Characterization of kinetochores in multicentric chromosomes

Long-term cultures of certain rat and mouse cell lines carry several dicentric and some multicentric chromosomes. Using antikinetochore antibodies obtainable from serum of scleroderma (var. CREST) patients we studied the number of kinetochores formed along the length of these chromosomes. The rat cells displayed as many kinetochores as there were centromeres. However, mouse cells showed the synthesis of only one kinetochore in dicentric and multicentric chromosomes which had been in the culture for a period of 1 year or more. When translocations were induced by bleomycin in mouse L cells, the newly formed dicentric chromosomes showed the formation of two kinetochores. It is not known when the accessory centromeres lose their capacity to assemble kinetochore proteins. Possibly, in the rat the latent kinetochores lack a specific component which renders them ineffective for microtubule binding. The reason for the formation of only one kinetochore in mouse multicentric chromosomes is not clear. It may be due to the accumulation of mutations, modification of the kinetochore protein so that it lacks the antibody binding component, or a more effective regulatory gene than in the rat.     

Chromosomal distribution of P and I transposable elements in a natural population of Drosophila melanogaster

The chromosomal distribution of P and I transposable elements was studied, by in situ hybridisation, in 25 isofemale lines of Drosophila melanogaster collected at Nasr'Allah in Tunisia. An important interline variability for the number of copies of both elements was revealed. The mean number of copies per line was 31.3 for P and 21.0 for I. Certain chromosome arms had a higher frequency of copies than others: arm 3R had the highest frequency of I elements; the X chromosome had the highest frequency of P elements and the lowest frequency of I elements. For both P and I elements the number of copies on the different chromosome arms is independent. Furthermore, there is no significant correlation between the number of copies of P and the number of copies of I for a given line. A study of the localisation of hybridisation sites on the X chromosome revealed the existence of preferred regions for each family. The population studied was of type M' in the P-M system of hybrid dysgenesis. There is no direct relationship between the M potential of an isofemale line and its number of copies of P elements. These results are compared with those of other investigators and the consequences for cytotype determination are discussed.     

Cellular Localization of the Molecular Forms of Acetylcholinesterase in Primary Cultures of Rat Sympathetic Neurons and Analysis of the Secreted Enzyme

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.     

Contribution of increasing and decreasing daylength to the photoperiodic control of LH secretion in the Ile-de-France ram

Sexually mature Ile-de-France rams were exposed to an 8-month light regimen in which the daily light increment and decrement were constant and equal to 7 min/day. Daylength therefore varied from 6 to 20 h. The animals were allotted to two groups of 12 rams each and submitted to the same light regimen but 4 months out of phase. Blood was collected every 40 min for 10 h, on 15 occasions at various intervals during the second light cycle. Plasma LH and on some occasions plasma testosterone concentrations were measured by radioimmunoassay. In both groups the number of LH pulses increased significantly as daylength increased from 11:40 to 20:00 h (P less than 0.01) but because their amplitude was low the mean plasma LH increase, although significant, was moderate. As daylength started to decrease (from 20:00 to 18:30 h), the frequency of LH pulses further increased (P less than 0.05) and the pulse amplitude doubled so that mean plasma LH values increased abruptly and remained high until the decreasing light photoperiod reached 11:40 h. Thereafter, the frequency and amplitude of LH pulses decreased and the mean plasma LH dropped to one-fourth of its maximal value. Mean plasma testosterone concentrations were low during most of the 8-month cycle but increased steeply when daylength decreased from 11:40 to 6:00 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

Summary Using the Southern hybridization technique, homologies were examined between restricted DNA of four methanogenic bacteria (Methanobacterium ivanovi, Methanobacterium thermoautotrophicum, Methanococcus voltae, Methanosarcina barkeri) and the nif (nitrogen fixation) genes of Klebsiella pneumoniae and Anabaena strain 7120. With K. pneumoniae probes, no hybridization was observed with nifA, nifNE, and nifJ but positive results were obtained with the nifHDK genes coding for nitrogenase. Homology was detected, in the four strains, with K. pneumoniae and Anabaena nifH probes. In M. voltae and M. ivanovi, the homology found with nifH was estimated to be about 70% and a weaker hybridization was observed also with nifD and nifK. In M. voltae, the sequence homologous to nifH was found on a 3.0 kbp HindIII fragment and sequences homologous to nifD and nifK on a 3.8 kbp HindIII fragment. The 3.0 kbp fragment was cloned and the region homologous to nifH was localized more precisely. When this fragment was used as a probe against other DNAs, it behaved as a K. pneumoniae and Anabaena nifH probe. The results suggest that the structural genes for nitrogenase may be present in archaebacteria and raise interesting questions regarding their evolution.     

Mitochondrial DNA of the basidiomycete Polyporus ciliatus

Summary Mitochondrial (mt) DNA of the white rot fungus Polyporus ciliatus was isolated and characterized. As a result of detailed restriction enzyme analysis, a physical map was established showing that this circular DNA has a molecular weight of 88.2 kb. By heterologous cross hybridization the sites of three mt genes were recognized. By nonselective cloning of mt DNA fragments in Saccharomyces cerevisiae, an autonomously replicating sequence (ars) was identified which has potential application in the development of a prokaryotic/eukaryotic shuttle vector.     

Binding of 5-methoxypsoralen to human serum low density lipoproteins

5-methoxypsoralen (5-MOP) binds to human serum low density lipoproteins (LDL) according to a two-step process. Scatchard analysis of the first step yields K = 1.4 × 10⁵ M^?1 and 4 binding sites. It involves the LDL apoprotein. The second step corresponds to a solubilization, in the lipidic core, of ? 45 molecules of 5MOP per LDL molecule. It is accompanied by a large blue shift of the 5MOP fluorescence. The ability of LDL to bind 5MOP and to carry it into various cells may explain some biological effects sometimes encountered during PUVA therapy.     

Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland

The distribution of epidermal growth (EGF) binding sites was studied in the pituitary gland using light and electron microscope autoradiography which was performed at different time intervals (2 to 60 min) after intravenous (IV) injection of [125I]EGF into adult rats. At the light microscopic level, the labeling was found over cells of the anterior pituitary gland. The time-course study performed by light microscope autoradiography showed that the maximal values were reached at the 2 min time interval. At this time interval, most silver grains were found at the periphery of the target cells. After, the number of silver grains decreased progressively and the localization of silver grains in the cytoplasm indicated the internalization of [125I]EGF. Electron microscope autoradiography showed that labeling was mostly restricted to mammotrophs and somatotrophs. Control experiments indicated that the autoradiographic labeling was due specific interaction of [125I]EGF with its binding site. These results indicate that EGF binding sites are present in at least two anterior pituitary cell types and suggest that EGF can exert a physiological role in the pituitary gland.     

Influence of very low doses of ionizing radiation on Synechococcus lividus metabolism during the initial growth phase

Previous results from this laboratory have shown that very low chronic doses of gamma radiation can stimulate proliferation of the Cyanobacterium Synechococcus lividus. This modification of cell proliferation occurred during the first doubling. In this paper, we have compared the metabolism of cells cultivated in a normal environment or under chronic irradiation. Incubation of the cells in a new medium induced a high superoxide dismutase (EC 1.15.1.1, SOD) activity at the 18th hour and a degradation of phycocyanin, thus demonstrating that cells were submitted to a photooxidative stress. This increase in superoxide dismutase activity was followed by concomittant peaks of glutathione reductase (EC 1.6.4.2, GR) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6P-DH) at the 24th hour. Irradiated cultures at a dose of 53.5 mGray/year show an earlier and higher peak of SOD, GR, and G6P-DH. In a second stage, cultures showed an earlier onset of photosynthesis under irradiation, as evidenced by an increase in pigment content and an enhancement of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, GAP-DH). These results show that the radiostimulation is related to the activation of enzymes protecting against peroxides that were induced under oxidative circumstances and to the activation of a glucose catabolism via the oxidative pentose phosphate pathway.Abbreviations mGy milli-Gray - SOD superoxide dismutase - G6P-DH glucose-6-phosphate dehydrogenase - GAP-DH glycer-aldehyde-3-phosphate dehydrogenase - GSSG oxidized glutathione