Repair of single nucleotide DNA mismatches transfected into mammalian cells can occur by short-patch excision

Synthetic DNA linkers containing a single mismatched nucleotide (C:A) are repaired without bias at high efficiency when introduced into mammalian cells on a SV40 shuttle vector. From the pattern of repair in vectors containing multiple linkers, it appears that DNA synthesis following mismatch excision can replace a length of DNA as short as 40 nucleotides. Furthermore, results from the introduction of linker molecules containing combinations of single-strand nicks suggest that transient unsealed nicks do not drive the direction of mismatch repair in mammalian cells, as has previously been proposed.     

Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

"Protistan" nomenclature: analysis and refutation of some potential objections

The nomenclature used for higher taxonomic categories of protista arose under the influence of the two-kingdom system, and is widely recognized as being evolutionarily misleading. The occurrence and promulgation of multiple, contradictory taxonomic systems have added to the confusion. Recently, we proposed a solution to this problem which involves identifying the largest taxa that are widely recognized to be monophyletic, and naming them using a commonly recognizable prefix and the suffix "protista". Thus, nomenclatural prejudice is eliminated, and, by keeping the system informal and thus circumventing the Linnaean system, our system remains flexible in accommodating new data yet should retain a great degree of stability. Here we discuss possible criticisms of our proposal, such as whether the introduction of more terms could lead to further taxonomic and nomenclatural instability, and conclude that our proposal remains a most reasonable solution to the problem of protistan nomenclature.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author     

Ultrastructural characterization of the interphase nucleus of Gonyostomum semen.

The interphase nucleus of the chloromonadophycean alga, Gonyostomum semen (Ehrenberg) Diesing, has a highly distinctive appearance. Interphase chromatin is readily distinguishable in both light and electron microscope preparations. It extends throughout the neucleus and frequently makes contact with the nucleoli and with the nuclear envelope. Among the chromatin filaments are large numbers of 35-46 nm diameter granules which occur singly or in clusters. The nucleoli are characteristically located in the posterior half of the nucleus and are composed of granular and non-granular components. Nuclear pores occur in slight depressions of the nuclear surface; their lumen has a diameter of approximately 75 nm and contains electron-dense material. The chromatin and the large numbers of nuclear granules are unusual and warrant further investigation.     

The metabolism of human mesenchymal stem cells during proliferation and differentiation

Human mesenchymal stem cells (MSCs) reside under hypoxic conditions in vivo, between 4% and 7% oxygen. Differentiation of MSCs under hypoxic conditions results in inhibited osteogenesis, while chondrogenesis is unaffected. The reasons for these results may be associated with the inherent metabolism of the cells. The present investigation measured the oxygen consumption, glucose consumption and lactate production of MSCs during proliferation and subsequent differentiation towards the osteogenic and chondrogenic lineages. MSCs expanded under normoxia had an oxygen consumption rate of ～98 fmol/cell/h, 75% of which was azide-sensitive, suggesting that these cells derive a significant proportion of ATP from oxidative phosphorylation in addition to glycolysis. By contrast, MSCs differentiated towards the chondrogenic lineage using pellet culture had significantly reduced oxygen consumption after 24 h in culture, falling to ～12 fmol/cell/h after 21 days, indicating a shift towards a predominantly glycolytic metabolism. By comparison, MSCs retained an oxygen consumption rate of ～98 fmol/cell/h over 21 days of osteogenic culture conditions, indicating that these cells had a more oxidative energy metabolism than the chondrogenic cultures. In conclusion, osteogenic and chondrogenic MSC cultures appear to adopt the balance of oxidative phosphorylation and glycolysis reported for the respective mature cell phenotypes. The addition of TGF-β to chondrogenic pellet cultures significantly enhanced glycosaminoglycan accumulation, but caused no significant effect on cellular oxygen consumption. Thus, the differences between the energy metabolism of chondrogenic and osteogenic cultures may be associated with the culture conditions and not necessarily their respective differentiation.