The in vitro lifespan of MRC-5 cells is shortened by 5-azacytidine-induced demethylation

The minor base 5-methylcytosine (5mC) in DNA may be important for the regulation of gene expression. Random loss of 5mC may occur during pre-replicative DNA synthesis in mortal cell strains, and thus give rise to biochemical aberrations in aging cells. 5-Azacytidine (5azaC) was used to induce loss of 5mC in DNA of human diploid fibroblasts (MRC-5) in an attempt to accelerate in vitro senescence. The 5mC content of DNA was measured by incorporation of [3H]uridine into dividing cells, hydrolysis of DNA and separation of bases by HPLC. In untreated MRC-5 cells, 5mC was 3.6% of the total cytosine (C+5mC) at population doubling (PD) 20 (28% of lifespan) and fell to 1.6% at PD 67 (97% of lifespan). A single pulse treatment with 5azaC (1 microgram/ml) induced demethylation and shortened the lifespan by 10% (6.8 PDs loss). Pulse-treated cells showed temporary growth inhibition, though they subsequently regained normal growth rate and morphology. However, uniform treatment with 0.1 microgram/ml 5azaC between PD 20 and 23 produced no immediate growth inhibition, but a 22% loss of 5mC and 25% decrement in lifespan (16.6 PDs loss). The present results indicate that 5mC levels fall during normal aging of MRC-5 cells and accelerated 5mC loss shortens the in vitro lifespan of these cells. Hypomethylation may thus be responsible for some aspects of in vitro aging.     

Synthetic peptides used to locate the alpha-bungarotoxin binding site and immunogenic regions on alpha subunits of the nicotinic acetylcholine receptor

Synthetic peptides corresponding to 57% of the sequence of alpha subunits of acetylcholine receptors from Torpedo californica electric organ and extending from the NH2 to the COOCH terminus have been synthesized. The alpha-bungarotoxin binding site on denatured alpha subunits was mapped within the sequence alpha 185-199 by assaying binding of 125I-alpha-bungarotoxin to slot blots of synthetic peptides. Further studies showed that residues in the sequence alpha 190-194, especially cysteines-alpha 192, 193, were critical for binding alpha-bungarotoxin. Reduction and alkylation studies suggested that these cysteines must be disulfide linked for alpha-bungarotoxin to bind. Binding sites for serum antibodies to native receptors or alpha subunits were mapped by indirect immunoprecipitation of 125I-peptides. Several antigenic sequences were identified, but a synthetic peptide corresponding to the main immunogenic region (which is highly conformation dependent) was not identified.     

Larval host plant affects fitness consequences of egg size variation in the seed beetle Stator limbatus

Egg size variation often has large effects on the fitness of progeny in insects. However, many studies have been unable to detect an advantage of developing from large eggs, suggesting that egg size variation has implications for offspring performance only under adverse conditions, such as during larval competition, periods of starvation, desiccation, or when larvae feed on low-quality resources. We test this hypothesis by examining the consequences of egg size variation for survivorship and development of a seed-feeding insect, Stator limbatus, on both a low-quality (Cercidium floridum) and a high-quality (Acacia greggii) host plant. Our results are consistent with the hypothesis. S. limbatus larval performance was affected by egg size only when developing on the poor-quality host (C. floridum); larvae from large eggs survived better on C. floridum than those from small eggs, while there was no evidence of an effect of egg size on progeny development time, body weight, or survivorship when larvae developed on A. greggii. These results indicate intense selection for large eggs within C. floridum-associated populations, but not in A. greggii-associated populations, so that egg size is predicted to vary among populations associated with different hosts. Our results also support this hypothesis; females from a C. floridum-associated population (Scottsdale) laid larger eggs than females from an A. greggii-associated population (Black Canyon City).     

A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time.     

Repair replication and unscheduled DNA synthesis in mammalian cell lines showing differential sensitivity to alkylating agents

Repair replication has been measured by CsCl density gradient centrifugation in cell lines showing differential sensitivity to mono- and bifunctional alkylating agents. A correlation between cellular sensitivity as measured by the D₀ value and amount of repair replication was demonstrated after exposure of Yoshida cells to nitrogen mustard (HN2) and methylene dimethanesulphonate (MDMS). No differences in the amount of repair replication after methyl methanesulphonate (MMS) were observed in two L5178Y cell lines which differed in sensitivity by virtue of the shoulder size only. The Yoshida cell lines showed no difference in sensitivity to MMS and no difference in amount of repair replication. Incorporation of tritiated thymidine 9[³H]TdR) after drug treament was also measured by autoradiography. The qualitative differences observed between the two cell lines were similar to those obtained in density gradient experiments. The temporal pattern of [³H]TdR uptake indicated that the reduced repair replication observed in the sensitive line after HN2 and MDMS is not due to slower synthesis. The kinetics of [^3H]TdR incorporation differed for all three mutagens suggesting that different enzymes may be involved in each case.     

Changes in body mass and organ size during remigial moult in common scoter Melanitta nigra

The “cost‐benefit” hypothesis states that avian body organs show mass changes consistent with the trade‐off between their functional importance and maintenance cost, which may vary throughout the annual cycle. Flightless moulting common scoter Melanitta nigra in Danish marine waters select rich undisturbed offshore feeding areas lacking predators, suggesting active feeding during moult. We tested four predictions relating to organ size during flightlessness in moulting male common scoter under this hypothesis. Namely that (i) pectoral muscles would show atrophy followed by hypertrophy, but that there would be no change in (ii) leg muscles and heart (the locomotory architecture required to sustain diving for food), (iii) digestive organs and liver (required to process food), or (iv) fat deposits (because birds could fulfil daily energy requirements from locally abundant food resources). Dissection of scoters collected at different stages during wing moult south of the Danish island of Læsø provided data on organ size that were consistent with these predictions. Pectoral muscle mass showed a c.23% atrophy during the middle of the flightless period relative to that at the end of moult. There was no significant loss in leg muscle, heart, digestive organs (except gizzard mass), liver, fat reserves or body mass with remigial growth. These findings are consistent with the hypothesis that common scoter moult in a rich feeding area, and rely on their diet to meet the nutritional requirements of remigial moult. These results differ in detail from those of a similar study of terrestrial feeding moulting greylag geese Anser anser, but because of the widely differing ecology of the species concerned, both sets of findings provide strong support for the hypothesis that variations in phenotypic plasticity in size of fat stores, locomotor and digestive organs can be interpreted as evolutionary adaptations to meet the conflicting needs (feather growth, nutritional challenges and predator avoidance) of the flightless moult period in different Anatidae species.     

A Genetic Link between an mRNA-Specific Translational Activator and the Translation System in Yeast Mitochondria

Translation of the Saccharomyces cerevisiae mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the products of at least three nuclear genes, PET122, PET494 and PET54. pet122 mutations that remove 24-67 amino acid residues from the carboxyterminus of the gene product were found to be suppressed by unlinked nuclear mutations. These unlinked suppressors fail to suppress both a pet122 missense mutation and a complete pet122 deletion. One of the suppressor mutations causes a heat-sensitive nonrespiratory growth phenotype in an otherwise wild-type strain and reduces translation of all mitochondrial gene products in cells grown at high temperature. This suppressor maps to a newly identified gene on chromosome XV termed PET123. The sequence of a DNA fragment carrying PET123 contains one major open reading frame encoding a basic protein of 318 amino acids. Inactivation of the chromosomal copy of PET123 by interruption of this open reading frame causes cells to become rho- (sustain large deletions in their mtDNA). This phenotype is characteristic for null alleles of genes whose products are essential for general mitochondrial protein synthesis. Thus our data strongly suggest that the PET123 protein is a component of the mitochondrial translation apparatus that interacts directly with the coxIII-mRNA-specific translational activator PET122.     

Dietary and microtopographical selectivity of Greenland white-fronted geese feeding on Icelandic hayfields

The feeding ecology of Greenland white-fronted geese Anser albifrons flavirostris was studied during .spring staging in Iceland 1997. Geese feeding on Poa pratense dominated hayfields (> 80% cover) were highly selective, selecting for Deschampsia caespitosa which comprised only 10% of the sward. Geese fed most on the south-facing fringes of Deschampsia tussocks. Subsequent analysis showed that the southern fringes of Deschampsia tussocks supported significantly greater biomass (27% greater mass of green material) and that leaves growing on the southern faces had significantly higher protein content than those on the northern faces (33.9% vs 30.5%)_- It appears that the geese maximise their nutritional intake in spring by selecting the grass species of highest quality and taking the most nutritious parts of the plants.     

Wrapping of genomic polydA.polydT tracts around nucleosome core particles.

Five human clones containing genomic regions of polydA have been isolated by their ability to form intermolecular triple helices with agarose cross-linked polyU. All of these clones contain Alu repetitive DNA sequences. End-labelled DNA fragments containing these sequences have been successfully reconstituted onto nucleosome core particles by salt exchange. The structure of these has been examined by digesting with DNase I, hydroxyl radicals or diethylpyrocarbonate. DNase I cleavage of the polydA tracts is poor in the free DNA but is markedly enhanced at certain positions when complexed with nucleosome cores. Phased digestion patterns are observed which continue through the (A)n blocks and reveal an average helical periodicity of about 10 base pairs. The distance between adjacent maxima varies between 8-12 base pairs, suggesting that the exact helical repeat is not necessarily constant. One fragment containing the sequence (TA)11T34 reveals a 12 base pair repeat within the (AT)n region. A pUC19 polylinker fragment containing a block of A69.T69 cloned into the Smal site could also be reconstituted onto nucleosome cores and reveals the same phased DNaseI digestion pattern. The DNase I cleavage pattern is not identical at each of the maxima, suggesting that the structural distortions imposed by the core particles are not constant along the DNA.