary gland to hormones when cultured in collagen gel

End buds from 4- to 5-week-old rat mammary glands were isolated and cultured within a rat tail tendon collagen gel matrix. Media containing equine serum or porcine serum and cholera toxin promoted growth, but not the production of casein or thioesterase II, nor did they induce a state of differentiation as assessed by cell ultrastructure. Medium supplemented with only 5% porcine serum, insulin and cholera toxin did not support growth or differentiation. However, when prolactin, estradiol, progesterone and hydrocortisone were added to this medium, growth was stimulated greatly and a differentiated state was induced as assessed by the production of casein and thioesterase and by the appearance of a highly secretory ultrastructure.     

Mutational analysis of the RNase-like domain in subunit 2 of fission yeast RNA polymerase II

Local sequence similarity exists between the subunit 2 of eukaryotic RNA polymerases II and the barnase-type bacterial RNases. The RNase-like domain from the Rpb2 ofSchizosaccharomyces pombe was expressed inEscherichia coli as a GST fusion protein and examined for its RNase activity. When the GST fusion protein was incubated in vitro with³²P-labeled RNA, the RNA degradation activity was less than 0.1%, if any, of the level of synthetic barnase. In order to check the in vivo function of this region, we constructed two mutantrpb2 alleles,rpb2 ^E357A andrpb2 ^H3a6L , each carrying a single amino acid substitution at the site correponding to one of the three essential amino acid residues forming the catalytic site in barnase (mutation of barnase at the corresponding sites results in complete loss of RNase activity) and five other mutantrpb2 alleles, each carrying a single mutation at various positions within the RNase-like domain but outside the putative catalytic site for RNase activity. When these mutantrpb2 alleles were expressed in anrpb2-disruptedS. pombe strain, all the mutants grew as well as the wild-type parent and did not show any clear defective phenotypes. These results suggest either that the RNase-like domain in Rpb2 does not function as an RNase in vivo or that the RNase activity of this domain, if present at all, is not essential for cell growth.     

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min^–1 at 100g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 × 10⁶ cells ml^–1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process.     

Mouse {beta}-galactoside {alpha}2,3-sialyltransferases: comparison of in vitro substrate specificities and tissue specific expression

Four types of ß-galactoside     

A Continuum theory for the prediction of lateral and rotational positioning of α-helices in membrane proteins: Bacteriorhodopsin

We have developed a new method for the prediction of the lateral and the rotational positioning of transmembrane helices, based upon the present status of knowledge about the dominant interaction of the tertiary structure formation. The basic assumption about the interaction is that the interhelix binding is due to the polar interactions and that very short extramembrane loop segments restrict the relative position of the helices. Another assumption is made for the simplification of the prediction that a helix may be regarded as a continuum rod having polar interaction fields around it. The polar interaction field is calculated by a probe helix method, using a copolymer of serine and alanine as probe helices. The lateral position of helices is determined by the strength of the interhelix binding estimated from the polar interaction field together with the length of linking loop segments. The rotational positioning is determined by the polar interaction field, assuming the optimum lateral configuration. The structural change due to the binding of a prosthetic group is calculated, fixing the rotational freedom of a helix that is connected to the prosthetic group. Applying this method to bacteriorhodopsin, the optimum lateral and rotational positioning of transmembrane helices that are very similar to the experimental configuration was obtained. This method was implemented by a software system, which was developed for this work, and automatic calculation became possible for membrane proteins comprised of several transmembrane helices. © 1995 Wiley-Liss, Inc.