Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

Summary A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tyl^r) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tyl^r phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyl^s) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyl^s mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyl^s mutant obtained by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tyl^r gene is not closely linked to tylosin biosynthetic genes.     

Growth, dry matter partitioning, and diurnal activities of RuBP carboxylase in citrus seedlings maintained at two levels of CO2

The long-term response of citrus rootstock seedlings to CO₂ enrichment was examined in Carrizo estrange ( Poncirua trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck] and Swingle citrumelo ( P. trifoliate x C. parodist Macf.]. Plaotlets 14 weeks old were transferred to outdoor controlled-environment chambers and maintained for 5 months from Feb. 14 to July 21. During this period, new growth (cm) of citrange and citrumelo shoots at 660 μl1⁻¹ was 94 and 69% greater, respectively, than at 330 μ1 1⁻¹. Total dry weight of both rootstock shoots had increased by over 100%. Growth of few species is affected this markedly by elevated CO₂ levels.
More carbon was partitioned to above-ground organs in CO₂-enriched citrus seedlings. Stem dry matter per unit length was also 32 and 44% greater in citrange and citrumelo, respectively. Total leaf area was increased by 124% in citrange and 85% in citrumelo due to greater leaf number and size. Variations in overall relative growth rate appeared to be related to the rapid, sequential, flush-type growth in citrus, in which an entire shoot segment with its associated leaves remains an active sink until fully expanded. RuBP carboxylase (EC 4.1.1.39) activity in leaves of recently-expanded flushes was higher in citrumelo plants grown at 660 vs 330 μ1 1⁻¹ CO₂ and changed diurnally for citrange (but not citrumelo) leaves at both CO₂ levels. The results are consistent with the hypothesis that positive long-term effects of CO² enrichment may be greater in species or during growth periods where sink capacity for carbon utilization is high.     

31P nuclear magnetic resonance study of enzymatically phosphorylated glycophorin A

Glycophorin A was phosphorylated using protein kinases and the new protein was investigated using³¹P NMR spectroscopy. Most of these ～30 moles of phosphate were found to be attached to Ser and Thr. Some of these phosphate residues appear to be affected by the carbohydrate residues present. The phosphorylated protein appears to be in a severe state of aggregation, with the degree of aggregationpH-dependent.     

Anisotropic rotational motions of poly(L-glutamic acid) in the alpha-helix conformation

The anisotropic rotational motion of the backbone and the side chains of poly(L -glutamic acid) in the α-helical structure was investigated using the ¹³C-T₁ and T₂ relaxation times of all carbon atoms with directly attached protons, obtained at a ¹³C-Larmor frequency of 67.89 MHz. The evaluation of the nmr data was carried out according to the previously derived anisotropic diffusion model, in which the macromolecule is considered a rigid rod. The rotation of the backbone is characterized by two diffusion constants, D₁ and D₃, describing the rotation perpendicular to and around the symmetry axis. The additional internal motion of the C^β-methylene group is described as a jump process with a jump rate, k₁, between two allowed rotametric states. Steric considerations indicate that the occupation of the third rotameric position is forbidden. The rotation of the C^γ-methylene group is decribed as a one-dimensional diffusion process around the C^β–C^γ bond. Investigation of the temperature dependence of the relaxation parameters led to the temperature dependence of the dynamic parameters. Activation energies were determined from these data. The dynamic parameters obtained for poly(L -glutamic acid) at 291 K are compared with the corresponding results of a previous study of poly(L -lysine). The development of an anisotropic diffusion model for the motions of the rod-shaped poly(L -lysine) α-helix and its application to the interpretation of the ¹³C-relaxation data of this molecule have already been published previously. In this model, both the overall molecular tumbling and the various internal motions have been characterized by diffusion constants or jump rates typical for each process. These dynamic parameters can be calculated from the spin–lattice relaxation times, the spin–spin relaxation times and the NOE factors of the C^α, C^β, and C^γ nuclei of the polypetide. In the present paper, we describe the application of the above-mentioned dynamic model to the interpretation of ¹³C-relaxation studies of a further homopolypeptide, poly(L -glutamic acid), in the α-helical structure. Furthermore, we studied the temperature dependence of the relaxation times of this polymer and determined the anisotropic diffusion parameters at each temperature. From their temperature dependence and from comparison of our present results with the data of our previous study of poly(L -lysine), we were able to derive new insights into the intramolecular diffusion processes and the excitation of various motions.     

Radioimmunoassay for human type VI collagen and its application to tissue and body fluids

A liquid phase radioimmunoassay (RIA) was developed for pepsin-solubilized human type VI collagen, allowing quantitative analysis of this protein down to a concentration of 3 ng/ml. No cross-reactivity was observed with human collagens type I, III, IV (triple helical portion and 7-S domain), and V, nor with laminin fragment Pl and plasma fibronectin. Significant amounts of closely related antigenic material were detected in serum, bile, ascites, and mesenchymal cell culture media. Type VI collagen could be completely solubilized from several tissues by a repeated pepsin digest, and its content as determined by RIA was found to be less than 0.1% of total collagen (55-70 micrograms/g protein). In fibrotic liver tissue type VI collagen was elevated up to 10-fold (620 micrograms/g protein) when compared to normal liver. Sera of patients with fibrotic liver disease, however, revealed antigen levels usually below the narrow normal range of 22 +/- 7.8 ng/ml (mean +/- 2.5 SD). We conclude that, although type VI collagen represents a minor fraction of the interstitial collagens, its comparatively high serum levels point to a considerable turnover in the normal individual. Our data suggest that in fibrosis as exemplified in fibrotic liver disease, the metabolism of this collagen is down-regulated, while at the same time, it accumulates in the interstitial matrix.     

Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to mutagenesis

The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents. The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical). We have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis. pGW1975 increases UV mutagenesis less than pKM101 in a uvrA⁺ strain but more than pKM101 in a uvrA⁻ strain. muc⁻ point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis. We have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB.     

Nucleotide sequence analysis of a variant human T-cell leukemia virus (HTLV-Ib) provirus with a deletion in pX-I.

A variant of human T-cell leukemia virus subgroup I (HTLV-I), designated HTLV-Ib, has been isolated from a transformed T-lymphocytic cell line established from a Zairian patient with adult T-cell lymphoma. A recombinant phage clone of the variant provirus, denoted lambda MC-1, hybridizes under high stringency to HTLV-I DNA probes, but 17 of 43 restriction enzyme sites differ from those of HTLV-I, 10 of them clustering within 1.5 kilobases in the env-pX region. Since this variant virus retains its capacity to transform T-cells in vitro, and since a pX product is suspected to be important in transformation, we have determined the nucleotide sequence of the entire pX region of this virus for comparison to the prototype HTLV-I. In addition, the region between the gag and pol genes, parts of the pol and env genes, and a portion of the U3 region of the long terminal repeat sequence were also analyzed. We noted 141 single-base-pair changes among 3,897 base pairs, which were relatively well distributed over those portions of the provirus that were examined. In addition, an 11-base-pair deletion was found which included the potential initiator ATG codon of the first open reading frame of pX (pX-I). The next potential initiator codon predicted by the sequence is followed by 10 codons and then a termination codon. An identical deletion was also demonstrated in the only provirus present in another cell line established from the same patient on a different occasion after transformation in vitro of normal human umbilical cord blood cells. These results indicate that pX-I is not required for transformation.     

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.     

1H NMR study of the interaction of bacteriophage λ Cro protein with the O R 3 operator

The 17 base pair operator O _R ³ oligonucleotide, which is the preferential binding site for the Cro repressor of phage , was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exhangeable base protons and the H1 and the H2-H2 sugar protons of the O _R ³ operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the O _R ³ operator — Cro repressor interaction.Abbreviations COSY correlated spectroscopy - FID free induction decay - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect spectroscopy - RD relaxation delay - TSP sodium 3-trimethylsilyl-(2,2,3,3-²H₄)propionate - EDTA sodium ethylendiamine tetraacetate