Molecular and cytological characteristics of nuclear DNA and chromatin for angiosperm systematics: DNA diversification in the evolution of four orchids

The species- and genus-specific DNA content, average base composition of nuclear DNA, presence or absence of satellite DNA, the percentage of heterochromatin and other characteristics of nuclear DNA and nuclear structure allow to deduce the molecular changes which accompanied, or more probably caused, cladogenesis in the orchids studied. It is suggested that saltatory replication (generative amplification) of certain DNA sequenes, diversification of reiterated DNA sequences, and loss of DNA play an important role in the evolution of orchids.—The relationship between changes of genome composition and of nuclear structure and ultrastructure is discussed on the basis of cot curves, heterochromatin staining with Giemsa (C banding), electron microscopy of nuclei, and molecular hybridization in situ.Some aspects of this paper have been presented at the Helsinki Chromosome Conference, August 1977 (Nagl & Capesius 1977).     

Some Factors Influencing the in vitro Infectivity and Replication of Encephalitozoon cuniculi*

SYNOPSIS. Rabbit Encephalitozoon cuniculi were propagated in vitro using rabbit choroid plexus (RCP) cells. The organisms reached maximum titer and numbers by 15 days. The source and in vitro passage level of RCP cells moderately influenced the sensitivity of the cells to infection. Cells less than 1 week old were significantly less sensitive than older cells. A moderate increase in infectivity for RCP cells was demonstrated with increasing organism passage level in vitro. Rabbit E. cuniculi were not affected by penicillin-streptomycin or gentamicin in the culture medium. The organism survived more than 9 days in buffer at 37 C and least 24 days at 4 and 20 C. Storage at -70 C or in liquid nitrogen was successful for at least 6 months. Encephalitozoon cuniculi survived 60 but not 120 min at 56 C. They were killed after 10 min of autoclaving and by 2% (v/v) Lysol, 10% (v/v) formalin and 70% (v/v) ethyl alcohol. The organisms survived at least 24 h at pH 9 or pH 4 and were not affected by sonication, freezing and thawing, or distilled water but lost significant infectivity after 24 h in CsCl or 40% (w/v) sucrose.     

Isoelectric focusing following by electrophoresis of proteins for visualizing their titration curves by zymogram and immunofixation

After isoelectric focusing followed by electrophoresis at right angles in the same gel slab, it is possible to visualize the titration curve of proteins by zymograms or immunofixation even of an unpurified sample. This information can be very useful for the selection of the proper purification strategy by charge-dependent methods, e.g. ion-exchange chromatography, zone and disc electrophoresis and isotachophoresis. The titration curve also gives information on the stability of the protein as a function of the prevailing pH of the medium, in the pH 3-10 range. A region of instability is found for most proteins in acidic conditions, below pH 4.5, while most proteins are stable in the alkaline pH region, at least up to pH 10. The best method for developing zymograms and immunoprints appears to pH 10. The best method for developing zymograms and immunoprints appears to be the 'sandwich technique', by which a thin agarose slab, cast on an hydrophilic polyester sheet, and impregnated with appropriate reagents, is left in contact with a polyacrylamide gel thin layer used to generate the titration curves.     

pK determinations via pH-mobility curves obtained by isoelectric focusing-electrophoresis: Theory and experimental verification

Basic equations have been derived linking the electrophoretic migration in a stationary pH gradient of simple, singly charged cations or anions and of mono- mono- valent ampholytes with the pKs of their ionizable groups. In the case of diprotic ampholytes, an equation and a curve are described calculating a correction factor to be applied to the mobility measurements, accounting for the influence of the opposite charge species on the mobility curve of the ion being measured. This correction factor is a function of ΔpK and increases exponentially with decreasing values of ΔpK. These theoretical considerations have been experimentally verified by running pH-mobility curves of colored compounds, such as methyl red, neutral red and dexorubicin. The pKs thus measured were in excellent agreement with the pKs obtained independently by spectrophotometric titrations.     

In vitro construction of a recombinant adenovirus Ad2:Ad5

A hybrid virus containing the left half of the Ad5 genome and the right half of the Ad2 genome has been constructed by ligating together in vitro the BamHI.-A fragment of Ad5 (map co-ordinates 0–59.5) to the-SawHI-A fragment of Ad2 (map coordinates 59.5–100), and using this DNA to transfect susceptible cells. Viable progeny virus has been obtained which grows as well as the parental virus without any requirement for helper virus, and probably contains a hybrid hexon polypeptide consisting of the major part of the Ad5 hexon with an Ad2 carboxy terminus.     

Recombination between satellite phage P4 and its helper P2. I. In vivo and in vitro construction of P4: :P2 hybrid satellite phage

P4::P2 hybrid satellite phages which carry a portion (including the P2 head gene Q and the cohesive end) of the left end of the P2 chromosome linked to the essential part of the P4 chromosome have been isolated by in vivo as well as in vitro recombination. These hybrids express gene Q and grow in the presence of a P2 helper even if defective in gene Q.     

Construction of plasmids that produce phage P22 repressor

In a series of plasmid constructions, the c2 (repressor) gene of phage P22 was cloned in a multicopy plasmid and expressed at increasing level. The final result of these constructions is a plasmid that maintains a level of approx. 200 times as much repressor as is found in a lysogen. A series of increasingly virulent phage mutants was isolated by plating sequentially on host cells with increasing levels of repressor. The methods used in the constructions should be applicable to obtaining elevated expression of cloned genes in other systems.