A detailed physical map of HeLa cell mitochondria DNA and its alignment with the positions of known genetic markers

Twenty-one fragments have been identified among the products of digestion of HeLa cell mtDNA with the restriction enzyme Hpa II. The sum of the molecular sizes of these fragments, estimated from their mobility relative to that of known markers, accounts, within experimental error, for the total length of HeLa cell mtDNA. The 21 fragments have been ordered in a physical map by two approaches: (1) sequential digestion with Hpa II of the fragments produced by Eco RI, Hind III, andHpa I enzymes, and (2) fragment-primed DNA synthesis. The Hpa II map has been aligned with the maps constructed with the other three enzymes and with the unique cutting site produced by Bam I. The combined map thus obtained has resolved HeLa cell mtDNA into 27 recognizable segments in the molecular size range between 75 and 1950 base pairs. This physical map has been aligned with the known positions of the rRNA and 4 S RNA genes on the two mtDNA strands by RNA-DNA hybridization experiments utilizing purified ³²P-labeled 12 and 16 S rRNA.     

Precise localization of the origin of replication in a physical map of HeLa cell mitochondrial DNA and isolation of a small fragment that contains it

The precise positions of the origin of replication³ and of the D-loop within the HpaII restriction map of HeLa cell mitochondrial DNA have been investigated. For this purpose, 7 S DNA, which is the heavy-chain initiation sequence, was used as a template for fragment-primed DNA synthesis by Escherichia coli DNA polymerase I. The results indicate clearly that the origin of replication lies in HpaII fragment 8 at about 80 base-pairs from the border with fragment 17, and that the D-loop region extends from this site, through fragment 17, to a position in fragment 10 which is about 365 base-pairs from the border with fragment 17. Sequential digestion of fragment 8 with HaeIII enzyme has allowed the isolation of a subfragment, about 200 base-pairs long, that contains the origin of replication.     

Novel myosin isoform in nuclear chain fibers of rat muscle spindles produced in response to endurance swimming.

With the use of myosin adenosinetriphosphatase (ATPase) and immunofluorescence staining methods, the adaptive responses of intrafusal and extrafusal fibers to endurance swimming were studied in frozen sections of rat soleus (SOL) and extensor digitorum longus (EDL) muscles. Glycogen depletion confirmed muscle fatigue at the end of a standardized bout of exercise. No significant age-dependent changes in myosin isoforms were detected in any fibers. The 12-wk training increased type I fibers by 10.9% in the SOL and type IIa fibers in the EDL by 16.6%. In trained muscle sections, both staining methods identified a permuted chain fiber, expressed the same as the myosin isoform in the bag2 fiber. However, no exercise-induced change of myosin isoform profile was found in the bag1 and bag2 fibers. Myosin ATPase (and immunofluorescence) staining showed the percentage of permuted chain fibers increased from 0 to 6.7% (5.6%) after 6 wk of training and to 19.2% (14.1%) after 12 wk of training and that it was still at 6.1% (4.2%) 10 wks after training. A novel myosin isoform may thus be expressed in nuclear chain fibers by repetitive recruitment of muscle spindles.     

Conjugation is necessary for a bacterial plasmid to survive under protozoan predation

Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.     

The effect of past changes in inter‐annual temperature variability on tree distribution limits

Aim The northern limits of temperate broadleaved species in Fennoscanndia are controlled by their requirements for summer warmth for successful regeneration and growth as well as by the detrimental effects of winter cold on plant tissue. However, occurrences of meteorological conditions with detrimental effects on individual species are rare events rather than a reflection of average conditions. We explore the effect of changes in inter‐annual temperature variability on the abundances of the tree species Tilia cordata, Quercus robur and Ulmus glabra near their distribution limits using a process‐based model of ecosystem dynamics. Location A site in central Sweden and a site in southern Finland were used as examples for the ecotone between boreal and temperate forests in Fennoscandia. The Finnish site was selected because of the availability of varve‐thickness data. Methods The dynamic vegetation model LPJ‐GUESS was run with four scenarios of inter‐annual temperature forcing for the last 10,000 years. In one scenario the variability in the thickness of summer and winter varves from the annually laminated lake in Finland was used as a proxy for past inter‐annual temperature variability. Two scenarios were devised to explore systematically the effect of stepwise changes in the variance and shape parameter of a probability distribution. All variability scenarios were run both with and without the long‐term trend in Holocene temperature change predicted by an atmospheric general circulation model. Results Directional changes in inter‐annual temperature variability have significant effects on simulated tree distribution limits through time. Variations in inter‐annual temperature variability alone are shown to alter vegetation composition by magnitudes similar to the magnitude of changes driven by variation in mean temperatures. Main conclusions The varve data indicate that inter‐annual climate variability has changed in the past. The model results show that past changes in species abundance can be explained by changes in the inter‐annual variability of climate parameters as well as by mean climate. Because inter‐annual climatic variability is predicted to change in the future, this component of climate change should be taken into account both when making projections of future plant distributions and when interpreting vegetation history.     

Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin

Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.     

The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers

Twinfilin is a ubiquitous and abundant actin monomer-binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K(D) = 0.05 microM) than ATP-G-actin (K(D) = 0.47 microM) under physiological ionic conditions and forms a relatively stable (k(off) = 1.8 s(-1)) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.