Prediction of the coding sequences of mouse homologues of KIAA gene: III. the complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have conducted a human cDNA project to predict protein-coding sequences (CDSs) in large cDNAs (> 4 kb) since 1994, and the number of newly identified genes, known as KIAA genes, already exceeds 2000. The ultimate goal of this project is to clarify the physiological functions of the proteins encoded by KIAA genes. To this end, the project has recently been expanded to include isolation and characterization of mouse KIAA-counterpart genes. We herein present the entire sequences and the chromosome loci of 500 mKIAA cDNA clones and 13 novel cDNA clones that were incidentally identified during this project. The average size of the 513 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 816 amino acid residues. By comparison of the predicted CDSs between mouse and human KIAAs, 12 mKIAA cDNA clones were assumed to be differently spliced isoforms of the human cDNA clones. The comparison of mouse and human sequences also revealed that four pairs of human KIAA cDNAs are derived from single genes. Notably, a homology search against the public database indicated that 4 out of 13 novel cDNA clones were homologous to the disease-related genes.     

Tag-mediated isolation of yeast mitochondrial ribosome and mass spectrometric identification of its new components.

Mitochondrial ribosomal proteins (mrps) of the budding yeast, Saccharomyces cerevisiae, have been extensively characterized genetically and biochemically. However, the list of the genes encoding individual mrps is still not complete and quite a few of the mrps are only predicted from their similarity to bacterial ribosomal proteins. We have constructed a yeast strain in which one of the small subunit proteins, termed Mrp4, was tagged with S-peptide and used for affinity purification of mitochondrial ribosome. Mass spectrometric analysis of the isolated proteins detected most of the small subunit mrps which were previously identified or predicted and about half of the large subunit mrps. In addition, several proteins of unknown function were identified. To confirm their identity further, we added tags to these proteins and analyzed their localization in subcellular fractions. Thus, we have newly established Ymr158w (MrpS8), Ypl013c (MrpS16), Ymr188c (MrpS17) and Ygr165w (MrpS35) as small subunit mrps and Img1, Img2, Ydr116c (MrpL1), Ynl177c (MrpL22), Ynr022c (MrpL50) and Ypr100w (MrpL51) as large subunit mrps.     

Structural alteration of linear plasmids encoding the genes for polychlorinated biphenyl degradation in Rhodococcus strain RHA1

Polychlorinated biphenyl (PCB) tolerant derivatives of a strong PCB degrader, Rhodococcus strain RHA1, were selected after growth in the presence of 100 g/ml PCBs. Some of the derivatives did not grow on biphenyl but accumulated a yellow coloured metabolite suggesting a defect in the meta-ring-cleavage compound hydrolase step encoded by the bphD gene. Other derivatives failed to grow on biphenyl and exhibited little PCB transformation activity suggesting a defect in the initial ring-hydroxylation dioxygenase step encoded by the bphA gene. These organisms had a structural alteration in the linear plasmids coding for the bph genes in RHA1, which included the bph gene deletion. When a bphD containing plasmid was introduced into a tolerant derivative, RCD1, which was shown to have a bphD deletion, the defect in the growth on biphenyl of RCD1 was overcome. The bph gene deletion seems to play a key role in these tolerant derivatives thereby suggesting that the toxic metabolic intermediate would be a main cause of the growth inhibition of RHA1 in the presence of high concentration PCBs.     

Isolation and characterization of cesium-accumulating bacteria.

Cesium-accumulating bacteria, strains CS98 and CS402, were isolated from soil by a radioactive autoradiographic method using 137Cs. These strains displayed the rod-coccus growth cycle and contained mesodiaminopimelic acid, mycolic acids, and tuberculostearic acids. The major menaquinone of CS98 was MK-8(H2). On the basis of these characteristics, strain CS98 was identified as Rhodococcus erythropolis and strain CS402 was classified in the genus Rhodococcus. The maximum values of cesium removal efficiencies in the liquid culture containing 10 mumol of cesium per liter for strains CS98 and CS402 were 90 and 47%, respectively. The maximum cesium contents in strains CS98 and CS402 were 52.0 and 18.8 mumol/g (dry weight) of cells, respectively. Maximum values of cesium concentration factors for strains CS98 and CS402 were 3.5 x 10(4) and 3.6 x 10(3), respectively.     

Photocycle of phoborhodopsin from haloalkaliphilic bacterium (Natronobacterium pharaonis) studied by low-temperature spectrophotometry.

Phoborhodopsin (pR) is the fourth retinal pigment of Halobacterium halobium and works as a photoreceptor for the negative phototactic response. A similar pigment was previously found in haloalkaliphilic bacterium (Natronbacterium pharaonis) and also works as the receptor of the negative phototactic response; this pigment is called pharaonis phoborhodopsin (ppR). In this paper, the photocycle of ppR was investigated by means of low-temperature spectrophotometry. The absorption maximum of ppR is located at 498 nm, while that of pR is at 487 nm. The absorption spectra of the two have similar vibrational structures. Irradiation of ppR below -100 degrees C produced a K-like intermediate (ppRK) which was a composite of two components. The original ppR and ppRK were perfectly photoreversible. On warming, ppRK was directly converted to an M-like intermediate without formation of the L-like intermediate. The M-like intermediate was converted to the O-like intermediate at pH 7.2, but the O-like intermediate was not detected at pH 9.0. The O-like intermediate then reverted to the original pigment. On the basis of these findings, the photocycle and the primary photochemical process of ppR are presented.     

Detection of multiple hormones and their mRNAs in human neuroblastoma cell line NB-1 using in situ hybridization,immunocytochemistry and radioimmunoassay

The production and secretion of multiple peptide hormones and tyrosine hydroxylase by the human neuroblastoma cell line NB-1 and the effects of dibutyryl cAMP (Bt₂cAMP) and phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on them were investigated. The presence of messenger RNA_s (mRNAs) of vasoactive intestinal peptide (VIP)/peptide histidine methionine (PHM), preprotachykinin, and tyrosine hydroxylase was detectable in the cytoplasm of cultured NB-1 cells by in situ hybridization. Treatment with Bt₂cAMP and TPA markedly increased the number of cells immunoreactive to VIP, PHM, neuropeptide Y, Met-enkephalin, substance P and tyrosine hydroxylase and also the contents of VIP and Met-enkephalin in the culture medium. Bt₂cAMP and TPA induced morphological changes characteristic of endocrine differentiation, such as an increase in neuroendocrine granules and the development of rough endoplasmic reticulum and Golgi apparatus. The results indicated that treatment with Bt²cAMP and TPA induces the expression of multiple genes of peptide hormone and tyrosine hydroxylase and increases hormone production and secretion through morphological changes into endocrine cells.     

The primary structures of helices A to G of three new bacteriorhodopsin-like retinal proteins.

The primary structures of helices A to G of all bacteriorhodopsin (BR)-like retinal proteins identified in newly isolated halobacteria have been determined from the nucleotide sequence of the BR-like protein genes. Using PCR methods, gene fragments encoding the A- to G-helix region of BR-like proteins were directly amplified from the total genomic DNA of the seven new halobacterial strains. Oligonucleotide primers corresponding to highly conserved regions in the helices A to G were designed from the nucleotide sequences of bacterioopsin (bop) and archaeopsin-I (aop-I), and some primers were effective for the amplification of the gene encoding C- to G-helix region of all new BR-like proteins. The primer corresponding to A-helix region was designed either from the nucleotide sequence of bop and aop-I or from the N-terminus amino acid sequence of a BR-like protein. Three new BR-like proteins were identified from the amino acid sequence, which was deduced from the nucleotide sequence of the genes encoding A- to G-helix region of the BR-like proteins. It was found that not only the amino acid sequence, but also the nucleotide sequence of the gene encoding the C- and G-helix region, in which a number of important residues for proton translocation are located, is highly conserved in three new BR-like proteins. Analysis of the primary structures of the A- to G-helix region of new BR-like proteins revealed that one has about 85% homology with aR-I and aR-II, and the rest have about 55% homology with halobium BR, aR-I and aR-II.(ABSTRACT TRUNCATED AT 250 WORDS)     

The glycine-rich sequence of the beta subunit of Escherichia coli H(+)-ATPase is important for activity

A short sequence motif rich in glycine residues, Gly-X-X-X-X-Gly-Lys-Thr/Ser, has been found in many nucleotide-binding proteins including the beta subunit of Escherichia coli H(+)-ATPase (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residues 149-156). The following mutations were introduced in this region of the cloned E. coli unc operon carried by a plasmid pBWU1: Ala-151----Pro or Val; insertion of a Gly residue between Lys-155 and Thr-156; and replacement of the region by the corresponding sequence of adenylate kinase (Gly-Gly-Pro-Gly-Ser-Gly-Lys-Gly-Thr) or p21 ras protein (ras) (Gly-Ala-Gly-Gly-Val-Gly-Lys-Ser). All F0F1 subunits were synthesized in the deletion strain of the unc operon-dependent on pBWU1 with mutations, and essentially the same amounts of H(+)-ATPase with these mutant beta subunits were found in membranes. The adenylate kinase and Gly insertion mutants showed no oxidative phosphorylation or ATPase activity, whereas the Pro-151 mutants had higher ATPase activity than the wild-type, and the Val-151 and ras mutants had significant activity. It is striking that the enzyme with the ras mutation (differing in three amino acids from the beta sequence) had about half the membrane ATPase activity of the wild-type. These results together with the simulated three-dimensional structures of the wild-type and mutant sequences suggest that in mutant beta subunits with no ATPase activity projection of Thr-156 residues was opposite to that in the wild-type, and that the size and direction of projection of residue 151 are important for the enzyme activity.