Transfer of genes to Chinese hamster ovary cells by DNA-mediated transformation

We have transferred DNa to Chinese hamster ovary (CHO) cells by DNA-mediated transformation. CHO tk- cells were transformed with the clones gene for herpes simplex virus thymidine kinase (HSV-tk) and were found to have a 50-fold lower frequency of transformation than mouse Ltk- cells at the same DNA dosage. By altering the amount of tk gene and carrier DNA present, frequencies of up to 5 x 10(-5) were obtained. CHO HSV-tk+ transformants were very stable, and in several clones the HSV-tk gene copies integrated in higher-molecular-weight DNA. These cells also exhibited cotransformation for unselected markers. CHO lines were also transformed at a frequency of 10(-4) with the bacterial gene Ecogpt in a SV40-pBR322 vector. CHO tk-cells could be transformed at a frequency of 10(-7) with cellular DNA isolated from CHO tk+ cells. CHO cells offer a well-defined genetic system within which to transfer either cloned or whole cellular DNAs.     

Polypeptide synthesis by mitoplasts isolated from mouse liver

Polypeptide synthesis by mouse liver mitochondria was studied by incubating purified mitoplasts (mitochondria treated with digitonin) with [³⁵S]methionine. The products were separated either by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, or by isoelectric focusing, followed by SDS polyacrylamide gel electrophoresis. At least 14 distinct bands with molecular weights (mol. wt) ranging from about 8 000 to about 70 000 were found upon radioautography of the gels. When the samples were incubated in the presence of chloramphenicol, only a single weak band was found, whereas the protein pattern was unaffected by the presence of cycloheximide in the medium. The newly synthesized proteins were all acidic and evidence was obtained that they were hydrophobic in nature. Virtually all the labelled polypeptides were present in the membrane fraction, whereas the matrix showed little radioactivity. The data indicate that the proteins synthesized by mammalian mitochondria, like those in yeast, are components of the inner mitochondrial membrane. One protein of mol. wt 22 000 D was detected in the incubation medium. Since more of this component was present in the medium than in the pelleted mitoplasts and since this protein was not found in the matrix fraction of sonicated mitoplasts, it is believed that it had been excreted from the inner mitochondrial membrane. The finding that the number of proteins synthesized in mitoplasts isolated from mouse liver is considerably higher than that synthesized in yeast mitochondria reflects a most efficient utilization of the mammalian mitochondrial genome.     

On the chromatographic heterogeneity of human fetal hemoglobin.

Minor fetal hemoglobins in red cell hemolysates of newborn and adults with elevated levels of Hb F have been separated and quantitated by Biorex 70 column chromatography. In addition to Hb F1, other minor hemoglobin zones eluting before F1, pre-F1, and after F1, post-f1 have been observed. The relative amounts of the two pre-F1 zones and F1 are higher in the red cells of adults with 97--100% Hb F (homozygous hereditary persistence of fetal hemoglobin, homozygous deltabeta-thalassemia and homozygous beta0-thalassemia) than in the red cells of an adult with homozygous beta+-thalassemia with 66% Hb F, a child with a trisomy-D-13 having 38% Hb F, and in two newborn. Hb F was glycosylated in vitro with [14C]glucose or [14C] glucose 6-phosphate, and was acetylated using chicken reticulocyte lysate or a crude acetyltransferase preparation isolated from the same lysate with [14C]acetyl-CoA as substrate. Chromatographic analyses indicated that the Hb F1 zone can be formed both by glycosylation and acetylation of Hb F, and that pre-F1 zones can be products of the reaction of Hb F with phosphorylated glycolytic intermediates. Biosynthesis of minor hemoglobins in reticulocytes was studied with [14C]leucine in the presence and absence of cycloheximide and by pulse-chase. The resulting data indicate that Hb F1 synthesis is dependent upon Hb F synthesis and that the posttranslational modification may take place at an early stage in Hb F synthesis.     

Incorporation of 3H from delta-(L-alpha-amino (4,5-3H)adipyl)-L-cysteinyl-D-(4,4-3H)valine into isopenicillin N.

1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.     

The synergism of cardiotoxin and phospholipase A2 in hemolysis

The synergistic effect of exogenous cobra phospholipase A₂ on the hemolysis rate of guinea pig erythrocytes by highly purified snake venom cardiotoxins was investigated. In the presence of phospholipase A₂ the reaction was not only faster and had a lower activation energy but followed a sigmoidal instead of a linear time course. Similar results were obtained using porcine pancreatic phospholipase A₂. Significantly, addition of even a trace of cobra phospholipase A₂ (approx. 0.1%, w/w) was sufficient to bring about the full synergistic effect, emphasizing the stringent purity requirements for any meaningful investigation of cardiotoxin's own action. The possibility that the action of cardiotoxin on its own may involve the stimulation of an endogenous phospholipase is discussed in the light of the results obtained with exogenous cobra enzyme.     

Increased water drinking induced by sodium depletion in sheep.

Forty-eight hours of sodium depletion by acute cannulation of a parotid duct, via the buccal papilla, in the sheep, resulted in a progressive decrease in salivary secretion rate, salivary, urinary and plasma [Na] and no change in plasma [K]. In the first 24 h of Na depletion water intake was significantly increased. As normal sheep parotid saliva [Na] is higher than plasma [Na] and salivary loss over the first 24 h represented Na loss in excess of water relative to extracellular proportions, increased water intake was not osmotically induced. However, the animals did not replace their water deficit on either of the 2 days of Na depletion. This would appear to be valuable experimental model of increased water intake probably induced by hypovaolaemia, but uncomplicated concurrent osmotic stimuli, or any other factors which might result with the other commonly used experimental stimuli of thirst such as haemorrhage.     

Substrate-binding ability of Escherichia coli ribonucleic acid polymerase in relation to its protein composition.

The metabolism of [3H]progesterone in the rabbit endometrium and myometrium was studied in vitro. The major metabolities identified were 5alpha-pregnane-3,20-dione, 20alpha-hydroxypregn-4-en-3-one, 3beta-hydroxy-5alpha-preganan-20-one and 5alpha-pregnane-3beta,20alpha-diol. Other minor metabolites tentatively identified were 3alpha-hydroxy-5beta-pregnan-20-one,20alpha-hydroxy-5beta-pregnan-3-one and 5beta-pregnane-3alpha,20alpha-diol. The ability of the endometrium to metabolize progesterone on a unit weight bais was about 2.7 times that of the myometrium. The metabolism of [3H]progesterone in the rabbit uterus under the influnce of oestradiol-17beta and progesterone was studied. The ability of the oestradiol-treated rabbit uterus to metabolize progesterone was increased to 3.47 times that of the overiectomized control uterus, whereas the oestradiol-progesterone-treated rabbit uterus metabolized only 1.86 times that of the control. Study of the metabolism of progesterone with uterine subcellular preparations revealed that the 5alpha-reductase enzyme was present mainly in the nuclear fraction; 20alpha-hydroxysteroid dehydrogenase was found in the cytosol fraction and 3beta-hydroxysteroid dehydrogenase in the particulate fraction of the uterus. The metabolic pathways of progesterone in the rabbit uterine tissue are discussed.     

Effect of maternal diet on fetal hepatic lipogenesis.

The effects of: a, maternal diet; b, cyclic-3',5'-adenosinemonophosphate (cyclic AMP) and c, clofibrate on hepatic lipogenesis in fetal rats were studied. The experimental diets contained 22% protein, 40--50% carbohydrate, adequate vitamins, and minerals. In addition, the fat-containing diets were supplemented with either 15% corn oil, 25% corn oil, or 5% cholesterol + 10% oleic acid. In the clofibrate feeding studies, 0.3% (w/v) of the ethyl ester was added to a stock ration or to fat-free diet. Lipogenesis was measured in liver slices incubated with [2-14C]pyruvate, [1-14C]acetate, or 3H2O. In addition, activities of lipogenic enzymes were measured in cytosol fractions from liver homogenates. The effec-s of the experimental diets on liver composition were also examined. Lipogenic activity was higher in fetal than in maternal liver. When 15% corn oil was added to the maternal diet, fatty acid synthesis in fetal liver did not decrease as it did in maternal liver. Maternal fasting decreased fetal fatty acid synthesys by 50% when measured with 14C and less than 10% when measured with 3H2O. Although the addition of cholesterol to the maternal diet decreased cholesterol synthesis in maternal liver, no such decrease was observed in fetal liver. Changes in enzyme activities paralleled alterations in lipogenesis in maternal but not in fetal liver. Corn oil feeding or fasting increased the rate of transfer of linoleate from the dam to the fetus. However, accumulation of linoleate in fetal liver did not correlate with a decreased rate of fatty acid synthesis as it did in maternal liver. Maternal hepatic glycogen stores were depleted by fasting, but glycogen levels in fetal liver remained high under these conditions.