Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase

Summary The Bacillus subtilis cdd gene encoding cytidine/2-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order rpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in D69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis 43 and 37 RNA polymerase holoenzymes during growth and stationary phase.Abbreviations SDS sodium dodecyl sulphate - Ap ampicillin resistance - Tet^r tetracycline resistance - Km^r kanamycin resistance     

Stages of B-lymphocyte differentiation in acute lymphoblastic leukemia

Blasts phenotype was determined in 61 children with the acute lymphoblastic leukemia. Non-T-cell acute lymphoblastic leukemia was diagnosed in 51 children. Stages of blasts differentiation were determined with the aid of monoclonal antibodies set using alkaline phosphatase-anti-alkaline phosphatase technique. Blasts in 50 patients belonged to B subpopulation confirmed by the presence of panB CD19 and CD22 antigens. Common antigen was seen in 76.5% of the examined patients with non-T-cell acute lymphoblastic leukemia. Cases of non-T-cell acute lymphoblastic leukemia were divided into 8 subgroups depending on the antigens of B-cells differentiation. An identification of pre-B subgroups of the acute lymphoblastic leukemia indicates heterogenicity of the acute lymphoblastic leukemias in childhood and enables their classification into groups corresponding to the early stages of lymphoblasts maturation.     

The association of H-2Ld with human beta-2 microglobulin induces localized conformational changes in the alpha-1 and- 2 superdomain

We have analyzed changes in the antigenicity of major histocompatibility complex class I molecules resulting from the association of human beta-2 micro-globulin (B2m) with the mouse class I heavy chain. In particular, the H-2L^d molecule exhibited enhanced crossreactivity for the 34-1-2 monoclonal antibody. In order to assess the nature of this structural alteration induced by human B2m, we utilized H-2 class I hybrid molecules in the mapping of the 34-1-2 determinant to the helical region of the alpha-1 domain. H-2L^d class I hybrid molecules were then used to establish the importance of the alpha-2 and- 3 domains in the observed increase of 34-1-2 cross-reactivity following exchange with human B2m. The H-2L^d hybrids suggest that alterations in interdomain contact are responsible for enhanced 34-1-2 cross-reactivity on the H-2L^d molecule. It is likely that this alteration arises through changes in class I conformation at regions of the molecule distant from points of contact between B2m and the class I molecule. This suggests that perturbations induced by association of human B2m with H-2L^d can affect the conformation of the alpha-1 and- 2 superdomain. That class I antigenic determinants are altered by the association of human B2m with mouse class I further suggests that the class I molecule is structurally flexible and may reflect the ability of the class I molecule to bind and present a vast array of disparate peptides to the T-cell receptor.     

Forskolin stimulation of pepsinogen secretion from frog esophageal mucosa is partly mediated by intrinsic cholinergic neurons

Forskolin was found to stimulate pepsinogen secretion from frog esophageal mucosa. The stimulation was dose-dependent and was accompanied with a great increase in tissue cAMP content. The response to forskolin mimicked the action of bethanechol and was not additive with bethanechol. The stimulatory effect of forskolin was inhibited by 50% in the presence of either atropine or tetrodotoxin. On the other hand, incubation in a calcium-free medium not only reduced the response to forskolin by 45% but also eliminated the influence of atropine and tetrodotoxin. These results indicate that forskolin may stimulate pepsinogen secretion from the frog esophageal mucosa via activating adenylate cyclase, and part of its effect may arise from eliciting acetylcholine release from the intrinsic neurons.     

Reclustering of scattered Golgi elements occurs along microtubules

Depolymerization of the interphase microtubules by nocodazole results in the scattering and apparent fragmentation of the Golgi apparatus in Vero fibroblast cells. Upon removal of the drug, the interphase microtubules repolymerize, and the scattered Golgi elements move back to the region around the microtubule-organizing center (MTOC) within 40 to 60 min. Using a fluorescent lipid analogue (C6-NBD-ceramide) as a vital stain for the scattered Golgi elements, their relocation was visualized by video-enhanced fluorescence microscopy in Vero cells maintained at 20 degrees C. The NBD-labeled structures were identified as Golgi elements by their colocalization with galactosyltransferase in the fixed cells. During reclustering, NBD-labeled Golgi elements were observed to move by discontinuous saltations towards the MTOC with velocities of 0.1 to 0.4 micron/s. Paths along which Golgi elements moved were super-imposable on microtubules visualized by indirect immunofluorescence. Neither the collapse of intermediate filaments caused by microinjection of antibodies to vimentin nor the disruption of microfilaments by cytochalasin D had an effect on the reclustering of Golgi elements or the positioning of the Golgi apparatus. These data show that scattered Golgi elements move along microtubules back to the region around the MTOC, while neither intact intermediate filaments nor microfilaments are involved.     

Citric acid production by Aspergillus niger immobilized on polyurethane foam

Summary Citric acid was produced using Aspergillus niger immobilized on polyurethane foam in a bubble column reactor. Most of the adsorbed cells remained on the support and, as a result, high oxygen tension was maintained during the reactor operation. However, uncontrolled growth of the pellets made continuous reactor operation difficult. The citric acid productivity obtained from 15 vol.% foam particles containing immobilized cells was 0.135 g/l per hour. This productivity of immobilized cells was almost the same as that of free cells. The oxygen level dropped to half saturation in 5 days in the immobilized cell culture in contrast to 2 days in the free cell culture.     

Quantitative analysis of DNA secondary structure from solvent-accessible surfaces: the B- to Z-DNA transition as a model

Solvent structure and its interactions have been suggested to play a critical role in defining the conformation of polynucleotides and other macromolecules. In this work, we attempt to quantitate solvent effects on the well-studied conformational transition between right-handed B- and left-handed Z-DNA. The solvent-accessible surfaces of the hexamer sequences d(m5CG)3, d(CG)3, d(CA)3, and d(TA)3 were calculated in their B- and Z-DNA conformations. The difference in hydration free energies between the Z and the B conformations (delta delta GH(Z-B] was determined from these surfaces to be -0.494 kcal/mol for C-5 methylated d(CG), 0.228 kcal/mol for unmethylated d(CG), 0.756 kcal/mol for d(CA)-d(TG), and 0.896 kcal/mol for d(TA) dinucleotides. These delta delta GH(Z-B) values were compared to the experimental B- to Z-DNA transition energies of -0.56 kcal/mol that we measured for C-5 methylated d(CG), 0.69-1.30 kcal/mol reported for unmethylated d(CG), 1.32-1.48 kcal/mol reported for d(CA)-d(TG), and 2.3-2.4 kcal/mol for d(TA) dinucleotides. From this comparison, we found that the calculated delta delta GH(Z-B) of these dinucleotides could account for the previous observation that the dinucleotides were ordered as d(m5CG) greater than d(CG) greater than d(CA)-d(TG) greater than d(TA) in stability as Z-DNA. Furthermore, we predicted that one of the primary reasons for the inability of d(TA) sequences to form Z-DNA results from a decrease in exposed hydrophilic surfaces of adjacent base pairs due to the C-5 methyl group of thymine; thus, d(UA) dinucleotides should be more stable as Z-DNA than the analogous d(TA) dinucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure and androgen regulation of a 20-kilodalton protein specific to rat ventral prostate

Nuclear and cytosolic forms of a 20-kdalton rat ventral prostate protein were purified and partially sequenced from their N-termini. Isolated nuclei were treated with micrococcal nuclease and extracted in 0.6 M NaCl, and proteins were separated by affinity chromatography on Matrex gel green A, ammonium sulfate fractionation, and fast protein liquid chromatography on Superose 12. The 43 amino acid N-terminal sequence of the nuclear 20-kdalton protein was identical with the cytosolic protein except it lacked 7 N-terminal amino acids present in the cytosolic form. The DNA sequence of a full-length complementary DNA clone isolated from a ventral prostate gt11 library extended the N-terminal sequence of the cytosolic form by an additional nine amino acids from the predicted initiation methionine. The cDNA included the nucleotide sequence for the 43 amino acid N-terminal sequence of the purified 20-kdalton protein and predicted molecular weights of 16,686, 17,521, and 18,650, respectively, for the nuclear, cytoplasmic, and nonprocessed proteins. Northern blot analyses of reproductive tract tissue RNAs using the 20-kdalton protein cDNA as probe revealed a single mRNA species of 0.92 kb detectable only in extracts of rat ventral prostate. Expression of the 0.92-kb mRNA was androgen dependent since the mRNA was undetectable in extracts obtained 4 days after castration and was restored 16 h after restimulation with androgen.     

Chemical modification of bovine transducin: probing the GTP-binding site with affinity analogues

The structure of the GTP-binding site of transducin, a signal-transducing G-protein involved in the visual excitation process, was studied by affinity labeling. Radioactive GTP analogues with reactive groups attached to different moieties of the GTP molecule were obtained and include 8-azido-GTP, P3-(4-azidoanilino)-P1-5'-GTP (AA-GTP), 5'-[p-(fluorosulfonyl)benzoyl]guanosine (FSBG), 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)-GTP (ANPAP-GTP), the 2',3'-dialdehyde derivative of GTP (oGTP), and a bifunctional cross-linking analogue, 8-azido-P3-(4-azidoanilino)-P1-5'-GTP (8-azido-AA-GTP). With the exception of FSBG, all of the analogues were found to bind to transducin specifically and serve as a cofactor to activate the retinal cGMP cascade or act as a competitive inhibitor for the GTPase activity of transducin. The labeling sites of these analogues were localized by tryptic peptide mapping. ANPAP-GTP and oGTP were unable to covalently modify transducin, suggesting that the 2'- and 3'-hydroxy groups on the ribose ring of GTP are not in direct contact with the protein. AA-GTP only labeled the T alpha subunit of transducin and was localized on the 21-kDa tryptic fragment of T alpha. This indicates that the phosphate moiety of the bound GTP is in direct contact with this peptide. On the other hand, 8-azido-GTP labeled both the T alpha and T beta gamma subunits of transducin. The labeling on T alpha was on the 12-kDa tryptic fragment, suggesting that the guanine ring binding site is composed of a different peptide fragment than the phosphate binding region. Treatment with the bifunctional analogue 8-azido-AA-GTP generated the cross-linked products of T alpha and T beta gamma. This observation implies that the guanine ring of the bound GTP on T alpha could be in close proximity with T beta gamma.(ABSTRACT TRUNCATED AT 250 WORDS)