Effects of dnats genes on the replication of plasmids in Bacillus subtilis

An essential region (2.3 kb) for the replication of a low-copy-number plasmid, pBS-2, has been identified and cloned into plasmid pHV60 in Bacillus subtilis. The resultant plasmid, pKW1, and two other plasmids, pC194 (medium copy number) and pTP5 (high copy number), were examined by double radio-labelling and gel electrophoresis to determine which host functions are required for their replication in B. subtilis. Replication of pKW1 requires the functions of most dna genes, in particular dnaB, C, E, F, G and H; pC194 requires only dnaG and H; and pTP5 requires dnaE, F, G and H. Thus dnaG and dnaH are required for the replication of all three plasmids tested, even though each plasmid showed a different spectrum of dependency on other host functions. Because of its greater dependence on host functions and its low copy number, pKW1 should be a useful model with which to investigate the function of host genes in the replication of DNA in B. subtilis. pKW1 should also be a useful shuttle vector for cloning of genes in B. subtilis in cases when high gene dosage might be a problem.     

Energy-dependent formation of free ATP in yeast submitochondrial particles, and its stimulation by oligomycin

Yeast submitochondrial particles, in a P_i- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5–10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.     

Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.     

Dependence of magnetic resonance image (MRI) intensity values on relaxation times, pulse intervals and other signal attenuation factors

Magnetic resonance image (MRI) pixel intensities were investigated using a phantom containing several uniform size chambers filled with solutions of known relaxation times, as well as head scans of patients and volunteers. Intensities were measured with a variety of pulse intervals typically used for imaging with spin echo, (SE) and inversion recovery (IR) sequences at 0.15 Tesla using the back projection (R-THETA) method, and at 0.27 Tesla using the 2-dimensional Fourier transform (2DFT) technique. The results were compared with the calculated dependence of MRI signal intensity on relaxation times and pulse interval parameters using the well known functions containing exponential forms. The experimental and the calculated pixel intensity time dependence did not always agree. We infer that factors other than the conventional functions for T1 and T2 signal decay are important. These factors may include the attenuation of the radiofrequency (RF) signals through inhomogenious lossy dielectric materials (e.g., tissues and organs), the location (coordinate) of the portion of the sample to be imaged relative to the RF coils, and the timing and amplitude of gradient pulses relative to the RF input and the detected signals. The flow velocity and diffusions are also important determinants of the signal from blood vessels and body fluids. We point out the necessity for further investigation toward more comprehensive understanding of MRI intensities.     

Time course adaptations in rat skeletal muscle isomyosins during compensatory growth and regression

The purpose of this study was to ascertain the time course of change during both compensatory growth (hypertrophy) and subsequent growth regression on myosin isoform expression in rodent fast-twitch plantaris muscle in response to functional overload (induced by removal of synergists). Peak hypertrophy of the plantaris muscle (92%) occurred after 9 wk of overload. After 7 wk of overload regression (induced by a model of hindlimb unweighting), muscle weight returned to within 30% of control values. Myofibril protein content (mg/g muscle) remained relatively constant throughout the overload period but became significantly depressed relative to control values after 7 wk of regression. However, when expressed on a per muscle basis (mg/muscle) no differences existed at this time point (t = 7 wk regression). The distribution of native myosin isoforms in the myofibril protein pool of the overloaded plantaris muscle reflected a progressive increase (23% at t = 9 wk; P less than 0.001) in the relative proportion of slow myosin (Sm). This change was also accompanied by increases in intermediate myosin (Im) as well as the repression of the fast myosin one (Fm1) isoform (P less than 0.001). These shifts in Sm and Fm1 isoform expression were gradually reversed during the regression period, whereas Im remained elevated relative to control values. These adaptive changes in myosin isoform expression during both hypertrophy and regression were further supported by concomitant shifts in both myosin adenosinetriphosphatase (ATPase) activity (decreased during overload) and slow myosin light chain (SLC) expression. However, during regression the changes in myosin isoform expression and myosin ATPase were not as synchronous as they were during overload. Estimation of the mixed myosin heavy chain (MHC) half-life (t 1/2), using a linear model that assumes zero-order synthesis and first-order degradation kinetics, revealed t 1/2 values of approximately 19 and 10 days for the overload and regression periods, respectively. Collectively these data suggest that 1) skeletal muscle myosin isoforms and corresponding ATPase activity are in a dynamic state of change, although not completely synchronous, in response to altered muscle stress, and 2) the kinetics of change in the mixed MHC protein pool are slower during compensatory growth compared with regression of growth.     

Subunit composition of rodent isomyosins and their distribution in hindlimb skeletal muscles

Three adult skeletal muscle sarcomeric myosin heavy chain (MHC) genes have been identified in the rat, suggesting that the expressed native myosin isoforms can be differentiated, in part, on the basis of their MHC composition. This study was undertaken to ascertain whether the five major native isomyosins [3 fast (Fm1, Fm2, Fm3), 1 slow (Sm), and 1 intermediate (Im)], typically expressed in the spectrum of adult rat skeletal muscles comprising the hindlimb, could be further differentiated on the basis of their MHC profiles in addition to their light chain composition. Results show that in muscles comprised exclusively of fast-twitch glycolytic (FG) fibers and consisting of Fm1, Fm2, and Fm3, such as the tensor fasciae latae, only one MHC, designated as fast type IIb, could be resolved. In soleus muscle, comprised of both slow-twitch oxidative and fast-twitch oxidative-glycolytic fibers and expressing Sm and Im, two MHC bands were resolved and designated as slow/cardiac beta-MHC and fast type IIa MHC. In muscles expressing a mixture of all three fiber types and a full complement of isomyosins, as seen in the plantaris, the MHC could be resolved into three bands. Light chain profiles were characterized for each muscle type, as well as for the purified isomyosins. These data suggest that Im (IIa) consists of a mixture of fast and slow light chains, whereas Fm (IIb) and Sm (beta) isoforms consist solely of fast- and slow-type light chains, respectively. Polypeptide mapping of denatured myosin extracted from muscles expressing contrasting isoform phenotypes suggests differences in the MHC primary structure between slow, intermediate, and fast myosin isotypes. These findings demonstrate that 1) Fm, Im, and Sm isoforms are differentiated on the bases of both their heavy and light chain components and 2) each isomyosin is distributed in a characteristic fashion among rat hindlimb skeletal muscles. Furthermore, these data suggest that the ratio of isomyosins in a given muscle or muscle region is of physiological importance to the function of that muscle during muscular activity.     

Analyse der schlagauslösenden Bewegungsparameter einer punktförmigen Beuteattrappe bei der Aeschnalarve

Zusammenfassung Bei der Libellenlarve Aeschna cyanea M. werden die einzelnen, für die Auslösung des Fangschlags relevanten Bewegungsparameter einer punktförmigen Beuteattrappe und die wirksamste Kombination dieser Parameter bestimmt.Als Beiz dient der beliebig bewegbare Leuchtpunkt eines Oszillographen, der auf die ebene Mattscheibe eines Versuchsaquariums projiziert wird. Die Schläge der frei beweglichen Larve werden vom Beobachter gezählt oder elektrisch registriert.Die Bahngeschwindigkeit von kontinuierlich gebotenen Bewegungsreizen wirkt sich stark auf die Schlagzahl aus: Die Schläge nehmen von 0,005–2,5 cm/sec zu, nehmen von 5,1 cm/sec an wieder ab und hören bei 41,0 cm/sec ganz auf. Die Veränderung der mittleren Geschwindigkeit von Sinusschwingungen und Zufallsbewegungen bewirkt ähnliche Reaktionskurven wie die Veränderung der gleichmäigen Geschwindigkeit von Dreiecksschwingungen und kreisförmigen Bewegungen; eine gleichförmige Optimalgesohwindigkeit wird jedoch stärker beantwortet als eine periodisch schwankende.Bietet man eine Folge von diskontinuierlichen Bewegungsreizen, die nach einer einmaligen Durchquerung eines begrenzten Feldes der Projektionsfläche verschwinden, so spielt die Dauer einer Einzelbewegung eine Rolle. Um die Schläge voll in Gang zu bringen, müssen eindimensionale Schwingungen 3–6 sec dauern, ein viel intensiver wirkender zweidimensionaler Reiz (Zickzackbewegung) jedoch nur 0,8 sec.Im optimalen, relativ hohen Geschwindigkeitsbereich läßt die Erhöhung der Bewegungsamplitude von 0,25 auf 2,0 cm die Schlagzahl progressiv absinken. Der Vergleich zwischen diesen Amplituden und dem Öffnungswinkel des frontalen, die Beute fixierenden Ommatidienfeldes zeigt, daß der Leuchtpunkt nur bei sehr kleinen Ablenkungen die frontalen Rezeptoren kontinuierlich reizt. — Die Bevorzugung von raschen Bewegungsreizen mit kleiner Amplitude besteht nicht bei Larven, die durch prompte Fixier- und Folgereaktionen den Leuchtpunkt in ihrer frontalen Fixierebene bewahren.Der Vergleich zwischen den 4 in dieser Untersuchung erzeugten Bewegungsmustern (ein- und zweidimensionale Schwingungen, Kreis- und Zufallsbewegungen) zeigt, daß eine Bewegung um so mehr Schläge auslöst, je vollständiger sie auf dicht aneinanderliegenden, zweidimensionalen Bahnen das frontale Ommatidienfeld abtastet.Die optimale Reizkombination (Zickzackbewegung) besteht aus einer kleinen (0,2–0,4 cm) Vertikalschwingung mit optimaler Geschwindigkeit, die sich langsam (0,32 cm/sec) seitlich verschiebt. Dieser Reiz stellt die Verbindung der optimalen Werte aller Bewegungsparameter dar und bewirkt, daß pro Zeiteinheit eine möglichst große Zahl frontaler Rezeptoren mit der optimalen Bahngeschwindigkeit gereizt wird.

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Analysis of the parameters of a moving lightspot which release the predatory strike in dragonfly larvae

Summary This study analyses the predatory strike response of the dragonfly larva Aeschna cyanea M. towards a moving spot of light. Its aim is to determine the single parameters of movement of the spot which release the strike and the most effective combination of these parameters.As the velocity of a continuously moving lightspot is increased the number of strikes rises to a maximum (at 2.5 cm/sec) and then declines to 0 (at 41.0 cm/sec). Both uniform and non uniform velocities give curves of similar shape but different magnitudes.In the presentation of a sequence of discontinuous movements (where the spot moves across a part of the screen and then disappears) the duration of a single movement is important: Unidimensional oscillations must last 3 to 6 sec in order to release predatory strikes; twodimensional zigzag movements, much more effective, need last only 0.8 sec.In the optimal velocity range, increasing the amplitude of a movement from 0.25 to 2.00 cm produces a progressive decrease of the response rate. The comparison between these amplitudes and the size of the field of the frontal ommatidia, which fixate the prey, suggests that the spot stimulates these receptors continuously only when it moves with small amplitudes. — However, this preference for small amplitudes does not exist in those individuals which have rapid fixation- and following-reactions and which thus can track the stimulus.Comparison between the four patterns of movement which have been presented (one- and two-dimensional oscillations, circular and random movements) suggests that the spot releases more strikes the more exactly its movement covers the frontal fixation plane.The most effective stimulus for eliciting strikes was found to be a twodimensional zigzag oscillation. This movement consists of a small (0.2–0.4 cm), rapid (2.5 cm/ sec), vertical oscillation, which progresses slowly (0.3 cm/sec) sideways. This movement combines the optimal values of all parameters and stimulates with the optimal velocity the greatest possible number of frontal receptors in a given time.

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Diese Arbeit wurde in Seewiesen mit Dr. H. C. Howland begonnen und dank der fortwährenden, großzügigen Unterstützung von Herrn Dr. H. Mittelstaedt beendet. Frau L. Dinnendahl fertigte die Zeichnungen an und durchsah das Manuskript, Dr. E. Kramer und Herr P. Heinecke standen mir ständig in technischen Fragen bei, Herr E. Butenandt leistete wertvolle Kritik am Manuskript. — In Genf gewährte mir Prof. J. Piaget vollkommene Freiheit in der Gestaltung meiner Arbeit. — Ihnen allen sei an dieser Stelle herzlichst gedankt.     

Development of sensory innervation in chick skin: comparison of nerve fibre and chondroitin sulphate distributions in vivo and in vitro

In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using themonoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.