Regulation of product synthesis in cell cultures of Catharanthus roseus (L) G. Don

Cell suspension cultures of the Madagascan Periwinkle Catharanthus roseus (L) G. Don were maintained on Gamborg's B5 medium and their growth monitored by measuring cellular fresh and dry weight, cell number and mitotic activity. Samples of cells of different ages and physiological states were subcultured onto an alkaloid production medium and their rates of growth and alkaloid accumulation measured over a period of 30–45 days. In two experiments the rate of biomass accumulation was directly related to the rate of cellular serpentine accumulation. Possible mechanisms underlying this phenomenon are discussed in relation to the properties of cells comprising the inocula.     

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Cultured fetal rat myoblasts release peptide growth factors which are immunologically and biologically similar to somatomedin

The production of immunologically and biologically active somatomedin activity from isolated myoblasts and fibroblasts from fetal rats of 21 days gestational age was investigated. Myoblast-rich cell populations were derived from primary cultures of dispersed muscle cells by the tendency of myoblasts to become detached from the culture dish in the presence of cytochalasin B. Fibroblasts were obtained from fetal muscle. Culture medium conditioned by exposure to myoblasts for 48 hours produced an increased incorporation of both [35S]sulphate and [3H]thymidine by explants of fetal rat costal cartilage in vitro compared to fresh medium. Myoblast-conditioned medium also contained somatomedin-C-like immunoreactivity which diluted in parallel with partially purified human somatomedin-C (3,271 +/- 446 mU/mg cell protein; mean +/- SEM, seven experiments). Medium conditioned by exposure to fetal rat fibroblasts did not promote isotope uptake by fetal rat cartilage above control values, and contained only low levels of somatomedin-C-like immunoreactivity (343 +/- 89 mU/mg cell protein, three experiments). The release of both somatomedin bioactivity and immunoreactivity into conditioned medium was significantly reduced by the incubation of myoblasts in the presence of rat growth hormone (100 ng/ml and 500 ng/ml). We conclude that fetal rat myoblasts released growth factor activity during culture which exhibited biological and immunologic characteristics of somatomedin. Since the bioactivity was demonstrated on skeletal tissues from rat fetuses of the same gestational age as those that yielded myoblasts such growth factor release may be physiological.     

The active site regions of lacZ and ebg beta-galactosidases are homologous

The active site-directed inhibitor 4-nitrophenyl-beta-D-galactopyranosylmethyltriazene, previously shown (Fowler, A. V., Zabin, I., Sinnott, M. L., and Smith, P. J. (1978) J. Biol. Chem. 253, 5283-5285) to alkylate methionine 502 in lacZ beta-galactosidase, was used to label the second naturally occurring beta-galactosidase of Escherichia coli (ebgo). The reagent was also used to label two mutant forms of the enzyme (ebga and ebgb) selected for enhanced lactase activity. In the case of ebgo and ebga, 75 and 85% of the label, respectively, was incorporated into a tryptic peptide which is homologous (38% identity) to residues 483-503 of the lacZ beta-galactosidase sequence. In the ebgo and ebga enzymes, a serine probably is alkylated. In the case of the ebgb enzyme, 61% of the label is found on a tryptic peptide homologous (69% identity) with residues 457-468 of the lacZ beta-galactosidase. In this peptide, a glutamic acid and a tyrosine residue are both alkylated.     

Purification, structure, and properties of hybrid beta-galactosidase proteins

Structural studies are reported on seven hybrid proteins produced by gene fusions that contain a "foreign" amino acid sequence substituting for part of the NH2-terminal region of the beta-galactosidase polypeptide. All of these hybrid proteins retain beta-galactosidase enzyme activity. A simple and rapid purification scheme for the hybrid beta-galactosidase is described, involving ammonium sulfate fractionation, DEAE-Bio-Gel, and Bio-Gel A-1.5 chromatography. The proteins are tetramers and have activity equivalent to that of wild type enzyme. Their amino acid sequences were determined by isolation and sequence determination of the cyanogen bromide peptide containing the joining site. The subunit sizes vary from 1009 to 1355 residues compared to 1023 for wild type. Up to 26 amino acid residues at the NH2 terminus of beta-galactosidase can be substituted by the new sequence. The nature of the new sequence apparently has no influence on stability or activity of the hybrid, but those hybrids with more of the beta-galactosidase sequence deleted are less stable to heat or urea treatment and tend to dissociate to dimeric form. All hybrids are less stable to heat and urea than wild type. Antipeptide antibodies raised against peptides derived from the NH2-terminal region of wild type beta-galactosidase were found to bind to the hybrid proteins, although they do not bind to the normal enzyme. These results indicate that the quaternary structure is disturbed but not disrupted by substitution of the different sequence, and these results help to localize one of the intersubunit contact regions in beta-galactosidase.     

The influence of sucrose, 2,4-D,and kinetin on the growth,fine structure,and lignin content of cultured sycamore cells

Summary When suspensions of sycamore cells are cultured in a synthetic medium containing 1.0 mg/l 2,4-D and 0.25 mg/l kinetin, maximum cell yield is obtained with an initial concentration of 6 per cent sucrose. There is a progressive increase in dry weight per cell, decline in extractive-free weight as a percentage of cell dry weight and increase in lignin content per cell as the initial sucrose concentration is increased from 1 per cent to 15 per cent. The percentage of lignin in the extractive-free cell residue is further enhanced by increasing the level of 2,4-D to 10 mg/l or by growing the cells in an auxin-free medium containing 10 mg/l kinetin.When the cell suspensions are treated with phloroglucinol/HCl it is found that only a proportion of the cells contain lignin, that this lignin occurs in the protoplasts and in plates between the cells, and that lignin is present in the culture medium.Electron micrographs confirmed the absence of any secondary wall such as is characteristic of tracheary elements. Cells cultured in the presence of 6 per cent sucrose or higher levels showed numerous amyloplasts and frequently the presence of electron opaque material. This occurs in the irregular but not frequent wall thickenings, as droplets in the vacuoles and as amorphous sheets between the cells. Pictures showing such electron opaque droplets clustered on the inner face of the tonoplasts suggest that this material is formed in the cytoplasm and released into the vacuoles. In addition these cells are characterised by the presence of fine electron opaque granular material in their vacuoles and external to their protoplasts. Cultures richest in lignin showed the highest content of electron opaque globules in, and amorphous sheets between, the cells and it is suggested that these correspond to lignin or a lignin-hemicellulose complex. In the presence of 15 per cent sucrose many cells showing breakdown of organised structure were observed; they were characterised by the persistence of mitochondria and particularly of the amyloplasts and by their high content of the electron opaque material equated with lignin. This material was also present in the dead cells.