Identification and characterisation of PmaCI an endonuclease of novel specificity from Pseudomonas maltophila.

We report the use of MonoQ FPLC (Fast Protein Liquid Chromatography) for the rapid purification of a novel Type II restriction endonuclease PmaCI, from Pseudomonas maltophila, which recognises the sequence 5'-CAC decreases GTG-3'. The resulting enzyme is free of other nucleases to a level suitable for its characterisation by multiple-substrate digestion and DNA sequencing techniques. This method appears to be widely applicable and we have used it for the isolation of restriction endonucleases of comparable purity from a range of other organisms. Also described is a rapid method for screening a library of small inserted regions in recombinant M13 molecules for the presence and subsequent screening of restriction sites of interest.     

A Bacillus subtilis plasmid that can be packaged as single-stranded DNA in Escherichia coli: use for oligodeoxynucleotide-directed mutagenesis

A Bacillus subtilis/Escherichia coli shuttle vector was modified to contain the origin of DNA replication of the E. coli filamentous phage f1, in both orientations. Upon superinfection with and f1-related phage of an E. coli strain containing either of the modified vectors, the single-stranded (ss) form of the plasmid was packaged in virions and released to the culture medium. Each of these ss DNAs has been purified from the virions and used as a template for oligodeoxynucleotide-directed mutagenesis. The resulting mutations were demonstrated by DNA sequencing. The capacity of these vectors to be isolated as phage ss DNA from E. coli and to replicate as plasmids in B. subtilis makes them convenient substrates for the production of oligodeoxynucleotide-directed mutations for studies in B. subtilis.     

Stability of tissue culture medium pH as a function of autoclaving,time, and cultured plant material

Autoclaving is a standard procedure for sterilizing nutrient media for plant tissue cultures. Most tissue cultures are grown at pH 5.2 to 5.8 with pH adjustments being made prior to autoclaving. This paper reports that there are significant differences between initial pH levels and pH levels following autoclaving, particularly in the pH range of 5.7 to 8.5. This effect is noted with and without agar. In addition, we report that with time the pH of the medium drifts into the acid range. When Cucumis callus was added to the medium, the pH was changed significantly within 48 hours. The amount and direction (increase or decrease of pH) was significantly correlated with the original pH. This suggests that researchers should be wary of the true pH situation in their medium. In addition, in publications authors should specify whether their medium pH value was determined before or after autoclaving.     

Efficient extraction of RNA from mammalian tissue

RNA extraction from mammalian tissue has been compared using the different deproteinizing agents: a) guanidine-HCl, b) guanidinium-thiocyanate, c) buffer-saturated phenol, or d) buffer-saturated phenol followed by a proteinase K digestion of the aqueous phase. Both solid tissues (first, second, and third trimester fetal bovine pancreas), and human white blood cell populations were studied. Degradation, as seen in citric acid-urea agarose gels, and the ability to serve as templates for cell-free protein synthesis were used as criteria to assess the efficiency of the different methods. We conclude that employing buffer-saturated phenol with proteinase K digestion is a superior method for consistent extraction of relatively undegraded RNA in quantitative amounts from mammalian tissue.     

The identification and quantitative analysis of abscisic acid in plant extracts by gas-liquid chromatography

Summary New techniques are described which permit the quantitative analysis of microgram quantities of abscisic acid in plant extracts by gas chromatography. Presumptive methyl abscisate peaks on gas chromatograms are positively identified by photosensitised isomerisation to methyl 2-trans-abscisate. Losses of abscisic acid during pre-purification are corrected by using 2-trans-abscisic acid as an internal standard.     

The Behaviour of Dasyuroides byrnei (Marsupialia) in Captivity

Das Verhalten des Beuteltiers Dasyuroides byrnei in Gefangenschaft wurde untersucht. Dasyuroides ist nachtaktiv und verbirgt sich tagsüber in einem Bau oder Schlupfwinkel. Körperhaltung, Fortbewegungsarten, Höhlengraben, Beutefang und andere Verhaltensweisen sind beschrieben. Das Duftmarkieren hat Mitteilungswert. Beide Geschlechter setzen Duftstoff ab, indem sie ihre Sternaldrüsen und Kloakengegend an Gegenständen reiben. Freundschaftliche, kämpferische, sexuelle und mütterliche Verhaltensweisen werden beschrieben. Dasyuroides scheint kein geselliges Tier zu sein; Angriffe gegen Artgenossen führen selten zu Verletzungen, weil das Angriffsverhalten stark stilisiert ist. Wachstum und Entwicklung der Jungen wurden verfolgt. Die durchschnittliche Wurfgröße beträgt fünf oder sechs; die Jungen bleiben an den Zitzen des ♀ bis zum 56. Lebenstag. Mit 100 Tagen können sie entwöhnt werden. Das Verhalten dieser Art wird mit anderen Arten der Familie Dasyuridae verglichen, auch mit Plazentaliern.     

Deoxyribonucleic Acid of Adeno-associated Satellite Virus

Staining patterns suggest that in the adeno-associated satellite virion there exist quasi single-stranded regions which are renatured after extraction to exhibit double strandedness.