Effect of the levels of dissolved oxygen on the expression of recombinant proteins in four recombinantEscherichia coli strains

Summary Four recombinant strains ofEscherichia coli were examined for the effects of the dissolved oxygen level on the level of biomass, the plasmid content, and the level of recombinant protein at the stationary phase of batch growth. Strains JM101/pYEJ001, and TB-1/pYEJ001 (encoding chloramphenicol acetyltransferase), and strain TB-1/p1034, and TB-1/pUC19 (encoding -galactosidase) were grown at the constant dissolved oxygen levels of 0, 50, and 100% air saturation, as well as in the absence of dissolved, oxygen control. The biomass of all strains under constant aerobic conditions was 12–36 times higher than that under anaerobic conditions, but was the same as or slightly higher than that without dissolved oxygen control. The plasmid content in all strains under anaerobic conditions was 2.9–11.7 times higher than that under aerobic conditions. The optimal dissolved oxygen concentration for the specific activity of recombinant proteins was dependent upon the strain. In no strain were constant aerobic conditions optimal. However, because of the effect on biomass, controlled aerobic conditions were optimal for the volumetric activity of recombinant protein in all but one strain.     

The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.     

Uptake and metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells. Effects of potassium cyanide and dinitrophenol

Cultured monkey hepatocarcinoma cell (NCLP-6E) were used to investigate the uptake and metabolism of thyroid hormones. Intracellular accumulation was shown by the failure to acutely release hormone from cells subsequently exposed to serum proteins, and by the metabolic trnasformation of the hormones to deiodinated products and their sulfates. When hepatocarcinoma cell monolayers were studied at hormone concentrations below 10(-10) M, neither KCN nor dinitrophenol inhibited uptake. Taken together with previous findings that uptake was neither saturable nor reduced at low temperature, these results indicate that this process was not active transport. Deiodination of both the phenolic and non-phenolic rings, however, was partially inhibited by KCN but not by dinitrophenol. Sulfation of 3,3'-diiodothyronine and 3'-monoiodothyronine was strongly inhibited by both KCN and dinitrophenol. Uptake of the hormones and their metabolites was also measured in suspended hepatocarcinoma cells and compared with the uptake by normal rat hepatocytes, human fibroblasts and human lymphocytes. In these experiments 1 micrometer triidothyronine and 0.47 mM dinitrophenol were used to inhibit deiodination and sulfation, respectively. Uptake was similar in all cell types. Accumulation was highest with 3,5,3'-triiodothyronine, intermediate with other compounds having iodines in both rings, lowest with compounds iodinated in only one ring, and absent with iodothronine sulfates. These findings help to explain the relative rates of metabolism of the iodothyronines and their release from the cells.     

Regulation of the Escherichia coli methylgalactoside transport system by gene mglD.

Constitutive activity of the methylgalactoside transport system of Escherichia coli K-12 is shown to result from mutation of a genetic locus distinct from the two previously described regulatory loci for this permease. Employing an autoradiographic procedure whereby constitutive and inducible cells can be differentiated, it is demonstrated that this locus, termed mglD, is 20% cotransducible with ptsF by bacteriophage P1. Selection for constitutive mutants among an inducible population yielded cells who mutations mapped in mglD. Cotransduction of mglD with mglB, minus C, and minus A, three genes required for activity of the methylgalactoside transport system, is 95, 88, and 81%, respectively. The results of recombination studies employing three and four factors indicate that the order of genes in this region is ptsF, mglD, B, C, A.     

Isolation of a human plasmin-derived, functionally active, light (B) chain capable of forming with streptokinase an equimolar light (B) chain-streptokinase complex with plasminogen activator activity.

A functionally active human plasmin light (B) chain derivative, stabilized by the streptomyces plasmin inhibitor leupeptin, was isolated from a partially reduced and alkylated enzyme preparation by an affinity chromatography method with a L-lysine-substituted Sepharose column. This light (B) chain derivative was found to be relatively homogeneous by electrophoretic analysis in both an acrylamide gel/dodecyl sulfate system and on cellulose acetate. It possessed approximately 3% of the proteolytic activity (casein substrate) of the original enzyme, and it incorporated 0.09 mol of [3H]diisopropyl phosphorofluoridate per mol of protein. It contained 3.1 +/- 0.3 carboxymethylated cysteines per mol of protein and can be designated as a CmCys5-light (B) chain (CmCys)3. When this isolated light (B) chain derivative was mixed in equal molar amounts with streptokinase, the mixture developed both human and bovine plasminogen activator activities; the bovine activator activity was approximately 66% of the bovine activator activity of the equimolar human plasmin-streptokinase complex. Although this complex now incorporated 0.50 mol of [3H]diisopropyl phosphorofluoridate per mol of protein, its proteolytic activity, on a molar basis, was the same as the proteolytic activity of the isolated light (B) chain derivative. It was shown by electrophoretic analysis in both an acrylamide gel/epsilon-aminocaproic acid system and on cellulose acetate that the light (B) chain derivative and streptokinase forms an equimolar light (B) chain-streptokinase complex, indicating that the binding site for streptokinase is located on the light (B) chain of the enzyme. A functionally active equimolar light (B) chain-streptokinase complex was also isolated from a partially reduced and alkylated equimolar human plasmin-streptokinase complex by the affinity chromatography method. The plasminogen activator activities (human and bovine) of this light (B) chain-streptokinase complex were similar to those of the plasmin-streptokinase complex from which it was derived. Although this complex incorporated 0.70 mol of [3H]diisopropyl phosphorofluoridate per mol of protein, its proteolytic activity, on a molar basis, was only 14% of proteolytic activity of the plasmin-streptokinase complex.     

Association of p60fyn with middle tumor antigen in murine polyomavirus-transformed rat cells.

Rabbit antisera raised against human FYN-specific peptides were used to evaluate the expression of the fyn gene product in normal and murine polyomavirus middle tumor antigen (MTAg)-transformed rat cells. The antisera were capable of detecting p60fyn in both normal and MTAg-transformed cells. Two different antisera directed against unique p60fyn sequences were found to detect p60fyn-MTAg complexes in cell lysates from the MTAg-transformed cells. The MTAg molecules immunoprecipitated by FYN antisera were phosphorylated on tyrosine during immune-complex kinase reactions at sites similar to those found on MTAg in complexes with pp60c-src. Whereas the abundance of p60fyn was estimated to be less in the MTAg-transformed cells than in their normal counterparts, the specific activities of p60fyn molecules in the normal and transformed cells were similar.     

Home Range and Frugivory Patterns of Mountain Gorillas in Bwindi Impenetrable National Park,Uganda

Mountain, western, and Grauer's gorillas exhibit broad differences in ecological patterns with western gorillas eating more fruit and having larger home ranges than their largely folivorous counterparts in the Virunga Volcanoes. We studied the home range and frugivory patterns of one group of Gorilla beringei beringei in the little-studied population of Bwindi Impenetrable National Park, Uganda, to compare with other populations and to investigate whether there was any relationship between patterns of frugivory and home range size. During the 3-year study, the gorillas ate 16 species of fruit on 27% of observation days. There was high variability in frugivory among the 3 years and no consistent seasonal pattern. Annual home range size was ca. 21 km² for Years 1 and 2, and it increased dramatically to 40 km² in Year 3. Home range size varied considerable between months and seasons, but there is no clear relationship between occurrence of fruit-eating and home range size. The group exhibited more fruit-eating and a larger home range size those ofthe gorillas in the Virunga Volcanoes. Their home range size is comparable to that of western gorillas, though Bwindi gorillas consumed less fruit. Home range size and utilization by all gorillas probably depends on a complex relationship between the distribution and abundance of both fruit and herbaceous vegetation and social factors such as male mating tactics.     

Genetic mapping of turnip mosaic virus resistance in Lactuca sativa

Presence of the dominant Tu gene in Lactuca sativa is sufficient to confer resistance to infection by turnip mosaic virus (TuMV). In order to obtain an immunological assay for the presence of TuMV in inoculated plants, the TuMV coat protein (CP) gene was cloned by amplification of a cDNA corresponding to the viral genome using degenerate primers designed from conserved potyvirus CP sequences. The TuMV CP was overexpressed in Escherichia coli, and polyclonal antibodies were produced. To locate Tu on the L. sativa genetic map, F₃ families from a cross between cvs Cobbham Green (resistant to TuMV) and Calmar (susceptible) were genotyped for Tu. Families known to be recombinant in the region containing Tu were infected with TuMV and tested by the indirect enzyme-linked immunosorbent assay (ELISA) using the anti-CP serum. This assay placed Tu between two random amplified polymorphic DNA (RAPD) markers and 3.2 cM from Dm5/8 (which confers resistance to Bremia lactucae). Also, bulked segregant analysis was used to screen for additional RAPD markers tightly linked to the Tu locus. Five new markers linked to Tu were identified in this region, and their location on the genetic map was determined using informative recombinants in the region. Six markers were identified as being linked within 2.5 cM of Tu.