Nucleotide sequence of the gene coding for four subunits of cytochrome c oxidase from the thermophilic bacterium PS3

The gene coding for four subunits of cytochrome aa3-type oxidase was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced. The N-terminus of each subunit was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequences contained 615 amino acid residues for subunit I (CO1/caaB product), 333 residues for subunit II (CO2/caaA product), 207 residues for subunit III (CO3/caaC product), and 109 residues for subunit IV (CO4/caaD product) after processing. Re-examination of the sequencing of caa revealed a longer open reading frame for CO1, which contains 14 transmembrane segments instead of 12 [Sone et al. (1988) J. Biochem. 103, 606-610], although the main portions of the sequences constituting cytochrome a (FeA), cytochrome a3 (FeB), and CuB are correct. PS3 CO2 has an additional sequence for cytochrome c after the CuA binding protein portion with 2 transmembrane segments, which is homologous to the mitochondrial counterpart. PS3 CO3 has DCCD-binding glutamyl residues but contains only 5 transmembrane segments, unlike the mitochondrial counterpart, which has 7 segments. The subunits of PS3 cytochrome oxidase (aa3-type) show clear similarity in amino acid sequences with those of cytochrome bo-type oxidase from Escherichia coli as well, in spite of the difference of hemes. PS3 CO3 and CO4 are much more similar to E. coli CO3 and CO4 than to mitochondrial CO3 and CO4, respectively.     

Molecular cloning and expression of the rat beta 1-adrenergic receptor gene.

Using the sequence homology approach for cloning related genes within the G-protein-coupled receptor gene family, we have cloned the gene for the rat beta 1-adrenergic receptor (beta 1-AR). The rat beta 1-adrenergic receptor gene was isolated from a lambda EMBL3 rat genomic DNA library using the hamster beta 2-adrenergic receptor (beta 2-AR) coding sequence as a probe under low stringency hybridization conditions. The rat beta 1-AR gene encodes a protein of 466 amino acids that contains one consensus site for N-linked glycosylation (Asn-15) and three consensus sites for cAMP-dependent protein kinase phosphorylation (Ser-296, Ser-301, and Ser-401). The encoded rat beta 1-AR is 98 and 91% similar at the amino acid level with the human beta 1-AR in the transmembrane domains and in the overall sequence, respectively. Genomic Southern blot and gene dosage analyses indicate that the rat beta 1-AR gene is a single copy gene. The tissue distribution of the rat beta 1-AR mRNA was highest in the pineal gland with other brain regions and peripheral tissues, including the heart, expressing the mRNA at moderate levels. The bacteriophage clone containing the rat beta 1-AR gene with its natural promoter was co-transfected with the selectable marker (pRSVneo) conferring neomycin resistance into beta 1-AR-deficient mouse L cells. Analyses of the selected transfectant demonstrates efficient expression of the beta 1-AR gene and functional receptor. 125I-Labeled iodocyanopindolol bound transfectant membranes with an affinity of KD = 24 pm; the beta 1-AR-selective antagonist ICI 89,406 displaced iodocyanopindolol binding with a Ki approximately 140 times lower than that for the beta 2-AR-selective antagonist ICI 118,551. In addition, in the transfectant cell line, adenylylcyclase was stimulated by beta-adrenergic receptor agonists with the rank order of potency of isoproterenol greater than norepinephrine = epinephrine, consistent with properties expected of the beta 1-AR subtype.     

Differential effect of UV irradiation on induction of intragenic and intergenic recombination during commitment to meiosis in Saccharomyces cerevisiae

A comparison was made between the induction of intragenic and intergenic recombinations during meiosis in a wild-type diploid of Saccharomyces cerevisiae. Under non-irradiated normal conditions, production of both intragenic and intergenic recombinants greatly increased in the cells with commitment to meiosis. The susceptibility of cells to the induction of both the spontaneous intra- and intergenic recombinations in meiotic cells was similar. However, under condition of UV irradiation, there were striking differences between intra- and intergenic recombinations. Susceptibility to induction of intragenic recombination by UV irradiation was not enhanced at meiosis compared with mitosis, and was not altered through commitment to meiotic processes. In contrast, however, susceptibility to the induction of intergenic recombination by UV irradiation was enhanced markedly during commitment to meiosis compared with mitosis. Genetic analysis suggested that the enhanced susceptibility to recombination during meiosis is specifically concerned with reciprocal-type recombination (crossing-over) but not non-reciprocal-type recombination (gene conversion). Hence it is concluded that the meiotic process appears to be intimately concerned with the mechanism(s) of induction of recombination, especially reciprocal-type recombination.     

Detection of IgG antibody to measles virus by radioimmunoassay technique

A highly sensitive procedure of solid-phase radioimmunoassay (RIA) was developed for the detection of measles IgG antibody. HeLa cells persistently infected with measles virus were used as a solid-phase antigen. This technique was applied to the detection of measles IgG antibody in patients with subacute sclerosing panencephalitis (SSPE) and multiple sclerosis. Normal subjects having experienced natural measles or measles vaccination and patients with various neurological diseases of non-virus nature were also examined as control groups. Measles antibody was detected at high titers in both the sera and cerebrospinal fluid of SSPE patients. Moreover, RIA/HI ratios of SSPE patients were significantly higher than those of normal subjects, suggesting the presence in the formers of antibodies to nucleocapsids at high titers as well as to viral envelopes. On the other hand, no significant difference was found in both RIA and HI titers between the sera of multiple sclerosis and those of various neurological diseases.     

Selective alkylation of cycloheptaamylose

The verification of a number of assumptions made about cycloamylose structure, substrate binding and catalysis is dependent on the ability to 3-O-alkylate selectively these oligosaccharides. In the presence of barium oxide and barium hydroxide octahydrate, cycloheptaamylose reacts with 3-bromopropene in dimethylformamide and dimethyl sulfoxide to produce a high yield of tetradecakis-2,6-O-allylcycloheptaamylose, a compound which can serve as a highly versatile intermediate in the preparation of heptakis-3-O-alkylated cycloheptamyloses. Depending on the nature of the substituent to be introduced at the C-3 hydroxyl position, the intermediate allyl ether can be 3-O-alkylated and the allyl ether can be isomerized to the vinyl ether and cleaved; or, alternatively, the allyl ether can be first subjected to isomerization followed by alkylation, and then cleavage.     

Peptidomimetic modification improves cell permeation of bivalent farnesyltransferase inhibitors

Bivalent enzyme inhibitors, in which a surface binding module is linked to an active site binding module through a spacer, are a robust approach for site-selectively delivering a minimally-sized agent to a protein surface to regulate its functions, such as protein–protein interactions (PPIs). Previous research revealed that these agents effectively disrupt the interaction between farnesyltransferase (FTase) and the C-terminal region of K-Ras4B protein. However, the whole cell activity of these peptide-based agents is limited due to their low membrane permeability. In this study, we tested a peptidomimetic modification of these bivalent agents using a previously developed inhibitor, FTI-249, and evaluated their cell permeability and biological activity in cells. Confocal cell imaging using fluorescently-labeled agents showed that the peptidomimetic 3-BODIPY penetrated cells, while the peptide-based 1-BODIPY did not. Cell-based evaluation demonstrated that peptidomimetic 3 at a concentration of 100 μM inhibited HDJ-2 processing in cells, indicating that this peptidomimetic modification improves cell permeability, thus leading to enhanced whole cell activity of the bivalent compounds.     

Purification and Properties of Phospholipase D from Actinomadura sp. Strain No. 362

An extracellular phospholipase D from Actinomadura sp. Strain No. 362 was purified about 430-fold from the culture filtrate. The purified enzyme preparation was judged to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point of the enzyme were estimated to be about 50,000—60,000 and 6.4, respectively. The enzyme was most active at pH 5.5 and 50°C in the presence of Triton X-100, but showed the highest activity at pH 7.0 and 60 — 70°C in its absence. The enzyme was stable up to 30°C at pH 7.2 and also stable in the pH range of 4.0 to 8.0 on 2 hr incubation at 25°C. With regard to substrate specificity, this enzyme hydrolysed lecithin best among the phospholipids tested. It was activated by Fe^{3 +}, Al³⁺, Mn^{2 +}, Ca^{2 +}, diethyl ether, sodium deoxycholate and Triton X-100, but was inhibited by cetyl pyridinium chloride and dodecylsulfate.     

Studies on the Accumulation of Orotic Acid by Escherichia coli K12

The mechanism whereby Escherichia coli K12 accumulates orotic acid in culture fluid was studied. Pyrimidine compounds were incorporated effectively into cells of E. coli K12, stimulated the growth, and depressed the accumulation; while purine compounds were not so much consumed by the microorganism for its growth, and affected the accumulation to a lesser extent. On the other hand, E. coli B unable to accumulate orotic acid utilized less effectively pyrimidine compounds for its growth than strain K12.

It is supposed, therefore, that in the de novo pathway for pyrimidine synthesis in E. coli K12 the step from orotic acid to 5′-UMP is genetically depressed so that orotic acid is accumulated when pyrimidine compounds, that would cause a feedback inhibition of orotic acid synthesis upon incorporation, are not supplemented.