Overlapping of the coding regions for alpha and gamma components of penicillin-binding protein 1 b in Escherichia coli

Summary The mode of biosynthesis of penicillin-binding protein(PBP)-1 b in Escherichia coli was investigated by use of the plasmid carrying the ponB(PBP-1 b) gene region. Analyses of the products synthesized in minicells and in vitro showed that PBP-1 b was synthesized as two molecular species corresponding to the and components of PBP-1 b. The coding regions for the and components were located within the ca. 3.7 kb MluI-HincII fragment and transcribed in the direction from the HincII to the MluI site. The capacity for producing the component was abolished by a deletion extending to the MluI site ca. 0.7 kb inward from the HincII end of the ca. 3.7 kb fragment; the remaining 3.0 kb region with the MluI site at both ends directed the production of the component alone. The production of the component was enough to correct all the known defects caused by a ponB mutation. In addition to these results, the analyses for cross-reacting materials produced in correspondence to the various deletions indicated that the coding regions for the and components overlapped and that the N-terminal portion was responsible for the difference between the two components. The distal region about 0.7 kb long inward from the MluI end of the MluI-HincII fragment was dispensable for producing the functional PBP-1 b, although the PBP-1 b produced was curtailed. By a larger distal deletion reaching almost to the middle of the MluI-HincII fragment, the polypeptide produced for PBP-1 b lost the ability to bind penicillin and still retained a low but significant activity for glycan synthesis. We suggest, therefore, that the polypeptide portion required for transglycosylase activity resides on the N-terminal half of PBP-1 b, followed by the middle portion necessary for penicillin-binding and the C-terminal part dispensable for the function of PBP-1 b.     

Evolutionary relationship of the Bacillus licheniformis macrolide-lincosamide-streptogramin B resistance elements

Summary Naturally occurring erythromycin (Em) resistance was found in 11 of the 18 Bacillus licheniformis isolates tested but was absent from a wide variety of other Bacillus strains. The Em resistance elements confer inducible macrolide-lincosamide-streptogramin B (MLS) resistance and are related to ermD an MLS resistance element previously cloned from the chromosome of B. licheniformis 749. The MLS sensitive B. licheniformis strains and the other sensitive Bacillus strains tested, lack sequences with detectable homology to ermD. The sensitive B. licheniformis strains do exhibit homology to sequences which flank ermD in B. licheniformis 749. The relative sizes of the homologous DNA fragments suggest that the sensitive strains are lacking a 3.6 kb segment which contains ermD. It is shown that ermD is homologous to chromosomal DNA from Streptomyces erythreus ATCC 11635, an Em producing organism. These observations suggest to us that MLS resistance may have arisen in the Streptomyces and spread to B. licheniformis another gram positive bacterium found in soil. It is further proposed that ermD is or was located on a transposon-like element and has spread and evolved further to yeild a variety of related Staphylococcal and Streptococcal MLS determinants.     

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora

Summary The organization of the ribosomal DNA (rDNA) repcat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, N. tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11+0.21 kb; standard error=0.038; coefficient of variation (C.V.)=2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5 end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.     

Three-dimensional structure of clathrin cages in ice.

We have collected tilt series of electron micrographs from unstained clathrin cages embedded in vitreous ice. From these micrographs we have generated three-dimensional reconstructions of individual hexagonal barrels, which show details of the internal structure. Four types of preparation have been examined: (i) coated vesicles; (ii) cages reassembled from clathrin heavy and light chains; (iii) reassembled cages treated with elastase to remove the light chains; and (iv) reassembled cages treated with trypsin to remove the light chains and the terminal domains of the clathrin heavy chains. In the intact and elastase-treated cages, the clathrin extends from the vertices into the interior of the polyhedron and forms an inner shell of material. Limited digestion with trypsin removes the inner shell, which indicates that this material corresponds to the terminal domains of the clathrin heavy chains.     

The structure of cruciforms in supercoiled DNA: probing the single-stranded character of nucleotide bases with bisulphite.

The single-stranded character of cytosine bases in three cruciform structures has been assessed by an examination of reactivity towards sodium bisulphite. Unpaired cytosine residues undergo deamination at C4 to give deoxyuracil, and propagation in an ung Escherichia coli host results in C-G----T-A transition mutations, detectable by restriction cleavage or sequence analysis. Very high frequencies of such mutations have been found at cruciform loops, confirming their unpaired character, with almost zero background mutation frequencies elsewhere. A low level of modification was observed at the four-way junction of a cruciform. The results indicate that the optimal cruciform loop size is four bases, with loose 'breathing' at the first base pair at the top of the cruciform stem at 37 degrees C, and little or no opening of base pairs at the four-way junction.     

Overproduction of human Cu/Zn-superoxide dismutase in transfected cells: extenuation of paraquat-mediated cytotoxicity and enhancement of lipid peroxidation.

The 'housekeeping' enzyme Cu/Zn-superoxide dismutase (SOD-1) is encoded by a gene residing on human chromosome 21, at the region 21q22 known to be involved in Down's syndrome. The SOD-1 gene and the SOD-1 cDNA were introduced into mouse L-cells and human HeLa cells, respectively as part of recombinant plasmids containing the neoR selectable marker. Human and mouse transformants were obtained that expressed elevated levels (up to 6-fold) of authentic, enzymatically active human SOD-1. This enabled us to examine the consequences of hSOD-1 gene dosage, apart from gene dosage effects contributed by other genes residing on chromosome 21. Human and mouse cell clones that overproduce the hSOD-1 had altered properties; they were more resistant to paraquat than the parental cells and showed an increase in lipid peroxidation. The data are consistent with the possibility that gene dosage of hSOD-1 contributes to some of the clinical symptoms associated with Down's syndrome.     

Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures.

The strength in vivo of 14 promoters was determined in a system which permits the quantitation of RNA synthesis with high accuracy. Up to 75-fold differences in promoter strength were measured and the most efficient signals are promoters from coliphages T7 and T5. Their activity approaches the strength of fully induced promoters of the rRNA operons which may be close to the functional optimum of a single sequence. By contrast, a synthetic 'consensus promoter' belongs to the less efficient signals. Our data show that optimal promoter function can be achieved by alternate structures and strongly suggest that information outside of the 'classical' promoter region contributes to promoter activity.     

Identification and characterization of a calcium-binding protein in the mouse chorioallantoic placenta.

Mouse chorioallantoic placenta contains a specific calcium-binding protein (MCaBP). A procedure involving gel filtration and ion-exchange chromatography was developed to purify the MCaBP. The MCaBP activity increased as a function of embryonic gestation and was highly specific for Ca2+. The MCaBP is a monomeric protein of Mr 57000, with pI 4.7. Specific antibodies were prepared against the MCaBP and were used to localize the MCaBP to syncytiotrophoblasts of the chorionic villi of mouse chorioallantoic placenta. These properties suggest that the MCaBP may be involved in transplacental calcium transport.     

Interactions of the neurotoxic amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine with monoamine oxidases.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a thermal breakdown product of a meperidine-like narcotic used by drug abusers as a heroin substitute, produces Parkinsonian symptoms in humans and primates. The nigrostriatal toxicity is not due to MPTP itself but to one or more oxidation products resulting from the action of monoamine oxidase (MAO) on this tertiary allylamine. Both MAO A and B catalyse the oxidation of MPTP to the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP+), which undergoes further oxidation to the fully aromatic 1-methyl-4-phenylpyridinium species (MPP+). These bio-oxidations are blocked by selective inhibitors of MAO A and B. Additionally, MPTP, MPDP+ and MPP+ are competitive inhibitors of MAO A and B. The A form of the enzyme is particularly sensitive to this type of reversible inhibition. Both MAO A and B also are irreversibly inactivated by MPTP and MPDP+, but not by MPP+. This inactivation obeys the characteristics of a mechanism-based or 'suicide' process. The inactivation, which is accompanied by the incorporation of radioactivity from methyl-labelled MPTP, is likely to result from covalent modification of the enzyme.     

Wavelength-dependence of the relative rate constants for the main geometric and structural photoisomerization of bilirubin IX alpha bound to human serum albumin. Demonstration of green light at 510 nm as the most effective wavelength in photochemical changes from (ZZ)-bilirubin IX alpha to (EZ)-cyclobilirubin IX alpha via (EZ)-bilirubin.

The kinetics for the quantitatively important reaction: (Formula: see text) that is, the photochemical interconversion between bilirubin and its geometric and structural photoisomers bound to human serum albumin in aqueous solution when various wavelengths of monochromatic light were used, were assayed by h.p.l.c. In order to clarify the wavelength-dependence of the relative rate constants in the individual steps, a light-source with a half-bandwidth of 10 nm was used at increments of 20 nm, in the range from 410 nm to 550 nm. We describe for the first time studies on the wavelength-dependence of rate constants in geometric and structural photoisomerization reactions in vitro of (ZZ)-bilirubin or (EZ)-bilirubin bound to human serum albumin, especially the relative rate constants of cyclization of (EZ)-bilirubin into (EZ)-cyclobilirubin. Because studies in vitro have demonstrated that the wavelengths from 350 to 450 nm are mutagenic, the results obtained indicated that the safest and ideal light-source for phototherapy is green light of 510 nm, which keeps (ZE)-bilirubin concentrations as low as possible, as shown by a maximal value of k2 at 510 nm and a relatively low value of k1 at 510 nm. This light-source still ensures the substantial absorption of (ZZ)-bilirubin, which is the precursor of (EZ)-bilirubin, the intermediate in (EZ)-cyclobilirubin formation and, furthermore, as shown by the maximal value of k5 and a considerable value of k4 at 510 nm, promotes the cyclization of (EZ)-bilirubin derived from (ZZ)-bilirubin even though k3 at 510 nm also shows a peak value.