Sequence motifs characteristic of DNA[cytosine-N4]methyltransferases: similarity to adenine and cytosine-C5 DNA-methylases

The sequences coding for DNA[cytosine-N4]methyltransferases MvaI (from Micrococcus varians RFL19) and Cfr9I (from Citrobacter freundii RFL9) have been determined. The predicted methylases are proteins of 454 and 300 amino acids, respectively. Primary structure comparison of M.Cfr9I and another m4C-forming methylase, M.Pvu II, revealed extended regions of homology. The sequence comparison of the three DNA[cytosine-N4]-methylases using originally developed software revealed two conserved patterns, DPF-GSGT and TSPPY, which were found similar also to those of adenine and DNA[cytosine-C5]-methylases. These data provided a basis for global alignment and classification of DNA-methylase sequences. Structural considerations led us to suggest that the first region could be the binding site of AdoMet, while the second is thought to be directly involved in the modification of the exocyclic amino group.     

Extrachromosomal circular DNAs in Drosophila melanogaster: Comparison between embryos and Kc0% cells

We established the size distribution of extrachromosomal covalently closed circular DNA molecules from embryos of various Drosophila melanogaster strains and from Kc0% tissue culture cells. In embryos, more than 80% of the circular DNA molecules are smaller than 2.5 kb and all the distributions show a peak of molecules of between 200 and 400 bp. The Kc0% cell distribution differs mainly from that of embryos in that 48% of the molecules have a size between 4 and 8 kb. Correlating with this, circular molecules homologous to copia, 412 and 297 were detected only in Kc0% cells. The three tandemly repeated families containing the 5S genes, the histone genes and the 240 bp repeat of the ribosomal DNA intergenic spacer, which had previously been identified in circular DNAs from embryos, were also found in cultured cells. A fourth tandemly repeated family corresponding to the 1.688 g/cm³ satellite DNA was detected, both in embryos and Kc0% cells. It consists of circular multimeric molecules containing multiple copies of the 359 bp repeated unit. No circular DNA molecules homologous to the actin genes, the type I ribosomal DNA insertion, or the F and I transposable elements were found in embryos or Kc0% cells. Thus it appears that the extrachromosomal circular DNA molecules from embryos and from tissue culture cells differ mainly in the presence of circular copies of the copia-like transposable elements.     

Copy number and distribution of P and I mobile elements in Drosophila melanogaster populations

The distribution of the number of copies of P and I transposable elements per genome was investigated by in situ hybridization for a large set of Drosophila melanogaster strains. These included the P, Q and M types of the P-M system of hybrid dysgenesis. P element copy number varied widely (range 5–59). P and Q strains had around 40 copies whereas M strains generally had lower numbers (between 5 and 35) with one extreme value (52). The copy number of I elements appeared to be precisely regulated, as no strains were found outside the 15±5 range. The number of copies of the two families were independent. An excess of P copies on the X chromosome compared with the autosomes was found for the P and Q strains, but not for M strains. Among X-inserted P sites, a very high frequency of occupation was found at the tip of the X chromosome (cytological site 1A), especially for P and Q strains. The possible regulatory role in the P-M system of X-inserted P sites is discussed.     

Methylation of ribosomal cistrons in diploid and tetraploid Odontophrynus americanus (Amphibia,Anura)

Odontophrynus americanus (Amphibia, Anura) genomic DNA from diploid and tetraploid specimens was treated with restriction enzymes sensitive to cytosine and adenine methylation (5 meC and 6 meA). In both diploids and tetraploids a high proportion of the total DNA was not cleaved by 5 meC-sensitive enzymes as observed on agarose gels stained with ethidium bromide. The DNAs were transferred to nitrocellulose filters and hybridized with cloned fragments containing sequences of Xenopus laevis 28S and 18S ribosomal DNA (rDNA). A high level of methylation of the ribosomal repeat units was revealed by 5 meC-sensitive enzymes in blood, liver, kidney and testis tissues. Adenine was methylated to a lesser degree and similarly in the rDNA from both germinative and somatic tissues. Comparison of the results obtained with DNA of diploids and tetraploids showed that methylation of ribosomal genes was increased in tetraploid genomes of adult frogs, but exact quantitative determinations could not be performed by this methodology. Cloning of the 28S region of the rDNA repeat unit was performed in the gtWESC vector. Restriction patterns obtained with methylation-sensitive enzymes using diploid and tetraploid derived clones confirmed the high level of methylation of the corresponding region of the ribosomal repeat unit in genomic DNAs. The implications of these results in the regulation of expression of the ribosomal genes in diploids and tetraploids are discussed.     

Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes

Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.     

GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs

Platelet GPIIbIIIa is only synthesized in megakaryocyte or in cell lines with megakaryocytic features. The sequence for GPIIb and GPIIIa have recently been derived from cDNAs obtained from HEL cells. The sequence of these proteins produced by the megakaryocyte, has however, not been determined yet. This study describes full length cDNAs for GPIIb and GPIIIa isolated from megakaryocyte cDNA libraries. The cDNA sequences indicate the presence of nucleotide differences, between the sequence of the GPIIIa cDNAs from HEL cells, endothelial cells and megakaryocytes. One difference was also observed between HEL and megakaryocyte GPIIb at position 633 where a cystein in the megakaryocyte GPIIb, is replaced by a serine in the HEL sequence. The mRNA species for GPIIb (3.4kb) and GPIIIa (6.1 kb) were of the same size in HEL cells and human megakaryocytes.     

Effects of calcium input/output on the stability of a system for calcium-regulated viscoelastic strain fields

The Goodwin and Trainor model of pattern generation in calcium-regulated strain fields is studied in the case where calcium input and calcium output processes are involved. It is shown that the properties of the original model may still remain provided that the input-output processes are not unstable. In this last case, despite the eventual stabilizing effect of the calcium exchange term, perturbations of the generalized system can grow and lead to inhomogeneous solutions. Applications to cell differentiation and cell growth are discussed.     

Impact of salt adaptation on esterified fatty acids and cytochrome oxidase in plasma and thylakoid membranes from the cyanobacterium Anacystis nidulans

During adaptation of photoautotrophically growing fresh water cyanobacterium Anacystis nidulans to high salinity the cells showed a pronounced increase of proton-sodium antiporter activity, and of cytochrome c oxidase in isolated and purified plasma membrane. At the same time the concentrations of plasma membrane-bound EDTA-resistant copper and iron (determined by inductively coupled plasma atomic emission spectrometry) rose proportionately, accompanied by an increase in whole cell respiration. In plasma membranes from salt adapted cells lipid/protein ratios were markedly higher than in control cells, levels of esterified saturated and long-chain fatty acids being significantly higher than the respective levels of unsaturated and short-chain fatty acids which explains the higher lipid-phase transition temperatures derived from Arrhenius plots. Immunoblotting of the membrane proteins with antisera raised against the cytochrome c oxidases from Paracoccus denitrificans and A. nidulans gave two cross-reacting bands with apparent molecular weights around 50000 and 30000 (subunits I and II, respectively) which were more pronounced in plasma membranes from salt adapted cells when compared to control cells. The protein pattern of plasma membranes from salt adapted cells also showed the appearance of bands at apparent molecular weights of 44000–48000 and 54000–56000 which might stem from the proton/sodium-antiporter in this membrane.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - cyt cytochrome - DCCD N,N-dicyclohexylcarbodiimide - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - ICP-AES inductively coupled plasma atomic emission spectrometry - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - EPR electron paramagnetic resonance spectrometry     

Cystic fibrosis typing with DNA probes and screening for ΔF508 deletion in families from Southern France

Summary A sample of 235 individuals from 49 French cystic fibrosis (CF) families with at least one living affected child was typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene, and was screened for the ΔF508 mutation. Using a combination of six probes, 44 out of the 49 families were sufficiently informative to enable prenatal diagnosis or carrier determination. As in many other populations, linkage disequilibrium was found between the CF locus and the haplotype B (XV2c: allele 1; KM19: allele 2), which accounts for about 78% of CF chromosomes in our families. The ΔF508 deletion was present in 64.3% of CF chromosomes.     

Somatic hybrid plants produced by electrofusion between dihaploid potatoes: BF15 (H1), Aminca (H6) and Cardinal (H3)

In order to regenerate somatic hybrids, mesophyll protoplasts from a dihaploid potato, BF15 (H1), were electrofused with those from two other dihaploid clones, Aminca (H6) and Cardinal (H3). Determination of the ploidy level by flow cytometry showed that 10% of plants regenerated from the fusion experiment with BF15 + Aminca were diploids, 14% triploids, 63% tetraploids and very few were mixoploids or had a higher ploidy level. Using morphological markers and vigour in plant growth, we were able to recover a total of 24 somatic hybrid plants, respectively 20 and 4 hybrids (accounting for 12% and 13% of regenerants) from the fusions BF15 + Aminca and BF15 + Cardinal. Most of the somatic hybrids were at the expected tetraploid level (2n=4x=48). The hybrid nature was confirmed by examining isoenzyme patterns for malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD).