Optimization of speed and accuracy of decoding in translation

The speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10⁻³, consistent with the values measured in vivo. Selectivity is predominantly due to the differences in k_cat values for cognate and near-cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near- and non-cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate-limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation.     

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

Pericentric inversion of chromosome 19 in three families

Summary Pericentric inversion of chromosome 19 has been found in several members of three unrelated families from a restricted geographical region. In one of the families, an additional pericentric inversion of chromosome 9 was observed. Reproductive problems, multiple abortions in two families and a neonatal death in the third, were present. A review of previously described cases is included, and the genetic risk connected with this type of rearrangement is also discussed.     

Initial reactions in the mineralization of 2-sulfobenzoate by Pseudomonas sp. RW611

Abstract Pseudomonas sp. strain RW611 utilized the ammonium salt of 2-sulfobenzoate as sole source of carbon, sulfur, nitrogen, and energy. The xenobiotic sulfo substituent was dioxygenolytically eliminated as sulfite, which was then slowly oxidized to sulfate. 2,3-Dihydroxybenzoate, which resulted from desulfonation underwent meta -cleavage, mediated by 2,3-dihydroxybenzoate 3,4-dioxygenase activity. This enzyme was inhibited by 3-chlorocatechol and 2,3,4-trihydroxybenzoate.     

Ein Beitrag zum histochemischen Verhalten der Kern-DNS in Interphase und Mitose,dargestellt durch kombinierte Säurehydrolyse und Akridinorangefluorochromierung

Zusammenfassung Aufbauend auf vorangegangenen Arbeiten über die unterschiedliche Darstellung von DNS durch Kombination einer Säurehydrolyse mit einer Akridinorange-fluorochromierung, sollte untersucht werden, ob sich, damit unter standardisierten Bedingungen Unterschiede der DNS an alkoholfixierten Kernen bei Tumoren, im Laufe der Mitose sowie bei Kernen nekrotischer Zellen darstellen lassen.Ein rascherer Fluoreszenzumschlag zum Langwelligen bereits nach kurzer Hydrolyse (1 min, n/HCl, 37° oder 60°) war zu beobachten: bei allen nekrotischen Zellkernen, bei den Kernen eines Teiles der untersuchten Tumoren (in 45 von 85 Fällen), innerhalb eines Interphasekernes im Heterochromatin und in allen Fällen an den Mitosechromosomen. Unter Berücksichtigung unserer heutigen Vorstellungen über den Bindungsmechanismus des Akridinorange an die Nukleinsäuren des Zellkernes und über die Veränderungen der DNS-Molekülstruktur nach Säurehydrolyse kommen als morphologisches Substrat für den Fluoreszenzumschlag in Betracht: eine geringere sekundärstrukturelle Festigkeit des DNS-Moleküls, ein geringerer Polymerisationsgrad und ein erhöhter Gehalt an ribonukleaseresistentem DNS-RNS-Komplex.Unabhängig von der noch problematischen Deutung der Befunde ist die angewandte Methode in der Lage, Unterschiede in der Struktur der Nukleinsäuren dieser Zellkerne zur Darstellung zu bringen, die sich bisher einem Nachweis entzogen haben.

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Summary Based on previous papers on the variable demonstration of DNA by means of a combination of acid hydrolysis and conjugation with acridine orange, a study was conducted to investigate if under standardized conditions alcohol fixed nuclei of tumours show differences in their DNA during mitosis and necrosis. A rather sudden change of the fluorescence to the long wave region after short hydrolysis (1 min, n/HCl, 37° or 60°) was observed: in all necrotic cell nuclei; in the nuclei of some of the tumours (45 out of 85); in the heterochromatine of an interphase nucleus, and in all cases of chromosomes in mitosis. Taking into consideration the current knowledge on the linking mechanism of acridine orange to nucleic acids of the cell nucleus, and on changes of the molecular structure of DNA following acid hydrolysis the morphologic substrate responsible for the change of the fluorescence could be: a lesser secondary structural stability of the DNA molecule; a lower degree of polymerisation and a higher level of a ribonuclease resistant DNA-RNA complex.Although it is rather difficult to interprete the findings, it can be said that the method described reveals differences in the structure of certain cell nuclei, which so far have not been demonstrable.

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Einige Ergebnisse dieser Arbeit wurden auf der 4. Arbeitstagung der Arbeitsgemeinschaft Morphologie in der DDR am 2./3. Oktober 1964 in Leipzig vorgetragen und sind Teil einer Habilitationsschrift, die dem Senat der Medizinischen Akademie Carl Gustav Carus, Dresden, vorgelegen hat.     

Quantification of Y chromosome bearing spermatozoa of cattle using in situ hybridization.

An easy assay for quantification of Y chromosome-bearing sperm (Y-sperm) is needed, especially to monitor sperm separation techniques. In the present study a tritiated bovine male-specific DNA fragment was tested for identification of Y-sperm by in situ hybridization. A protocol for in situ hybridization to bovine sperm was developed and used to study the proportion of Y-sperm of 12 bulls. The usefulness of the method in optimization of sperm separation procedures is illustrated through analysis of fractions of sperm separated by Percoll density gradient centrifugation.     

Rat hypothalamic extract inhibits vasopressin-stimulated Na+-K+-ATPase activity in the rat renal medulla

Arginine vasopressin stimulates Na⁺-K⁺-ATPase activity located in the rat thick ascending limb of s'Henle loop. Mammalian hypothalamus appears to produce a factor capable of inhibiting Na⁺-K⁺-ATPase activity in a variety of tissues. The effect of a purified rat hypothalamic extract with and without AVP on rat renal Na⁺-K⁺-ATPase activity was evaluated by a cytochemical technique. The hypothalamic extract alone failed to affect basal Na⁺-K⁺-ATPase activity throughout renal segments after 10 min exposure. Na⁺-K⁺-ATPase activity stimulated by AVP (1–10 fmol l^?1) for 10 min was inhibited by rat hypothalamic extract over the concentration range 10^?7–10^?3 U ml^?1 in a dose-dependent manner. Complete inhibition of AVP-stimulated Na⁺-K⁺-ATPase activity occurred at a hypothalamic extract concentration of 10^?3 U ml^?1. Only Na⁺-K⁺-ATPase activity located in the renal medullary thick ascending limb was influenced by the rat hypothalamic extract.