Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Duplicated region of the mouse genome containing a cytoplasmic gamma-actin processed pseudogene associated with long interspersed repetitive elements

The structures of two cloned recombinants of bacteriophage lambda and mouse genomic DNA (lambda mA14 and lambda mA36) were compared by electron microscopic analysis of various heteroduplex DNAs, restriction endonuclease mapping and nucleotide sequence determination. Each clone was shown to be derived from a distinct region of the mouse genome, but the two exhibited structural similarity over a region of at least 11,000 bases which included a cytoskeletal gamma-actin processed pseudogene of approximately 1800 bases. It is concluded that the two genomic regions were derived from a common ancestral region by duplication or amplification. The homologous regions of the two clones contained members of the long interspersed repetitive L1Md (long interspersed repeated sequence 1 of Mus domesticus) family lying in opposite orientation to one another, so that single-stranded DNA from the clones could form intra-molecular heteroduplexes. The complete nucleotide sequences of three L1Md members in lambda mA14 were determined. The longest of these (L1Md-14LH) had inserted into the gamma-actin processed pseudogene and, although it contained internal deletions, appeared to possess intact 5' and 3' ends. A second L1Md member (L1Md-14RH1) also appeared to have an intact 5' end but had lost most of its 3' portion, and a third member (L1Md-14RH2) was an internal fragment. The repeated sequence at the 5' ends of L1Md-14LH and L1Md-14RH1 showed these to be members of the L1Md-A family.     

Effects of hyperthermia and X-irradiation on mouse stromal tissue

The sensitivity of normal stroma to heat, irradiation and heat combined with irradiation has been studied using the tumour bed effect (TBE) assay. Irradiation before implantation led to a TBE. This TBE was dose dependent below 15 Gy, the TBE remaining relatively constant above 15 Gy. The interval (0-90 days) between irradiation and tumour implantation did not influence the magnitude of the TBE. Hyperthermia with large heat doses (45-60 min at 44 degrees C) before implantation may lead to a TBE. The interval between hyperthermia and tumour implantation proved to be very important. Our results show that the recovery from heat-induced stromal damage is very rapid. When the interval between hyperthermia and tumour implantation is 10 days or longer, no TBE could be observed. Irradiation combined with large heat doses (30-60 min at 44 degrees C) decreased the radiation-induced TBE. However, the combination of irradiation with mild heat treatments (15 min at 44 degrees C) could lead to a larger TBE than after irradiation alone. When hyperthermia was given prior to irradiation, the interval between heat and irradiation proved to be very important. With large intervals (21 days or longer) the TBE values were about the same as with irradiation alone. When heat was given after irradiation it always reduced the irradiation-induced TBE.     

Identification of FtsW and characterization of a new ftsW division mutant of Escherichia coli.

The product of the ftsW gene has been identified as a polypeptide that, like the related RodA protein, shows anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FtsW is produced at low levels that can be increased by altering the translation initiation region of the mRNA. Overproduction of FtsW strongly inhibits cell growth. A new mutant allele, ftsW201, causes a temperature-dependent block in the initiation stage of cell division which is similar to the division block in ftsZ mutants. The block in initiation of division in the ftsW201 allele is shown to be independent of FtsZ or the FtsZ inhibitor, SulA. In addition, the ftsW201 mutant is hypersensitive to overproduction of the division initiation protein FtsZ at the permissive temperature. Our results suggest a role for FtsW in an early stage of division which may involve an interaction with FtsZ.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement.     

Inhibition of cell division initiation by an imbalance in the ratio of FtsA to FtsZ.

Elevated levels of FtsA protein block cell division at a very early stage, similar to that caused by inhibition of the action of FtsZ. In contrast, overexpression of FtsA and FtsZ together does not block division. A specific ratio of FtsA to FtsZ protein, therefore, is required for cell division.     

Urbanisation versus agriculture: a comparison of local genetic diversity and gene flow between wood mouse Apodemus sylvaticus populations in human‐modified landscapes

Urbanisation and agriculture dramatically modify the landscapes available for use by wildlife, affecting key aspects of their ecology such as survival, foraging, predation, competition and reproductive success. Relatively little is known about the effects of urbanisation and agriculture on the genetic structure, gene flow and genetic diversity of wild species. Here, landscape genetic techniques were applied to compare local genetic diversity and gene flow between wood mouse populations in urban and arable landscapes. Using nine microsatellite markers, individuals were genotyped from six arable and seven urban sample sites. Inter‐population genetic differentiation was significantly greater in urban than arable habitat, while allele richness, private allele richness and heterozygosity were higher for arable sample sites, with varying degrees of significance. These suggest that urban habitat was sufficiently fragmented to limit gene flow. To test the effect of landscape features on gene flow, several cost‐distance measures were generated. Overland distance and Euclidean distance correlated best with inter‐population genetic differentiation in arable habitat, whereas distances that accommodated differences in habitat quality better explained differentiation in urban habitat. There was no evidence that margins adjacent to roads, rivers or railways facilitated gene flow. Together, the results indicate that urban landscapes expose wood mice to greater fragmentation in habitat quality than arable areas, leading to greater population isolation that is not mitigated by the presence of dispersal corridors.