Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12

Summary The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b ₅₆₁ and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20160. From its deduced amino acid sequence, cytochrome b ₅₆₁ is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b ₅₆₁ with those of other bacterial b-type cytochromes was observed.     

Hormone-Autonomous Suspension Culture from Leaf Explants of Sugar Beets in Liquid Medium

Hormone autonomous callus was institutioniated reproduciblyon MS agar medium with 0.25 mg/liter of BA as the sole planthormone (AI medium) from young leaf explants of sugar beets.When leaf explants were inoculated into AI medium and culturedon a reciprocal shaker, single cells began to be released fromthe cut surfaces of the leaf pieces after 6 days, followed byactive release. When the single cells which had been releasedwere transferred to fresh liquid MS medium without any planthormones, they could divide and grow autonomously, giving riseto hormone-autonomous suspension cultures. The effects of BAon induction of hormone-autonomous cells are discussed. (Received March 12, 1987; Accepted October 13, 1987)     

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)     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens II. IAA Biosynthetic Activity of Cultured Carrot Tissues Transformed with Wild-Type and Mutant Ti Plasmids

IAA biosynthetic activity was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens harboring wild-type,aux^– or cyt^– Ti plasmids. In vitro IAAM hydrolaseactivities in tissues transformed with wild-type, and cyt^–Ti plasmids were 3.09 and 19.82 nmol/g proteins/30 min, respectively,but not detectable when aux^– Ti plasmids were used. Theactivity of IAA biosynthesis, determined by the incorporationof radioactivity into IAA in tissues fed with [¹⁴C]-tryptophan,was 34.13, 10.92 and 32.47 pmol/g fr wt/30 min in tissues transformedwith wild type, aux^– and cyt^– Ti plasmids, respectively.The incorporation of radioactivity into the IAAM fraction wasdetected only in the tissues transformed with wild type andcyt^– Ti plasmids. These results showed that the T-DNAencoded pathway of IAA biosynthesis was active in tissues transformedwith wild-type and cyt^h Ti plasmids, and that the activity ofIAA biosynthesis in those tissues was higher than that in tissuestransformed with the aux^– Ti plasmid. (Received March 16, 1988; Accepted July 31, 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)     

Ca2+-activated K+ conductance causes membrane hyperpolarizations in a monkey kidney cell line (JTC-12)

Summary We have previously reported hyperpolarizing membrane potential changes in a monkey kidney cell line (JTC-12) which has characteristics resembling proximal tubular cells. These hyperpolarizations could be observed spontaneously or evoked by mechanically touching adjacent cells. In this report, we have shown further evidence that these hyperpolarizations are elicited by an increase in membrane conductance to K⁺ which is caused by an increase in cytosolic Ca²⁺ concentration. In addition, we have found another type of hyperpolarization which is evoked by applying flow of extracellular fluid to the cell. Intracellular injection of Ca²⁺ and Sr²⁺ evoked hyperpolarizations, while intracellular injection of Mn²⁺ and Ba²⁺ did not. Intracellular injection of EGTA suppressed both spontaneous and mechanically evoked hyperpolarizations. In Ca²⁺-free medium, both spontaneous and flow-evoked hyperpolarizations were not observed, while mechanical stimuli consistently evoked hyperpolarization. In Na⁺-free medium, the incidence of cells showing the spontaneous or flow-evoked hyperpolarization increased, and the amplitude and the duration of the mechanically evoked hyperpolarization became greater. Quinidine inhibited all types of hyperpolarization. These data suggest that hyperpolarizations in JTC-12 cells are due to an increase in Ca²⁺-activated K⁺ conductance.     

Nuclear location of hormone-free estrogen receptors by monoclonal antibodies could be a tissue-fixation dependent artifact

The 'two-step' model proposed by Jensen and his collaborators for explaining estrogen action conceptualized hormone-free estrogen receptors (ER) to be cytoplasmic, and hormone-filled, transformed ER to be nuclear. Applying monoclonal antibodies which recognized epitopes in ER and formaldehyde-fixed tissues, King et al demonstrated exclusively nuclear staining in target tissues utilizing immunoperoxidase technique. Recently these antibodies have become commercially available enabling other investigators to conduct studies. In this report, using these monoclonal antibodies we have demonstrated that a change in the concentration of formaldehyde alters the staining pattern yielding cytoplasmic instead of nuclear staining in calf uterus, MCF-7 cells, and ER(+) human breast cancer. In addition, neutralization of the antibody activity was not achieved with freshly prepared ER(+) cytosols. Formaldehyde-treated cytosols were essential. These results ought to caution investigators in determining in vivo location of antigens based on the staining pattern obtained in fixed tissues. Furthermore, this effect of formaldehyde on estrogen receptors may be applicable to other steroid hormone receptors.     

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.     

Human hybrid immunoglobulin M containing immunoglobulin A

Summary Some hybridoma clones made by fusion of a human lymphoblastoid cell line, HO323 with human B lymphocytes, secreted not only IgA but also IgM-like immunoglobulin molecules. The IgM-like immunoglobulin had a molecular size of 900 K which corresponded to that of IgM. Immunochemical analyses revealed that the IgM-like immunoglobulin contained two monomeric IgA and three monomeric IgM molecules. In the IgA moieties, half of original light chains were replaced withx chains derived from the IgM, and vice versa.     

Spawning behavior and early life history of the sharpnose puffer,Canthigaster rivulata,in the aquarium

Specimens ofCanthigaster rivulata (Temminck et Schlegel) were collected from Kominato and Hayama, central Japan, from May, 1985 to October, 1986. On the basis of the gonadosomatic index, gonadal histology and results of artificial fertilization of these specimens, the spawning season is considered to extend from late June to mid-September. The specimens exhibited the following dimorphic differences associated with sex: 1) The male is larger than the female. 2) Ventral side of the body is brownish orange in the male with vermiculated or reticulated patterns of bright violet, while it is white in the female. 3) The male has a well-developed skin fold along the mid-dorsal and mid-ventral lines, which is greatly elevated during courtship; whereas the female’s skin folds are not or slightly developed and conspicuous only during courtship. In an aquarium with the water temperatures of 22 to 26°C, a pair of fish spawned every four days late in the morning for three consecutive months. Courtship and spawning occurred in a pair. The male swam in front of the female, and elevated the skin folds both dorsally and ventrally, fully spreading the unpaired fins, with the ventral side of the body flashing bright blue and the dorsal side turning dark. Both fish swam in a circular fashion, elevating the skin folds. The male followed the female nudging her abdomen with his snout. Both fish turned upward, and released gametes. The eggs are spherical, 0.53–0.73 mm in diameter, demersal, adhesive, transparent, and pale yellowish orange in color, and contain a cross-shaped or asteroid cluster of oil globules. The egg membrane was thick and consisted of about 14 concentric layers. The incubation period ranged from 73.5 hours at 28.2–28.5°C to 145.0 hours at 22.1–22.4°C. The newly hatched larvae were 1.38–1.98 mm in total length (TL) with 84-11-13 = 19–21 myomeres. The yolk was absorbed when the larvae attained 1.49–2.22 mm TL, three days after hatching. The larvae were fed on oyster larvae, blue mussel larvae, sea-urchin larvae and rotifers, but all of them died in 16 days. During the embryonic and early larval stages, the only pigment cells that appeared on the body were the black chromatophores.