Cloning and restriction mapping of the alkaline phosphatase structural gene (phoA) of Escherichia coli and generation of deletion mutants in vitro

The structural gene for alkaline phosphatase (phoA) of Escherichia coli was cloned into the PstI site of pBR322, from a transducing bacteriophage, lambda p(phoA-proC). The restriction map of the plasmid was established. Based upon this information, several phoA deletion plasmids as well as a smaller phoA+ plasmid were constructed. The genetic map and restriction map were correlated by recombination analysis. Cells carrying one of the phoA+ plasmids overproduce alkaline phosphatase 10-fold upon phosphate limitation. However, both regulation and processing of the enzyme were found to be normal.     

Hormonally sensitive cyclic AMP phosphodiesterase in liver cells. An ecto-enzyme

Ecto-cyclic AMP phosphodiesterase activity was determined from freshly isolated and cultured liver cells. The cells were capable of hydrolyzing cyclic AMP in the medium. The ecto-phosphodiesterase represents a low Km phosphodiesterase which was activated by physiological concentrations of insulin. The product, 5'-AMP, was recovered in the medium and not with the cells. The enzyme was inhibited with aminophylline and trypsin. The ecto-phosphodiesterase activity was proportional to cell number, and total phosphodiesterase activity increased 5- to 10-fold when the cells were ruptured. About one-third of the ecto-phosphodiesterase activity from freshly isolated liver was due to phosphodiesterase in the medium. No phosphodiesterase was in the medium of cultured liver cells.     

Joint Segregation of Biochemical Loci in Salmonidae. II. Linkage Associations from a Hybridized Salvelinus Genome (S. NAMAYCUSHxS. FONTINALIS)

The results of more than 300 pairwise examinations of biochemical loci for joint segregation in brook trout (Salvelinus fontinalis) and in the hybridized genome of lake trout (S. namaycush) x brook trout are summarized. Nineteen loci have been assigned to the following eight linkage groupings on the basis of nonrandom assortment, including cases of both classical linkage and pseudolinkage: ODH with PMI with PGI-3, PGI-2 with SDH, ADA-1 with AGP-2, AAT-(1,2) with AGP-1 with MDH-1, MDH-3 with MDH-4, LDH-3 with LDH-4, IDH-3 with ME-2 and GUS with CPK-1. Pseudolinkage (an excess of nonparental progeny types) was observed only for male testcross parents. The results suggest that this phenomenon involves homeologous chromosome arms as evidenced by the de novo association of presumed duplicate loci in each case. Classical linkage has not been found for the five pairs of duplicate loci examined in Salvelinus, suggesting that not all of the eight metacentrics in the haploid complement involve fusions of homeologous chromosomes. Females consistently showed a greater degree of recombination.     

The toxin-agglutinin fold. A new group of small protein structures organized around a four-disulfide core

The three-dimensional structures of the snake venom postsynaptic neurotoxins and of the domains in wheat germ agglutinin show a remarkably similar overall folding pattern, consisting of equivalently placed, but variably sized loops which are held together by four similarly positioned disulfide bonds. Furthermore, occurrence of this wheat germ agglutinin-neurotoxin domain fold is predicted not only in the snake venom cardiotoxins and cytotoxins with neurotoxin-matched half-cystine sequence positions, but also for two small plant proteins, hevein and ragweed pollen allergen Ra5, on the basis of a nearly exact match of their half-cystine, sequence positions with those of the wheat germ agglutinin domain.     

Regulation of thymidine kinase synthesis during the cell cycle of Physarum polycephalum by the heat-sensitive system which triggers mitosis and S phase

Temperature shifts from 22 to 32 °C perturb one of the systems responsible for mitosis triggering in the plasmodia of Physarum (Myxomycetes). In order to determine if the same regulatory mechanism could also be involved in some other cell cycle events, the effects of temperature shifts on the peak of thymidine kinase (EC 2.7.1.21, ATP : thymidine 5′-phosphotransferase) synthesis have been studied. At 22 °C, the increase in thymidine kinase (tdk) activity begins shortly before mitosis and is thus always associated with the end of the G2 phase, the mitosis and the beginning of the S phase. The consequences of temperature shifts depend upon their position in the cell cycle. In all cases, a peak of tdk occurs concomitantly with the 32 °C mitosis. But, when the temperature shift is applied 90-15 min before the control metaphase at 22 °C, another peak of tdk is observed at 32 °C in absence of mitosis, but at the same time as the control mitosis at 22 °C. These results indicate that the increase in the synthesis of tdk is controlled by the heat-sensitive regulatory system which plays a role in the onset of mitosis and S phase. We further suggest that the increase in the synthesis of tdk and the triggering of mitosis are both controlled by the amount of a heat-sensitive effector. But the former takes place when the amount of the effector reaches a critical value lower than the value necessary to trigger mitosis.     

Relationships between cell-substratum interactions and the distribution of concanavalin A receptors of mouse embryo fibroblasts

The mobility of concanavalin A (ConA) receptors on the surfaces of mouse embryo cells are affected by cell-substratum interactions. When the cells are grown on a substratum from which they are easily detached by EDTA, their receptors have an increased mobility and are redistributed into patches after incubation with ConA at 37 °C.     

Glycosaminoglycans in the touch corpuscle of the dog

Synopsis Alcian Blue staining of the touch corpuscle of dog skin indicates that glycosaminoglycans are present as globular masses in some of the basal cells and are also seen in high concentration in the connective tissue core. The function of these basal cells is not clear but they may be responsible for the production of the large amount of glycosaminoglycan present in the underlying dermis.     

Isolation of the globular region of the subcomponent q of the C1 component of complement.

Digestion after heat treatment of the subcomponent q of the C1 component of complement by collagenase leads to the isolation of the globular region of the protein. This product ('heads') is composed of three chains giving an overall molecular weight of about 57000. About half of the collagen-like region present in C1 q is lost after digestion. The 'heads' are shown to be soluble and hemolytically active products.     

Mitotic factors from mammalian cells: A preliminary characterization

The objective of this study was the preliminary characterization of the factors from mitotic HeLa cells that can induce meiotic maturation in Xenopus laevis oocytes. We found that this factor is a heat-labile, Ca²⁺-sensitive, nondialyzable protein with a sedimentation value of 4-5S. Furthermore, no new protein synthesis was found to be required for this mitotic factor to induce maturation in the amphibian oocytes. These data suggest that the factors involved in the breakdown of nuclear membrane and the condensation of chromosomes that are associated with three different phenomena, mitosis, meiosis, and premature chromosome condensation, are very similar in different animal species.     

TESTOSTERONE-INDUCED CELL PROLIFERATION IN THE ACCESSORY SEX GLANDS OF MICE AT VARIOUS TIMES AFTER CASTRATION

The proliferative response to testosterone in the accessory sex glands (seminal vesicle and coagulating gland) of castrated male Balb/c mice has been examined by pulse and continuous thymidine-labelling experiments, and by the fraction of labelled mitoses technique. Progressive reductions in cellularity followed castration, and by varying the time elapsing between castration and the initiation of testosterone treatment, it was clear that the size of the response depended upon the number of cells in the tissue, relative to the normal complement. Interpretation of FLM data was difficult in periods where proliferative rates changed rapidly. We have attempted to explain the cell kinetic events by postulating a G₀ compartment, from which cells are stimulated to enter the proliferative cycle before subsequently returning to an out of cycle state. It was thought unlikely that substantial changes in cell cycle time occurred. In both the accessory sex glands, the overall form of the continuous thymidine labelling curves showed that most proliferative cells entered DNA synthesis in a shorter time after stimulation at 14 days after castration than they did at 3 days after castration. The data were not consistent with cells moving deeper into G₀ with time after castration. In the seminal vesicle almost all epithelial cells were potentially proliferative by 3 days after castration. In the coagulating gland only 30% were potentially proliferative at 3 days, increasing to 85% at 14 days after castration. However, such proportional increases represented much smaller changes in terms of absolute numbers of cells, because of a concomitant decline in cellularity from 3 to 14 days after castration.