Structure of rat liver nuclei after depletion and reassociation of histone H1

Chromatin in isolated rat liver nuclei was compared with chromatin in (i) nuclei depleted of H1 by acid extraction; (ii) nuclei treated at pH 3.2 (without removal of H1), and (iii) depleted nuclei following reassociation of H1. Electron microscopy and digestion by DNase I, micrococcal nuclease and endogenous Ca/Mg endonuclease were used for this comparative examination. Electron micrographs of H1-depleted nuclei showed a dispersed and finely granular appearance. The rate of DNA cleavage by micrococcal nuclease or DNase I was increased several-fold after H1 removal. Discretely sized intermediate particles produced by Ca/Mg endonuclease in native nuclei were not observed in digests of depleted nuclei. Digestion by micrococcal nuclease to chromatin particles soluble in 60 mM NaCl buffer appeared not to be affected in depleted nuclei. When nuclei were treated at pH 3.2, neither the appearance of chromatin in electron micrographs nor the mode or rate of nuclease digestion changed appreciably. Following reassociation of H1 to depleted nuclei, electron micrographs demonstrated the reformation of compacted chromatin, but the lower rate of DNA cleavage in native nuclei was not restored. Further, H1 reassociation produced a significant decrease in the solubility of nuclear chromatin cleaved by micrococcal nuclease or Ca/Mg endonuclease. In order to evaluate critically the reconstitution of native chromatin from H1-depleted chromatin we propose the use of digestion by a variety of nucleases in addition to an electron microscopic examination.     

Isoelectric focusing in immobilized pH gradients: Principle,methodology and some applications

A new technique for generating pH gradients in isoelectric focusing is described, based on the principle that the buffering groups are covalently linked to the matrix used as anticonvective medium. For the generation of this type of pH gradient in polyacrylamide gels, a set of buffering monomers, called Immobiline (in analogy with Ampholine), is used. The pH gradient gels are cast in the same way as pore gradient gels, but instead of varying the acrylamide content, the light and heavy solutions are adjusted to different pH values with the aid of the Immobiline buffers. Available Immobiline species make it possible to generate any narrow linear pH gradient between pH 3 and 10. The behaviour of these types of gradients in isoelectric focusing is described.Immobilized pH gradients show a number of advantages compared with carrier ampholyte generated pH gradients. The most important are: (1) the cathodic drift is completely abolished; (2) they give higher resolution and higher loading capacitu; (3) they have uniform conductivity and buffering capacity; (4) they represent a milieu of known and controlled ionic strenght.     

Distribution of perch (Perca fluviatilis L.) during their first year of life in Lake Constance

The distribution and behaviour of larval and juvenile perch (Perca fluviatilis L.) were studied for two years in large, deep Lake Constance. After hatching larvae were transported by water currents to the open water. The majority of larvae remained in the pelagic zone for about one month. In both years, their return to the littoral zone coincided with the decline of pelagic zooplankton abundance. After returning to the littoral zone, juveniles stayed among submerged macrophytes within 5 m depth and lived apart from larger perch which lived at depths of 6–20 m. By late summer, juveniles changed their distribution pattern: during the day they stayed intensively close to piers and ports, but increased their swimming activity at dusk, cruising among shallow and deep waters and feeding on zooplankton, and rested on the bottom at night. This behaviour appears to be related to the decrease of inshore food resources and to the presence of predators in deeper water. 0⁺ perch left the littoral zone and moved into deep waters when autumnal mixing began in late October. They overwintered near the bottom at depths of more than 30 m. During most of the year, juvenile and adult perch were separated from each other. But as soon as they occupied the same habitat, the occurrence of cannibalism increased.     

Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.) shoot apices: transformation patterns

Apical segments of embryonic axes of sunflower (Helianthus annuus L.) embryos were submitted to co-culture experiments with a disarmed strain of Agrobacterium tumefaciens, harbouring a plasmid coding for the marker enzyme -glucuronidase. The expression patterns of this marker were analysed at different developmental stages of the regenerated shoots. The results are consistent with the hypothesis that transformed shoots originate from transformation events that have occurred within the existing meristems. Two of the resulting chimaeric plants have been analysed in detail, and some representative gene integration patterns are presented.     

Familial adenomatous polyposis: A submicroscopic deletion at the APC locus in a family with mentally normal patients

Cytogenetically visible deletions that include the adenomatosis polyposis coli (APC) locus have repeatedly been reported in mentally handicapped polyposis patients. We report on a family with a submicroscopic deletion of about 200 kb including more than the 3 half of the APC gene and the adjacent DPI gene. The deletion was detected by linkage analysis with flanking and intragenic markers and proven by in situ hybridisation with intragenic cosmid clones. All the familial adenomatous polyposis (FAP) patients and persons at risk in the family show normal behaviour and intelligence. Thus, it is conceivable that at least some of the FAP patients in whom mutations could not be identified by routine methods may have large but submicroscopic deletions.     

Reduction of microtubule catastrophe events by LIS1, platelet-activating factor acetylhydrolase subunit.

Forming the structure of the human brain involves extensive neuronal migration, a process dependent on cytoskeletal rearrangement. Neuronal migration is believed to be disrupted in patients exhibiting the developmental brain malformation lissencephaly. Previous studies have shown that LIS1, the defective gene found in patients with lissencephaly, is a subunit of the platelet-activating factor acetylhydrolase. Our results indicated that LIS1 has an additional function. By interacting with tubulin it suppresses microtubule dynamics. We detected LIS1 interaction with microtubules by immunostaining and co-assembly. LIS1-tubulin interactions were assayed by co-immunoprecipitation and by surface plasmon resonance changes. Microtubule dynamic measurements in vitro indicated that physiological concentrations of LIS1 indeed reduced microtubule catastrophe events, thereby resulting in a net increase in the maximum length of the microtubules. Furthermore, the LIS1 protein concentration in the brain, measured by quantitative Western blots, is high and is approximately one-fifth of the concentration of brain tubulin. Our new findings show that LIS1 is a protein exhibiting several cellular interactions, and the interaction with the cytoskeleton may prove to be the mode of transducing a signal generated by platelet-activating factor. We postulate that the LIS1-cytoskeletal interaction is important for neuronal migration, a process that is defective in lissencephaly patients.     

Mapping of the Gene Encoding Human β-Defensin-2 (DEFB2) to Chromosome Region 8p22–p23.1

We recently reported the isolation of human β-defensin-2 (hBD-2), a novel epithelia-derived peptide antibiotic belonging to the β-defensin family. hBD-2 is expressed in skin and epithelia of the airway system, where it is believed to contribute to its antimicrobial defense. By fluorescencein situhybridization using a hBD-2 genomic DNA probe and subsequent fluorescence R-banding, the hBD-2 gene (HGMW-approved symbol DEFB2) was assigned to human chromosome region 8p22–p23.1. PCR with a set of CEPH YAC clones spanning this chromosomal region revealed CEPH YACs 773G4, 920D12, and 820B4 to contain the hBD-2 gene. Relying on the preexisting physical maps of 8p22–p23.1, the hBD-2 gene was mapped in close proximity to D8S1993 (WI-9956) within the interval flanked by D8S552 and D8S1130 (CHLC.GATA25C10). The fact that all currently described genes encoding defensins map to chromosome 8p21–pter suggests that a gene cluster in this chromosomal region may play a major role in antimicrobial defense.     

Limits for the detection of daily otolith increments in whitefish (Coregonus lavaretus L.) larvae

The formation of daily otolith increments in European lake white-fish was evaluated in the laboratory during 40 days at 4, 6, and 8 °C under limited and ad libitum food supply. Daily increments were easily identified in larvae reared at 8 °C. At 6 °C and, more, at 4 °C, an unstructured perinuclear zone was formed, and daily increments could only be recognized in the light microscope starting from 10 to 35 days after hatching. In some larvae from the 4 °C groups, no increments at all were found. Scanning electron microscopy (SEM) could not improve increment resolution. Only those increments could be viewed by SEM which were visible in the light microscope as well. We conclude that whitefish larvae which experience low (4–6 °C) temperatures during their first weeks of life, hence those which hatch in the lake, cannot be aged by the currently employed preparation techniques.