Telomere dynamics in an immortal human cell line.

The integration of transfected plasmid DNA at the telomere of chromosome 13 in an immortalized simian virus 40-transformed human cell line provided the first opportunity to study polymorphism in the number of telomeric repeat sequences on the end of a single chromosome. Three subclones of this cell line were selected for analysis: one with a long telomere on chromosome 13, one with a short telomere, and one with such extreme polymorphism that no distinct band was discernible. Further subcloning demonstrated that telomere polymorphism resulted from both gradual changes and rapid changes that sometimes involved many kilobases. The gradual changes were due to the shortening of telomeres at a rate similar to that reported for telomeres of somatic cells without telomerase, eventually resulting in the loss of nearly all of the telomere. However, telomeres were not generally lost completely, as shown by the absence of polymorphism in the subtelomeric plasmid sequences. Instead, telomeres that were less than a few hundred base pairs in length showed a rapid, highly heterogeneous increase in size. Rapid changes in telomere length also occurred on longer telomeres. The frequency of this type of change in telomere length varied among the subclones and correlated with chromosome fusion. Therefore, the rapid changes in telomere length appeared occasionally to result in the complete loss of telomeric repeat sequences. Rapid changes in telomere length have been associated with telomere loss and chromosome instability in yeast and could be responsible for the high rate of chromosome fusion observed in many human tumor cell lines.     

The localization of mercury and metallothionein in the cerebellum of rats experimentally exposed to methylmercury

Summary Rats were dosed with methylmercuric chloride, either by gastric gavage (5 × 10 mg kg^-1 body weight over a 15-day period), or in their drinking water (20 mg methylmercuric chloride l^–1 for 14 or 42 days). Localization of mercury within the cerebellum was performed with a silver physical development technique, and metallothionein with dinitrophenyl hapten-sandwich immunohistochemistry. Mercury was detected in structurally undamaged Purkinje neurones and adjacent Bergmann glial cells; no mercury was detected in granule cells even though these small cells nearest the Purkinje layer had a high incidence of pyknotic nuclei. In general, metallothionein was detected mainly in Bergmann glial cells, Purkinje cells, astrocytes and glial cells of white matter; no metallothionein was detected in granule cells. We hypothesized that the resistance of Purkinje cells to methylmercuric chloride reflects their ability to transform organic mercurials to inorganic mercury that, in turn, induces the synthesis of radical-scavenging metallothionein molecules.     

Morphological variability of geographically distinct populations of the estuarine copepod Acartia Tonsa

Variations in the number of spines on the left and right posterior dorsal and posterior margins of the prosome as well as the length of the prosome of Acartia tonsa from three estuaries, the upper western side of the Chesapeake Bay, Montauk Bay near the eastern end of Long Island Sound and the coast of Peru were determined. The length of the prosome and number of spines in each of the four locations were used as an indication of morphological similarity between the populations.     

The use of a perfused, whole-body preparation to measure the branchial and intestinal influx of 137-caesium in the rainbow trout (Oncorhynchus mykiss)

The branchial and intestinal influx of caesium (Cs) in the rainbow trout ( Oncorhynchus mykiss ) were measured using a perfused whole-body preparation. The branchial influx of Cs was small, 0–31 μmoles kg⁻¹ h⁻¹ at an external concentration of 1 mm. Branchial Cs influx was saturable, with a K_m of 1–92 mm and a J_max of l.05μmoles kg⁻¹ h⁻¹. Intestinal Cs influx was not saturable, but was directly proportional to the mucosal Cs concentration. Intestinal Cs influx was approximately 10–40 times greater than branchial Cs influx over a wide range of external Cs concentrations. These results are discussed with respect to mechanisms of Cs uptake and to the relative accumulation of radiocaesium from water and food in the environment.     

Chloroflexus-like organisms from marine and hypersaline environments: Distribution and diversity

We report the presence of a diverse number ofChloroflexus-like organisms in intertidal marine and submerged hypersaline microbial mats using light, infrared fluorescence, and electron microscopy. The intertidal organisms appear morphologically very similar to thermophilicC. aurantiacus while the 2 hypersaline strains are larger and have a more complex ultrastructure composed of chlorosome-bearing internal membranes that appear to arise as invaginations of the cell membrane. By comparing spectroradiometry of microbial mat layers with microscopic observations, we have confirmed that theChloroflexus-like organisms are major constituents of the hypersaline microbial mat communities. In situ studies on mat layers dominated byChloroflexus-like organisms showed that sulfide-dependent photoautotrophic activity sustained by near infrared radiation prevailed. Autoradiographic analyses revealed that autotrophy was sustained in the filaments by 750 nm radiation. Three morphologically distinct strains are now maintained in mixed culture. One of these appears to be growing photoautotrophically.     

Characterization of Marek's disease virus insertion and deletion mutants that lack US1 (ICP22 homolog), US10, and/or US2 and neighboring short-component open reading frames.

We report the characterization of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome. A deletion mutant (GA delta 4.8lac) lacks 4.8 kbp of US region DNA, the deleted segment having been replaced by the lacZ gene of Escherichia coli. This deletion results in the loss of the MDV-encoded US1, US10, and US2 homologs of herpes simplex virus type 1, as well as three putative MDV-specific genes, Sorf1, Sorf2, and Sorf3. Two mutants containing lacZ insertions in the US1 and US10 genes have been constructed, and we have previously reported a US2lac insertion mutant (J. L. Cantello, A. S. Anderson, A. Francesconi, and R. W. Morgan, J. Virol. 65:1584-1588, 1991). The isolation of these mutants indicates that the relevant genes are not required for growth of MDV in chicken embryo fibroblasts. The mutants had early growth kinetics indistinguishable from those of their parent viruses; however, 5 to 7 days after being plated, the US1 insertion mutant (US1lac) and the GA delta 4.8lac deletion mutant showed a 5- to 10-fold decrease in virus growth. This decrease in virus accumulation correlated with a 30 to 50% decrease in plaquing efficiency when these viruses were plated onto established versus fresh chicken embryo fibroblast monolayers compared with a 10 to 15% decrease seen for the parent viruses and for the US10lac or US2lac insertion mutants. Finally, GA delta 4.8lac could be reisolated from chickens, indicating that the deleted genes are not required for the infection of chickens following intra-abdominal inoculation of an attenuated serotype 1 MDV.     

Genetic Regulation of Development in Sorghum bicolor (VIII. Shoot Growth,Tillering, Flowering,Gibberellin Biosynthesis,and Phytochrome Levels Are Differentially Affected by Dosage of the ma3R Allele

Sorghum [Sorghum bicolor (L.) Moench] homozygous for ma3R lacks a type II, light-stable phytochrome of 123 kD and has a number of phenotypic characteristics consistent with the absence of functional phytochrome B. We have used plants heterozygous at Ma3 (Ma3/ma3R and ma3/ma3R) to determine the effect of dosage of ma3R on plant growth, flowering, gibberellin (GA) levels, and content of the 123-kD phytochrome. Both Ma3/ma3R and ma3/ma3R produced the same number of tillers per plant as their respective homozygous non-ma3R parents. Height of the heterozygotes was intermediate between the homozygous parents, although it was more similar to the non-ma3R genotypes. In both field and growth-chamber environments, the timing of floral initiation and anthesis in the heterozygotes also was intermediate, again more similar to non-ma3R plants. In Ma3/ma3R, levels of GA53, GA19, GA20, and GA1 were almost exactly intermediate between levels detected in Ma3/Ma3 and ma3R/ma3R plants. Immunoblot analysis indicated that there was less of the 123-kD phytochrome in Ma3/ma3R than in homozygous Ma3, whereas none was detected in ma3R/ma3R. The degree of dominance of Ma3 and ma3 over ma3R varies with phenotypic trait, indicating that mechanisms of activity of the 123-kD phytochrome vary among the biochemical processes involved in each phenotypic character. Although the heterozygotes were similar to homozygous Ma3 and ma3 plants in growth and flowering behavior, Ma3/ma3R contained 50% less of the bioactive GA (GA1) than non-ma3R genotypes. Thus, sensitivity to endogenous GAs also may be regulated by the 123-kD phytochrome. To fully regulate plant growth and development, two copies of Ma3 or ma3 are required to produce sufficient quantities of the light-stable, 123-kD phytochrome.     

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of chelators on iron uptake and release by the brain in the rat

The iron chelators desferrioxamine (DFO), pyridoxal isonicotinoyl hydrazone (PIH), 2,2-bipyridine, diethylenetriamine penta-acetic acid (DTPA) and 1,2 dimethyl-3-hydroxy pyrid-4-one (CP20) were analysed for their ability to change⁵⁹Fe uptake and release from the brain of 15- and 63-day rats either during or after intravenous injection of⁵⁹Fe-¹²⁵I-transferrin. DTPA was the only chelator unable to significantly reduce iron uptake into the brain of 15-day rats. This indicates that iron is not released from transferrin at the luminal surface of brain capillary endothelial cells. CP20 was able to reduce iron uptake in the brain by 85% compared to 28% with DFO. Only CP20 was able to significantly reduce brain iron uptake in 63 day rats. Once⁵⁹Fe had entered the brain no chelator used was able to mediate its release. All of the chelators except CP20 had similar effects on femur iron uptake as they did on brain uptake, suggesting similar iron uptake mechanisms. It is concluded that during the passage of transferrin-bound iron into the brain the iron is released from transferrin within endothelial cells after endocytosis of transferrin.