Characterization of the minimal origin required for replication of the streptococcal plasmid plP501 in Bacillus subtilis

By using deletional analysis the origin of replication, oriR, of the streptococcal plasmid pIP501 in Bacillus subtilis has been mapped at a position immediately downstream of the repR gene. Determination of both the right and left border of oriR allowed the definition of a sequence of a maximum of 52 nucleotides which theoretically constitutes the minimal origin of replication. Recently, the start point of leading-strand synthesis of the closely related plasmid pAM beta 1 has been mapped at a position which is located exactly in the middle of this sequence (Bruand et al., 1991). The function of oriR did not depend on its location downstream of the repR gene. Translocation of oriR-containing fragments to other regions of the plasmid proved to be possible. The smallest translocated fragment that still reconstituted autonomous replication was 72bp in size. This fragment was also active in directing the replication of an Escherichia coli plasmid in B. subtilis when the RepR protein was supplied in trans from a repR gene integrated into the host chromosome. The transformation efficiency of plasmids carrying translocated oriR fragments showed a certain dependence on the fragment length and orientation. The DNA sequence of oriR included an inverted repeat, both branches of which appeared to be essential for oriR function. The repeats of oriR shared sequence similarity with a repeat located upstream of promoter pII, which has been suggested to be involved in autoregulation of repR expression.     

DNA polymerase alpha-DNA primase from human lymphoblasts

The DNA polymerase alpha-DNA primase complex from the human lymphoblast line HSC93 has been enriched to near homogeneity by using an immunoaffinity purification protocol which was developed earlier for the purification of the calf thymus enzyme (Nasheuer, H.-P. and Grosse, F. (1987) Biochemistry 26, 8458-8466). Immunoaffinity purified polymerase-primase from human cells consisted of four subunits displaying molecular weights of 195,000 and 180,000 for the DNA synthesizing alpha-subunit, of 68,000 for the beta-subunit, and of 55,000 and 48,000 for the primase-carrying gamma- and delta-subunit, respectively. The isoelectric pH values for the individual subunits were estimated from non-equilibrium pH gradients to be between 5.9 and 5.7 for the alpha-subunit, at 5.5 for the beta-subunit, and at 7.5 and 8.0 for the gamma- and delta-subunit, respectively. The purified polymerase-primase converted single-stranded phi X174 DNA into the double-stranded form in a primase-initiated reaction. During this process, 3-10 RNA primers were formed. RNA primers were about 11 nucleotides long. Elongation of existing RNA primers by the human polymerase-primase was semi-processive; following primer binding the DNA polymerase continuously incorporated 20 to 50 nucleotides, then it dissociated from the template DNA.     

Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone synthase

Summary The c2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable element En (Spm) as a gene tag. The Spm element present at the c2 locus in the autonomously mutating c2-m1 line was isolated using En1 element specific probes. Sequences flanking the element were identified as c2 locus specific and were used to clone the nonautonomous c2-m2 and wild-type alleles. The cloning and analysis of a cDNA complementary to the c2 locus provided evidence that this gene encodes the enzyme chalcone synthase.     

Amino acid sequence of an active site peptide of avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase

Hydroxymethylglutaryl-CoA synthase is irreversibly inhibited by the active site-directed inhibitor 3-chloropropionyl-CoA. Enzyme modification has been postulated to involve alkylation of an active site cysteinyl sulfhydryl group. DEAE-Sephadex chromatography of tryptic digests prepared from enzyme inactivated using chloro[14C]propionyl-CoA suggested that bound radioactivity is localized on one peptide. Specificity of the modification was further demonstrated by reverse-phase high pressure liquid chromatography, which was used to isolate the radioactively labeled peptide in a chemically homogeneous form. Automated gas-phase Edman degradation techniques have been employed to confirm the assignment of cysteine as the inhibitor's target residue and to elucidate the sequence of amino acids which flank the 14C-carboxyethylated cysteine: Glu-Ser-Gly-Asn-Thr-Asp-Val-Glu-Gly-Ile-Asp-Thr-(Thr)- Asn-Ala-S-[14C]carboxyethylcysteine-Tyr-Gly-Gln-Thr-(Ala). These data represent the first assignment of active site structure for hydroxymethyl-glutaryl-CoA synthase.     

Immunoelectron microscopic studies on the localization of peptidoglycan peptide subunit pentapeptides in bacterial cell walls

The peptide subunit pentapeptide H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH₂ of peptidoglycan was localized in the cell walls of several Gram-positive bacteria employing the indirect immunoferritin technique. Specific antibodies to the D-alanyl-D-alanine moiety of non-crosslinked peptide subunit pentapeptide were raised in rabbits by immunization with synthetic immunogen albumin-(CH₂CO-Gly-L-Ala-L-Ala-D-Ala-D-Ala-OH)₃₉. Specificity of these antibodies for the peptide subunit pentapeptide and not for the peptide subunit tetrapeptide was corroborated in a Farr-type radio-active hapten binding assay. Specificity of labelling with ferritin was established by immunoelectron microscopic controls, and by the excellent correlation between specific labelling of cells with ferritin and the particular peptidoglycan primary structure of bacterial strains investigated. Cells of Lactobacillus gasseri, Streptococcus pyogenes and Staphylococcus aureus revealing non-crosslinked peptide subunit pentapeptides in their peptidoglycans could specifically be labelled. Lactobacillus acidophilus and Bacillus subtilis, on the contrary, missing such pentapeptides, failed in labelling.The implication of this method to possibly localize the points of attack of penicillin or cycloserine is discussed.Abbreviations used meso-A₂pm meso-diaminopimelic acid - DSM Deutsche Sammlung für Mikroorganismen, Göttingen, FRG This paper is dedicated to Professor Gerhart Drews on the occasion of his 60th birthday     

Deoxyribonucleic acid homologies among 96 strains of ammonia-oxidizing bacteria

DNA of 96 strains of the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio was isolated and analysed spectrophotometrically. Percentages of guanine plus cytosine (G+C) content, genome sizes, and DNA-DNA homologies were determined. The results indicated the presence of eight Nitrosomonas species, three or four Nitrosococcus species, five Nitrosospira species, and two species of both Nitrosolobus and Nitrosovibrio. DNA homologies between strains of a separate species ranged from 56–100%. Average homologies between strains of different species were 33% in Nitrosococcus, 36% in Nitrosomonas, 37% in Nitrosolobus, 40% in Nitrosospira, and 42% in Nitrosovibrio. Average homologies between species of different genera were 33% and thus not significantly above the background value of 30% detected between DNA of ammonia-oxidizing bacteria and Escherichia coli. Genome sizes ranged from 1.90–2.74×10⁹ dalton in Nitrosomonas, 2.09–2.37×10⁹ dalton in Nitrosococcus, 1.87–2.15×10⁹ dalton in Nitrosospira, 1.92–2.10×10⁹ dalton in Nitrosolobus, and 1.91–2.15×10⁹ dalton in Nitrosovibrio. Differences in genome sizes were in accordance with DNA homologies.     

The combination of photogrammetry and finite elements for a fine grained functional analysis of anatomical structures

Summary A new method of functional morphological analysis is presented. Combining stereophotogrammetry with the finite element technique, a new approach, permits a three-dimensional numerical stress analysis of arbitrarily shaped bodies to be performed. The stereophotogrammetric method which originated for three-dimensional calculations in the study of surfaces in land surveying is well suited for the determination of the nodal co-ordinates required for the finite element method, an engineering technique developed for behavioural analysis of solids and fluids responding to external forces. This approach was tested in a study of the functional morphology of the bill of an African wading bird, the shoebill Balaeniceps rex. A few findings of that study are given here in order to demonstrate the method. Advantages of the finite element method compared with other techniques for stress analysis of anatomical structures are also discussed. The method presents exciting possibilities for predicting displacement and stress responses more accurately and in much greater detail. The scope of this powerful computerized stress analysis technique is greatly enhanced with the introduction of stereophotogrammetry for determining the three-dimensional co-ordinates of complex anatomical structures. With the finite element method, the properties of the bone structure can be modelled as they occur in the life of the animal. This is not possible with physical models. Furthermore, rare specimens can be analysed non-destructively.     

Die Verbreitung der limnischen MeduseMedusina limnica Müller 1978 im Rotliegenden Mitteleuropas

The fresh water medusoid fossilMedusina limnica Müller, 1978 has a large regional, but a rather restricted vertical distribution within the continental Rotliegend facies. Its main occurrence is in the higher, but not highest part of the Rotliegend supergroup in red coloured claystones, siltstones, and finegrained sandstones of Artinskian age. Any marine influence can be excluded in Rotliegend sediments of this age in the European hercynian intramontane basin. Kurzfassung: Die fossile limnische MeduseMedusina limnica Müller 1978 hat innerhalb der kontinentalen Rotliegend-Fazies (Oberkarbon-Unterperm) eine große regionale, aber eine ziemlich begrenzte vertikale Verbreitung. Ihr Hauptvorkommen liegt im höheren, aber nicht höchsten Teil des Rotliegenden in rot gefärbten Tonsteinen, Schiuffsteinen und feinkörnigen Sandsteinen, die in das Artinskian eingestuft werden können. In den varistischen Intramontanbecken Europas kann in Rotliegend-Sedimenten dieses Alters jeglicher marine Einfluß ausgeschlossen werden.