Mapping and disruption of the cybB gene coding for cytochrome b561 in Escherichia coli

Abstract The cybB gene on a plasmid encoding cytochrome b ₅₆₁ in Escherichia coli was disrupted by insertion of Km^rl determinant DNA. The cromosomal cybB gene was replaced by the inactivated cybB gene on the plasmid by homologous recombination using λ phage lysogenization and heat-induction. The replacement was confirmed by Southern and Western blotting analyses. Deficiency on the cybB gene product did not affect the growth properties of the cells, and the oxidase activities of the cells dependent on various substrates were similar to those of the parental strain. Cytochrome b ₅₆₁ is concluded to be expressed in E. coli , but may not play a major role in cell growth. In the genetic map of E. coli , the cybB gene was determined by conjugational and transductional crosses to be at 31 min between trg and terC .     

Dendritic cell activating factor produced by mouse macrophage hybridoma

A mouse macrophage (M phi) hybridoma which produces a soluble factor responsible for the cooperation between M phi and spleen dendritic cells (DC) was constructed. The antigen-presenting activity and the stimulator cell activity in the allogeneic or syngeneic mixed leukocyte reaction of DC were significantly augmented when DC were incubated with the culture supernatant of the hybridoma treated with various stimulants including latex beads. The monokine present in the culture supernatant of the hybridoma, called dendritic cell-activating factor (DCAF), augmented the production of lymphocyte-activating factor by DC while Ia expression of DCAF-treated DC was not altered. DCAF had no effect on the antigen-presenting activity of peritoneal resident M phi or B cell blasts while the antigen-presenting activity of spleen M phi was enhanced, but the degree of the enhancement was much less than that of spleen DC. DCAF was found to have the following properties: its pI value is between pH 4 and 5; it is stable at pH 2 to 10; and it loses its activity on incubation at 75 C for 30 min. When the culture supernatant of the hybridoma stimulated with latex beads was subjected to gel filtration, the DCAF activity was detected in the 20 Kd to 25 Kd, 30 Kd to 40 Kd, and 50 Kd to 60 Kd molecular weight regions. The 30 Kd to 40 Kd fraction, which is the major peak fraction, was further purified by ion-exchange chromatography followed by gel-filtration chromatography. When each fraction was subjected to SDS-PAGE, a 30 Kd band corresponding to the DCAF activity was observed and DCAF was purified to about 90% purity.     

Construction of protocellular structures under simulated primitive earth conditions

We have developed experimental approaches for the construction of protocellular structures under simulated primitive earth conditions and studied their formation and characteristics. Three types of envelopes; protein envelopes, lipid envelopes, and lipid-protein envelopes are considered as candidates for protocellular structures. Simple protein envelopes and lipid envelopes are presumed to have originated at an early stage of chemical evolution, interaction mutually and then evolved into more complex envelopes composed of both lipids and proteins.Three kinds of protein envelopes were constructedin situ from amino acids under simulated primitive earth conditions such as a fresh water tide pool, a warm sea, and a submarine hydrothermal vent. One protein envelope was formed from a mixture of amino acid amides at 80 °C using multiple hydration-dehydration cycles. Marigranules, protein envelope structures, were produced from mixtures of glycine and acidic, basic and aromatic amino acids at 105 °C in a modified sea medium enriched with essential transition elements. Thermostable microspheres were also formed from a mixture of glycine, alanine, valine, and aspartic acid at 250 °C and above. The microspheres did not form at lower temperatures and consist of silicates and peptide-like polymers containing imide bonds and amino acid residues enriched in valine. Amphiphilic proteins with molecular weights of 2000 were necessary for the formation of the protein envelopes.Stable lipid envelopes were formed from different dialkyl phospholipids and fatty acids.Large, stable, lipid-protein envelopes were formed from egg lecithin and the solubilized marigranules. Polycations such as polylysine and polyhistidine, or basic proteins such as lysozyme and cytochromec also stabilized lipid-protein envelopes.     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens I. Auxin and Cytokinin Contents of Cultured Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Carrot and tobacco plants were transformed with Agrobacteriumtumefaciens harboring wild-type, aux^– or cyt^– Tiplasmids. In tobacco, these wild and mutant Ti plasmids inducedthe formation of non-morphogenic galls, galls with shoots, andgalls with roots, respectively. In carrot, however, transformationwith any of these plasmids resulted in only the formation ofamorphous tumors. Determination of IAA and cytokinin contentsshowed that in tobacco, significantly high amounts of cytokininsor IAA are present in the cells transformed with Ti plasmidspossessing cytokinin or IAA biosynthetic genes, respectively.In carrot, cytokinin contents were also high in the cells transformedwith Ti plasmids having cytokinin biosynthetic genes, whereasIAA contents of the cells were similar regardless of the plasmidsused for transformation. These results suggest that the mechanism regulating IAA metabolismmay be different in tobacco and carrot. (Received June 25, 1987; Accepted February 1, 1988)     

Nudeotide Pools and Purine Metabolism in Tissue Cultures of Wild-Type Datura innoxia and Adenine-Requiring Auxotroph

Cells of the auxotrophic mutant, Ad1, of Datura innoxia requiredadenine, adenosine, or inosine for their growth on solid agarmedium which contained Murashige-Skoog salts, 2,4-dichloro-phenoxyaceticacid, and sucrose. Thirteen purine and pyrimidine nucleotidesin extracts of wild-type and Ad1 cells were separated and quantifiedby HPLC. Levels of ADP-glucose and UMP were significantly higherin Ad1 than in wild-type cells, but those of other nucleotideswas found when Ad1 cells were transferred to fresh medium withoutadenine. The rate of the biosynthesis de novo of purines, asestimated from the rate of incorporation of ¹⁴C from [2-¹⁴C]-glycine and [¹⁴C]formate into adenine nucleotides, was reducedin Ad1 cells to 21 and 13% of the wild-type rate, respectively.The activities involved in the salvage of adenine and adenosinein Ad1 cells were similar to those in wild-type cells. Ad1 cellshad the capability to convert adenine to guanine nucleotidesand guanine to adenine nucleotides. ¹ Part 27 of the series, "Metabolic Regulation in Plant CellCulture". (Received March 7, 1988; Accepted August 3, 1988)     

Pigment types in sheep, goats, and llamas

Pigment types in various colors of fiber from sheep, goats, and llamas were assayed by a method using high performance liquid chromatography. In these three species the black/gray group is due to eumelanin, which is fully intense in all three species. Red phenotypes are due to pheomelanin and fade considerably with age in fiber from sheep and goats, but not in llamas. This phenomenon has implications on the genetic mechanisms used in generating white fiber. Brown phenotypes in sheep are due to eumelanin, in goats these phenotypes are equivocal, and they were not observed in llamas.     

Pigment types of various color genotypes of horses

Hair samples of various colors of horses were analyzed for content of both eumelanin and pheomelanin by a procedure using high performance liquid chromatography. The results are in accord with generally accepted genetic hypotheses accounting for the various colors. However, the results support the hypothesis that the chestnut/sorrel group of colors is conditioned by the extension locus, not the brown locus. The results also indicate that the brown locus is a likely contributor to some rare color phenotypes.     

Induction of LAK cells by high density dialyzing culture device and its cytotoxicity

Lymphokine activated killer (LAK) cells are generated by culture of lymphocytes with interleukin 2 (IL-2) in short term culture (3 to 5 days) and are used for adoptive immunotherapy for advanced cancer patients. The culture condition hitherto reported are essentially based on the rotating culture system, in which the maximum cell density was at 2 X 10(6) cell/ml and the cell recovery was usually less than 100%. The inability to induce LAK cells efficiently in vitro made the culturing of cells for therapy rather difficult and costly work because the mean infusion dose of LAK cells of one patient requires more than 1 X 10(10)/ml. We have therefore attempted to culture lymphocytes in 10 times higher concentration comparing with conventional methods. By using a new dialyzing culture system under continuous regulation of the amount of infused IL-2, nutrition medium, and pO2 and pCO2, we could culture cells at 2 X 10(7)/ml for more than 21 days and the resulted LAK cells showed a 100 times increase of activity on a per cell basis. By limiting dilution procedure, these killer cells mostly express T cell markers such as CD3 and CD8 but dose not express CD16.     

Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer

Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene. Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes. The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.