Silence of streptomycin 6-phosphotransferase gene derived by incubation at a high temperature inStreptomyces griseus

Summary The bald mutants from streptomycin (SM)-producingStreptomyces griseus 2247 obtained by incubation at high temperature (36° C), designated as HT strains, lost resistance to their own antibiotic and scarcely produced the antibiotic. Although SM susceptibility in the mutant was due to loss of SM 6-phosphotransferase activity produced in the cell, the gene coding for the enzyme cloned from an HT strain was surely expressed inS. lividans 1326 as a host. Northern blot analysis showed that the corresponding RNA is not detected in the mutant, indicating that though the gene encoding SM 6-phosphotransferase, at least, the structural gene is not deleted in the cell, the expression is silent.     

NH2-terminal processing of Bacillus subtilis alpha-amylase

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by removal of the NH2-terminal 41-amino acid sequence. To study the mechanism of this processing, the extracellular forms of alpha-amylase were analyzed for B. subtilis N7 alpha-amylase cloned and expressed in B. subtilis. The major form (form N34) isolated from log phase cultures in L-broth had an NH2 terminus corresponding to position 34 from the initiator Met but appeared to be microheterogeneous, as judged by native gel electrophoresis. The major forms from stationary phase cultures had NH2 termini at positions 40 (form N40) or 42 (form N42) and were homogeneous. The conversion of the larger to smaller forms could be achieved in culture supernatants or partially purified samples. The process N34----N40 was inhibited by EDTA; N40----N42 was facilitated by Ca2+. Phenylmethylsulfonyl fluoride inhibited the former but not the latter process. These results suggest that the signal peptidase cleavage site 30 decreases 35 is -Ala-Ala-Ala-Ser-Ala-Glu-Thr- (arrow or further upstream) and that proteolytic maturation occurs after secretion, which involves at least two different processing enzymes.     

Estrogen participation in induction of cervicovaginal adenosis-like lesions in immature mice exposed prenatally to diethylstilbestrol

The structure of human zygapophyseal joint synovial folds as seen by high-power light microscopy and transmission electron microscopy is described. Small myelinated nerves are demonstrated in association with some capillaries in the synovial folds. This may have clinical significance in the field of spinal pain.     

Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein

Penicillin-binding protein (PBP)-2 and the RodA protein are known to function in determining the rod shape of Escherichia coli cells. Peptidoglycan biosynthetic reactions that required these two proteins were demonstrated in the membrane fraction prepared from an E. coli strain that overproduced both of these two proteins and which lacked PBP-1B activity (the major peptidoglycan synthetase activity in the normal E. coli membranes). The cross-linked peptidoglycan was synthesized from UDP-N-acetylmuramylpentapeptide and UDP-N-acetylglucosamine in the presence of a high concentration of cefmetazole that inhibited all of PBPs except PBP-2. The peptidoglycan was synthesized via a lipid intermediate and showed up to 30% cross-linking. The cross-linking reaction was strongly inhibited by the amidinopenicillin, mecillinam, and by other beta-lactam antibiotics that have a high affinity for PBP-2, but not by beta-lactams that had very low affinity for PBP-2. The formation of peptidoglycan required the presence of high levels of both PBP-2 and the RodA protein in the membranes, but it is unclear which of the two proteins was primarily responsible for the extension of the glycan chains (transglycosylation). However, the sensitivity of the cross-linking reaction to specific beta-lactam antibiotics strongly suggested that it was catalyzed by PBP-2. The transglycosylase activity of the membranes was sensitive to enramycin and vancomycin and was unusual in being stimulated greatly by a high concentration of a chelating agent.     

The effect of concentration on the antioxidant effectiveness of alpha-tocopherol in lipid peroxidation induced by superoxide free radicals

Egg yolk phosphatidylcholine liposomes were rapidly oxidized in the presence of chelated iron and a superoxide-generating system. alpha-Tocopherol incorporated in the bilayer was oxidized at the same time. No lipid or alpha-tocopherol oxidation occurred in liposomes composed of dimyristoyl phosphatidylcholine. The antioxidant did not inhibit lipid peroxidation until its concentration reached a critical level, which depended on the effectiveness of the oxidative stress. Beyond this level, peroxidation was inhibited completely and, simultaneously, the rate of oxidation of tocopherol was lowered. The results suggest that the antioxidant efficiency of alpha-tocopherol depends on its ability to react mainly with the chain-initiating or chain-propagating lipid radicals. This, in turn, is closely tied to the tocopherol content of the membrane. Ascorbate inhibited the consumption of alpha-tocopherol, possibly by regenerating its reduced form.     

Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C4 Plants II. Peroxisomes of Panicum miliaceum L.

Peroxisomes were isolated by sucrose density gradient centrifugationfrom mesophyll and bundle sheath protoplasts of a C₄ plant,Panicum miliaceum L. The equilibrium density in the gradientwas 1.25 for bundle sheath peroxisomes and 1.23 for mesophyllperoxisomes, the former density being similar to that of peroxisomesof wheat mesophyll protoplasts. Photorespiratory and other microbody enzymes were assayed forthe peroxisomes of P. miliaceum to detect possible differentiationat an enzyme level. The specific activities of photorespiratoryenzymes, except for hydroxypyruvate reductase, in bundle sheathperoxisomes were 40–60% of those in wheat peroxisomes,when compared on a protein basis, and only 20–30% in mesophyllperoxisomes. However, peroxisomes from both cell types containedsignificant levels of all the enzymes involved in the photorespiratoryglycolate pathway, when compared with castor bean glyoxysomes.The activity of hydroxypyruvate reductase in the peroxisomesof P. miliaceum was comparable to or higher than that in wheatperoxisomes. Two ß-oxidation enzymes and urate oxidasewere detected in the peroxisomes in a similar level to thatin wheat peroxisomes. These results suggest that the peroxisomes of mesophyll andbundle sheath cells of P. miliaceum are essentially similarto those of C₃ plants, and that they cannot be differentiatedexcept for a difference in equilibrium density in a sucrosegradient. (Received December 24, 1984; Accepted April 9, 1985)     

Determination of the complete nucleotide sequence of pNS1, a staphylococcal tetracycline-resistance plasmid propagated in Bacillus subtilis

Abstract The complete nucleotide sequence of pNS1 (3879 bp), a tetracycline-resistance (Tc^R) plasmid drived from staphylococcal plasmid pTP5, has been determined and compared with that of the staphylococcal Tc^R plasmid pT181 [6]. The nucleotide sequences of the 2 plasmids are in agreement, except for 18 nucleotides, but these differences are significant in that they give rise to new open reading frames (ORFs). A short ORF-D is found in the copy control region, and the Tc^R region contains a single large ORF-A, that encodes the Tet protein (50 kDa). The upstream region of ORF-A contains 3 inverted repeat sequences, which can generate structures very similar in conformation of the structure of the control region of the inducible erythromycin-resistance gene of pE194.     

Characterization of a new fetal hemoglobin variant, Hb F Izumi A gamma 6Glu replaced by Gly, by molecular secondary ion mass spectrometry

Molecular secondary ion mass spectrometry has characterized the structure of a new fetal hemoglobin variant, Hb F Izumi, without separation of peptides or amino acid analysis. First, the mass spectrum of a tryptic digest of the abnormal gamma globin revealed a decreased by 72 mass units in the molecular mass of peptide T-1,2, indicating the presence of a Glu leads to Gly substitution. Next, the analysis of the digest produced by the addition of staphylococcal protease, which specifically cleaves glutamyl peptide bonds, determined the site of substitution at 6th glutamic acid residue in peptide T-1,2 which contains two glutamic acid residues. Since this mass spectrometric approach provides digitalized data on peptide analysis, we call it 'digit printing'. The high sensitivity of this technique is especially promising for the analysis of molecular abnormality in various genetic disorders.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

Early replicating DNA in heterochromatin ofPlagiochila ovalifolia (Liverworts)

The timing of DNA replication of heterochromatin in malePlagiochila ovalifolia was investigated by the use of³H-thymidine autoradiography. The estimated duration of the mitotic cycle was as follows: S period, 19 hr: G₂+prophase, 10 hr; G₁+meta-, ana-, telophase, 5 hr; total mitotic cycle, 34 hr. The first appearance of silver grains over the chromosomes was observed at 8 hr after the beginning of pulse labelling at which time the silver grains were only over the euchromatic regions, not over the heterochromatic regions. This labelling pattern was also observed at 10 to 15 hr. The heterochromatic regions having more grains than the euchromatic regions were observed at 20 to 25 hr. These results show that the DNA of the heterochromatin of this species is replicated earlier than the euchromatin.