New mutations fts-36, lts-33, and ftsW clustered in the mra region of the Escherichia coli chromosome induce thermosensitive cell growth and division.

Three new mutants of Escherichia coli showing thermosensitive cell growth and division were isolated, and the mutations were mapped to the mra region at 2 min on the E. coli chromosome map distal to leuA. Two mutations were mapped closely upstream of ftsI (also called pbpB), in a region of 600 bases; the fts-36 mutant showed thermosensitive growth and formed filamentous cells at 42 degrees C, whereas the lts-33 mutant lysed at 42 degrees C without forming filamentous cells. The mutation in the third new thermosensitive, filament-forming mutant, named ftsW, was mapped between murF and murG. By isolation of these three mutants, about 90% of the 17-kilobase region from fts-36-lts-33 to envA could be filled with genes for cell division and growth, and the genes could be aligned.     

Monoclonal antibodies that depress a specific subset of multiple components of the glutathione-induced response ofHydra

Summary Reduced glutathione evokes a feeding response, the tentacle-ball formation inHydra japonica. This response consists of at least 5 components (R1–R5). We raised 6 monoclonal antibodies (mAbs), each of which depressed a specific subset of these components, and we also examined the immunocytochemical localization of antigens with these mAbs at light microscopic level. The 2 mAbs that depressed R2 and R4 bound to the cnidocils of the desmoneme and the stenotele nematocytes; the 3 mAbs that depressed R5 bound to the apical surface adjacent to the cnidocils of the nematocytes; and the 2 mAbs that depressed R1 and R3 bound to the apical spot structures of unidentified cells in the ectoderm.Together with the specificity of the action of the mAbs on the behavioral response, the correspondence between the effects on the response and the structures visualized with these mAbs suggests that these structures include components of the receptor-effector system relevant to chemoreception.     

Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus.

A novel penicillin-binding protein, PBP-2' (Mr about 75,000), is known to be induced in excessively large amount by most beta-lactam compounds in cells of a clinically isolated strain of Staphylococcus aureus, TK784, that is highly resistant to beta-lactams and also most other antibiotics. This protein has very low affinities to most beta-lactam compounds and has been supposed to be the cause of the resistance of the cells to beta-lactams. A 14-kilobase DNA fragment was isolated from the cells that carried the gene encoding this penicillin-binding protein and also a genetically linked marker that is responsible for the resistance to tobramycin. This DNA was cloned on plasmid pACYC184 and was shown to cause both production of PBP-2' and resistance to tobramycin in Escherichia coli cells. However, the formation of PBP-2' in E. coli was only moderate and was independent of normal inducer beta-lactams. The PBP-2' formed in the E. coli cells showed slow kinetics of binding to beta-lactams similar to that of PBP-2' formed in the original S. aureus cells and gave a similar pattern of peptides to the latter when digested with the proteolytic V8 enzyme of S. aureus.     

Mutation in Pseudomonas aeruginosa causing simultaneous defects in penicillin-binding protein 5 and in enzyme activities of penicillin release and D-alanine carboxypeptidase.

Penicillin-binding protein 5 in Pseudomonas aeruginosa had moderately penicillin-sensitive D-alanine carboxypeptidase activity. As in Escherichia coli, a defect in this enzyme activity was not lethal.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 5 from the dacA mutant strain of Escherichia coli (TMRL 1222)

The localization of the active site of penicillin-binding protein 5 from the dacA mutant of Escherichia coli strain TMRL 1222 has been determined. The protein was purified to homogeneity and labeled with [14C] penicillin G. The labeled protein was digested with trypsin, and the active site tryptic peptide was purified by a combination of gel filtration and high-pressure liquid chromatography. Sequencing of the purified [14C]penicilloyl peptide yielded the sequence Arg-Asp-Pro-Ala-Ser-Leu-Thr-Lys, which corresponds to residues 40-47 of the gene sequence (Broome-Smith, J., Edelman, A., and Spratt, B. G. (1983) in The Target of Penicillin (Hakenbeck, R., Holtje, J.-V., and Labischinski, H., eds) pp. 403-408, Walter de Gruyter, Berlin). The catalytic amino acid residue that forms a covalent bond with penicillin was identified by treating the purified [14C]penicilloyl peptide with a mixture of proteases and then separating the radioactive products using high-pressure liquid chromatography. Analysis of the radioactive peaks by amino acid analysis confirmed that it is the serine residue that reacts with the beta-lactam ring of penicillin.     

Formation of regular packets of Staphylococcus aureus cells.

Staphylococcus aureus, which usually forms grape-like clusters, has the ability to form regularly arranged cell packets. These regular cell packets are formed when the activity of its separation enzyme(s) is lost either by treatment with detergents, such as sodium dodecyl sulfate or Trition X-100, or by mutation of the cells. These cell packets consisted of 8 to 64 spherical cells that have a three-dimensional arrangement. Some irregularity in the arragement of cells in packets, however, can be observed by scanning electron microscopy. It is concluded that S. aureus fundametally divides along three definitely oriented planes that are located at right angles to each other. After cell division, the cells usually become translocated due to the action of a separation enzyme(s) to form grape-like clusters.     

Monoclonal antibodies recognizing amino-terminal and carboxy-terminal regions of human aldolase A: probes to detect conformational changes of the enzyme.

Three monoclonal antibodies (MAbs1A2, 3C5, and 4C2) for human aldolase A [EC 4.1.2.13] were established. MAbs1A2, 3C5, and 4C2 were shown to belong to subclasses IgM, IgG1, and IgG2a, respectively. None of the MAbs inhibits aldolase A activity. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs to chimeric proteins between aldolases A and B [Kitajima et al. (1990) J. Biol. Chem. 265, 17493-17498] in ELISA and to the CNBr-cleaved fragments of aldolase A in immuno-blotting. MAbs1A2 and 3C5 reacted with sites located within amino acid residues 306-363 at the C-terminal region of the enzyme. MAb4C2 recognized an epitope of the enzyme present within amino acid residues 34-108 at the N-terminal region. In a competitive binding assay, MAbs1A2 and 3C5 competed with each other for binding to the antigen and also interfered with the binding of MAb4C2, whereas MAb4C2 failed to inhibit the binding of MAbs1A2 and 3C5 to the antigen. MAb3C5 showed a species-specificity in the reaction with the antigen; it reacted with human and rabbit aldolase A with similar reactivity but not at all with the rat and mouse enzymes, which differ from the human and rabbit enzymes in two amino acid residues at positions 328 and 348. Reactivities of MAbs to aldolase A were further examined with engineered enzymes containing an amino acid substitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fate of haploid parthenogenetic cells in mouse chimeras during development

The developmental capability of haploid parthenogenetic cells was investigated by studies on haploid parthenogenetic in equilibrium fertilized mouse chimeras. Two chimeras were born. One female chimera was smaller at birth and grew slower than its littermates. The distribution of haploid-derived cells in the chimeras was analyzed 11 months after their birth. Cells derived from haploid embryos were found only in the brain, eyes, pigment cells in hair follicles, and spleen, in which they constituted 30%, 20%, 10%, and less than 5%, respectively, of the cells. The correlation between the parthenogenetic contribution to the brain and growth retardation is discussed. All of the cells examined in these chimeric organs (brain and eyes) contained a diploid amount of DNA, suggesting that diploidization of the haploid parthenogenetic cells occurred during development. Possibly, the haploid state is not sufficient for cell growth, even in chimeras with fertilized embryos.     

Microbiological production of radioisotopically labelled 16 alpha-hydroxylated steroids in trace quantities.

[14-14C]16 alpha-Hydroxy-C-18- and C-19-steroid hormones were obtained in good yields by microbiological hydroxylation of correspondingly labelled steroids by Streptomyces roseochromogenes NRRL B-1233. Trace quantities of the labelled substrates were incubated on a rotary shaker (220 rpm) at 27 degrees C. The radioactive products were chromatographically separated, identified and the radiochemical purity was established by isotopic dilution analysis. The specific activities of 16 alpha-hydroxy-steroids obtained were assumed to be the same as those of the substrates, namely, 57.5 mCi/mmole for 16 alpha-hydroxy-4-androstene-3,17-dione, 57.5 mCi/mmole for 5-androstene-3 beta,16 alpha,17 beta-triol, 57.5 mCi/mmole for 16 alpha-hydroxy-dehydroepiandrosterone, 55.7 mCi/mmole for 16 alpha-hydroxy-estrone, and 57.5 mCi/mmole for 16 alpha-hydroxy-testosterone.