Evidence that replication initiates at only some of the potential origins in each oligomeric form of bovine papillomavirus type 1 DNA.

In a subclone of ID13 mouse fibroblasts latently infected with bovine papillomavirus type 1 (BPV-1) DNA, the viral genome occurred as a mixture of extrachromosomal circular monomers and oligomers. Multiple copies were also associated with the host cell genome, predominantly at a single site in a head-to-tail tandem array. We examined the replicative intermediates of extrachromosomal forms of BPV-1 DNA by using two-dimensional gel electrophoresis. The results obtained indicate that initiation of DNA replication occurred near the center of the EcoRI-BamHI 5.6-kilobase fragment. In some molecules, however, this fragment was replicated from one end to the other by means of a single fork initiated elsewhere. Termination also occurred within this fragment. The EcoRI-BamHI 2.3-kilobase fragment replicated as a DNA molecule containing a termination site for DNA replication and also by means of a single fork traversing the fragment from one end to the other. Thus, replication forks proceeded through these fragments in different manners, apparently depending on whether they were part of a monomer, a dimer, a trimer, or higher oligomers. These observations lead to the conclusion that initiation of DNA replication in BPV-1 DNA takes place at or close to plasmid maintenance sequence 1. From this point, replication proceeds bidirectionally and termination occurs approximately 180 degrees opposite the origin. The results obtained are consistent with one or more replication origins being quiescent in BPV-1 DNA oligomers.     

Nucleotide sequence of the metH gene of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2

The Escherichia coli K-12 metH gene, encoding the vitamin B12-dependent homocysteine transmethylase, is located between iclR and lysC in the 91-min region of the chromosome. The metH gene has been sequenced and reveals an open reading frame of 3600 bp encoding a polypeptide of 1200 amino acids (aa) with a calculated Mr of 132 628. The first 414 aa of the deduced polypeptide sequence are 92% identical to the 414 aa deduced from the partially sequenced Salmonella typhimurium LT2 metH gene. In-frame fusions of metH to lacZ were used to confirm the reading frame of the metH gene and to study its regulation. metH was repressed tenfold, presumably indirectly, by L-methionine and the metJ gene product, while vitamin B12 did not induce de novo synthesis of MetH.     

Identification of minor tightly bound H1 histone subfractions which fail to cleave their initiator methionine

Groups of CBA mice were administered [³⁵S] methionine (1 mCi/mouse). Non-histone proteins, H1 and H1⁰ histones and nucleosomal core histones were isolated from different issues by selective extractions. The measurements of radioactivity of individual bands and autoradiography of dry gels were used to identify methionine-containing and methionine-free histone variants. H1A and H1B histone variants extracted with 5% perchloric acid were methionine-free. However, minor sub-fractions of these histones which are more tightly bound to DNA (and which can be extracted only with 0.25 N HC1) contained [³⁵S] methionine and did show a higher specific activity than methionine-containing nucleosomal hitones. Cyanogen Bromide reaction which destroys non-histone proteins and methionine-containing nucleosomal histones removes radioactivity but does not alter the position of methionine-containing H1 minor bands. This indicates that the radioactive methionine occupies only the N-terminus of the H1 molecules. It is suggested that this methionine is an uncleaved initiator methionine. The presence of these methionine-containing minor H1 subfractions varies in different tissues.     

Effect of pectin on pectinases in autolysis of Botrytis cinerea

Pectic activity in autolyzed cultures of Botrytis cinerea in a medium with and without pectin was similar, but in the medium with pectin maximal activities occurred in younger cultures. The pectic activities found were polygalacturonase, polymethylgalacturonase, endo activity (pectin as substrate) and pectin lyase. The molecular weights of polygalacturonase, polymethylgalacturonase and endo activity (pectin as substrate) were 36000, 33000 and 30200 daltons respectively, and the molecular weight of pectin lyase was 18200 daltons. By gel electrophoresis four different pectic activities were detected, three in the top of the gel and one in the bottom. Two enzymes were characterized, the polygalacturonase activity (first band in the top) inhibited by Ca⁺⁺ and the pectin lyase activity (in the bottom) which was not inhibited by Ca⁺⁺. These enzymes are not induced by the presence of pectin in the medium during degradation of Botrytis cinerea.     

Isolation and biochemical characterization of Streptomyces clavuligerus mutants in the biosynthesis of clavulanic acid and cephamycin C

Summary Seven mutants of Streptomyces clavuligerus blocked in the biosynthesis of clavulanic acid, cephamycin C, or both antibiotics, have been isolated and characterized. Mutants nca1 and nca2 were unable to synthesize clavulanic acid but produced cephamycin C. Mutants nce1 and nce2 were completely blocked in cephamycin C production but formed clavulanic acid. A third group (mutants ncc1, ncc4 and ncc5) failed to produce both antibiotics. Arginase activity (forming ornithine) was very low in mutants ncc1 and ncc5. All the mutants blocked in clavulanic acid biosynthesis showed a normal ornithine--aminotransferase activity. Mutant ncc1, blocked in cephamycin biosynthesis, lacked completely lysine--aminotransferase (forming -aminoadipic acid) and isopenicillin N synthase. Two other mutants (nce2 and nce5) lacked isopenicillin N synthase. There was a good correlation between the isopenicillin N synthase and the lysine--aminotransferase activities of the nca mutants and the ability of those strains to produce cephamycin C. The condensing enzyme involved in the formation of the clavulanic acid nucleus appears to be different from the isopenicillin N synthase.Dedicated to Professor H.-J. Rehm on the occasion of his 60th birthday     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Effects of social isolation and crowding upon adrenocortical reactivity and behavior in the rat

The effects of social isolation and crowding on adrenocortical function and upon behavioral responsiveness to electric shock have been studied in male and female rats. All female experimental groups showed higher corticosterone levels and heavier adrenals than their male counterparts. The major effect of housing condition concerned the corticosterone response to stress, while basal hormone concentration was not modified. Socially housed rats showed a more intense adrenocortical response and also a greater behavioral reactivity to electric shock than the isolates.     

Lesion and regeneration in the medial cerebral cortex of lizards.

The cerebral cortex of Squamate reptiles (lizards and snakes) may be regarded as an archicortex or "reptilian hippocampus". In lizards, one cortical area, the medial cortex, may be considered as a true "fascia dentata" on grounds of its anatomy, connectivity and cyto- chemo-architectonics of its main zinc-rich axonal projection. Moreover, its late ontogenesis and postnatal development support this view. In normal conditions, it shows delayed postnatal neurogenesis and growth during the lizard's life span. Remnant neuroblasts in the medial cortical ependyma of adult lizards seasonally proliferate. The late-produced immature neurocytes migrate to the medial cortex cell layer where they differentiate and give off zinc-containing axons directed to the rest of cortical areas. This results in a continuous growth of the medial cortex and its zinc-rich axonal projection. Perhaps the most important characteristic of the lizard medial cortex is that it can regenerate after having been almost completely destroyed. Recent experiments in our laboratory have shown that chemical lesion of its neurons (up to 95%) results in a cascade of events; first, those related with massive neuronal death and axonal-dendritic retraction and, secondly, those related with a triggered neuroblast proliferation and subsequent neo-histogenesis, and the regeneration of an almost new medial cortex that shows itself undistinguishable from a normal undamaged one. This is the only report to our knowledge that an amniote central nervous centre may regenerate by new neuron production and neo-histogenesis. Perhaps the medial cortex of lizards may be used as a model for neuronal regeneration and/or transplant experiments in mammals or even in primates.     

Some peptide-like colocalizations in endocrine cells of the pyloric caeca and the intestine of Oncorhynchus mykiss (Teleostei)

Summary The coexistence of immunoreactivities to cholecystokinin, glucagon, glucagon-like peptide 1, salmon pancreatic polypeptide, neuropeptide tyrosine, and peptide tyrosine tyrosine was studied immunocytochemicaly, revealing for the first time in fish intestine the existence in the same cell of immunoreactivities to cholecystokinin-glucagon/glucagon-like peptide 1, cholecystokinin-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-neuropeptide tyrosine, salmon pancreatic polypeptide tyrosine tyrosine, and glucagon/glucagon-like peptide 1-peptide tyrosine tyrosine. Colocalization of cholecystokinin-salmon pancreatic polypeptide was observed only in the pyloric caeca of the rainbow trout Oncorhynchus mykiss, while the other colocalizations also occurred in proximal and middle intestinal segments. In all cases, endocrine cells immunoreactive to only one of the paired antisera were detected except for anti-glucagon and anti-glucagon-like peptide 1, which always immunostained the same cells.