Stable transformation of maize: the impact of feeder cells on protoplast growth and transformation efficiency

Summary The importance of cell culture conditions, including the use of feeder cells, on protoplast growth and transformation in maize (Zea mays L.) was investigated. Total GUS activity, measured two days after transformation, was five-fold higher in protoplasts cultured on feeder cells compared to those grown in the absence of feeder cells. Since the specific activity of GUS was only slightly higher in the transformed protoplasts plated over feeder cells, the stimulation in transient gene expression resulted mainly from the improved environment provided by the feeder system. For stable transformation, either PEG treatment or electroporation of protoplasts was used to introduce the neo gene. When PEG was used, over 85% of the putative transformants (resistant to kanamycin) contained the neo gene. The combination of PEG transformation and the optimized cell culture protocol using feeder cells enabled the selection of about 100 stably transformed lines per gFW of cells. Electroporation was less efficient.     

Phenotypic analyses of lymphokine-activated killer cells that release interferon γ and tumor necrosis factor α

Summary We recently reported that interleukin-2(IL-2)-activated peripheral blood lymphocytes and CD3⁺, lymphokine-activated killer (LAK) cell clones release tumor necrosis factor (TNF) and interferon (IFN) when stimulated with K562 erythroleukemia cells. We examined the phenotype of IL-2-activated peripheral blood leukocytes that secrete TNF and IFN when stimulated with K562 cells and demonstrated that TNF secretion is not due to the presence of contaminating mononuclear phagocytes. Further, we demonstrate that IL-2-activated natural killer (NK) cells release only IFN when stimulated with K562 cells while T lymphocytes exposed to monoclonal anti-CD3 and K562 cells secrete both TNF and IFN. However, T cells stimulated only with K562 cells did not release IFN or TNF while the admixture of these T cells with NK cells, when stimulated with K562 cells, released levels of TNF comparable to those produced by the unseparated cells. At present it is unclear whether only one or both effector cell types respond to K562 by releasing TNF or why the presence both cell types is needed.This work was supported by grants from the national Institutes of Health (CA 23074 and CA 17094) and the Arizona Disease Commission (8277-000000-1-0-YR-9301)     

NUCLEAR GENE DOSAGE EFFECTS ON MITOCHONDRIAL MASS AND DNA

In order to assess the effect of nuclear gene dosage on the regulation of mitochondria we have studied serial sections of a set of isogenic haploid and diploid cells of Saccharomyces cerevisiae, growing exponentially in the absence of catabolite repression, and determined the amount of mitochondrial DNA per cell. Mitochondria accounted for 14% of the cytoplasmic and 12% of the total cellular volume in all cells examined regardless of their ploidy or their apparent stage in the cell cycle. The mean number of mitochondria per cell was 22 in the diploid and 10 in the haploids. The volume distribution appeared unimodal and identical in haploids and diploids. The mitochondrial DNA accounted for 12.6 ± 1.2% and 13.5 ± 1.3% of the total cellular DNA in the diploid and haploid populations, respectively. These values correspond to 3.6 x 10^-15 g, 2.2 x 10⁹ daltons, or 44 genomes (50 x 10⁶ daltons each) per haploid and twice that per diploid cell. On this basis, the average mitochondrion in these cells contains four mitochondrial genomes in both the haploid and the diploid.     

New taxa of Apiaceae,Hydrophyllaceae and Saxifragaceae from Oregon and Idaho

One new taxon from Idaho,Saxifraga bryophora A. Gray var.tobiasiae, and two from Oregon,Cymopterus acaulis (Pursh) Raf. var.gree-Ieyorum andPhacelia lutea (Hook. & Arn.)J. T. Howell var.mackenzieorum are described and compared with their nearest relatives.     

Modification of Sindbis Virus Glycoprotein by Host-Specified Glycosyl Transferases

The amino acid sequence of the membrane glycoprotein of Sindbis virus is specified by the viral genome, but it has not been determined whether the carbohydrate portion of this molecule is specified by the cell or by the virus. We have examined two of the enzyme activities which catalyze transfer of monosaccharides to glycoprotein (sialyl and fucosyl transferases). Comparison of particulate enzyme preparations from infected and uninfected cells showed no difference in either the specific activity or acceptor specificity of these enzymes. This is impressive in view of the fact that the Sindbis membrane glycoprotein is the only glycoprotein synthesized in the infected cell. It was also determined that sialyl transferase from uninfected cells is capable of transferring ((3)H) sialic acid to acceptor prepared from Sindbis membrane glycoprotein. These results imply that at least some of the carbohydrate of the virus glycoprotein can arise by host modification.     

The bovine chromogranin A gene: structural basis for hormone regulation and generation of biologically active peptides.

The structure of the gene encoding bovine chromogranin-A has been determined by characterization of two isolated genomic clones. Chromogranin-A is encoded by eight exons, which organize the coding region into several distinct structural and functional domains. Exons 1-5 represent the highly conserved signal peptide and N-terminal domain, which are separated into regions corresponding to the signal peptide, N-terminal sequence, disulfide-bonded loop, and remainder of the conserved N-terminal domain. Exon 6 represents the variable domain and encodes a region that is identical to the novel chromogranin-A-derived peptide chromostatin. Exon 7 encodes the biologically active peptide pancreastatin as well as most of the conserved C-terminal domain, with the remainder found on exon 8. The mRNA sequence obtained from the gene contains five nucleotide differences from the consensus sequence of four reported bovine chromogranin-A cDNA clones. Two of the differences in the gene result in two amino acid changes in the region encoded by exon 6. The structural organization of the chromogranin-A gene resembles that of the chromogranin-B gene in the exons corresponding to the signal peptide, N-terminal sequence, disulfide loop, and C-terminal sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two new mimosaceous trees from the American Tropics

Piptadenia (sect.Pityrocarpa)leucoxylon is described from the lower Orinoco basin in Venezuela, andPithecellobium (sect.Samanea)foederale from Distrito Federal, Brazil. Figures, diagnoses, and discussion of kinship are presented for each.     

Testing for bacterial resistance to arsenic in monitoring well water by the direct viable counting method.

Direct viable counting of metal-resistant bacteria (DVCMR) has been found to be useful in both enumerating and differentiating metal-resistant and metal-sensitive strains of bacteria. The DVCMR bioassay was used to detect effects of low and high concentrations of arsenic and arsenicals on bacterial populations in groundwater. The level of resistance of the bacterial populations to arsenate was determined by the DVCMR bioassay, and the results showed a linear correlation with the total arsenic concentrations in the monitoring well water samples; no correlation was observed by culture methods with the methods employed. Bacteria resistant to 2,000 micrograms of arsenate per ml were isolated from all monitoring well water samples studied. Strains showed similar antibiotic and heavy-metal profiles, suggesting that the arsenic was not a highly selective pressure for arsenic alone. The monitoring well water samples were amended with arsenate and nutrients to determine the biotransformation mechanisms involved. Preliminary results suggest that bacteria indigenous to the monitoring well water samples did not directly transform, i.e., precipitate or volatilize, dissolved arsenic. It was concluded that arsenic contamination of the groundwater can be monitored by the DVCMR bioassay.