Relaxed circular, covalently closed simian virus 40 DNA molecules were associated with the four histones that are present in virions. In electron micrographs the resulting complexes appear twisted, with globular structures (nucleosomes) along the DNA. Incubation with an untwisting extract converts the twisted complexes to relaxed structures. Extraction of the DNA from the relaxed complexes yields supercoiled molecules. The number of superhelical turns in these molecules corresponds to the number of nucleosomes per DNA molecule in the complexes.In eukaryotic nuclei, the fundamental structure of chromatin fibers appears to be a flexible chain composed of globular particles connected by DNA filaments (1, 2). In these particles, termed nucleosomes (2), about 200 base pairs of DNA are associated with the four histonies F2a,, F2a2, F2b, and F3 (2-7). Such a repeating unit structure can be formed in vitro by association of the four histonies and linear l)NAs (2).In the nucleosomes the DNA is under constraint, since it is compacted about 5-fold compared to its length in the extended double helical form (2). The nature of this constraint can be studied by the association of histones to covalently closed circular DNA molecules, since supercoiling or unwinding of the DNA within the nucleosome (luring its formation would alter the supercoiling of the rest of the molecule (8). In addition, from the known thermodynamic prolerties of superhelical DNAs (8-10), the influence of the degree of superhelicity on the formation of nucleosome structures can provide information on whether the formation of a nucleosome is equivalent to an unwinding or winding of the double helix. Simian virus 40 (SV40) D)NA is particularly attractive for such a study for two reasons. First, two circular covalently closed allomorphic forms of this DNA are available, the superhelical DNA I and the relaxed circular DNA Ir which results from the incubation of DNA I with an untwisting extract (FE) (l1). This extract is thought to introduce a single-strand nick into superhelical DNA and to reseal the nick after the torsional tension in the double helix has been relieved. Second, SV40 DNA and the four histones are associated in vivo anld the complexes can be isolated from virions (12, 13) and from infected cells. In the latter case, the complex appears as a compactecl structure with about 20 nueleosomes (14).We

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In the liver of oviparous vertebrates vitellogenin gene expression is controlled by estrogen. The nucleotide sequence of the 5' flanking region of the Xenopus laevis vitellogenin genes A1, A2, B1 and B2 has been determined. These sequences have been compared to each other and to the equivalent region of the chicken vitellogenin II and apo-VLDLII genes which are also expressed in the liver in response to estrogen. The homology between the 5' flanking region of the Xenopus genes B1 and B2 is higher than between the corresponding regions of the other closely related genes A1 and A2. Four short blocks of sequence homology which are present at equivalent positions in the vitellogenin genes of both Xenopus laevis and chicken are characterized. A short sequence with two-fold rotational symmetry (GGTCANNNTGACC) was found at similar positions upstream of the five vitellogenin genes and is also present in two copies close to the 5' end of the chicken apo-VLDLII gene. The possible functional significance of this sequence, common to liver estrogen-responsive genes, is discussed.

We investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 10 9 to 10 10 PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-m membrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10 mM magnesium ions, phages showed no loss of titer over 1 month at 30°C.

Electron microscopic evidence indicates that Simian virus 40 (SV40) minichromosomes extracted from infected cells consist of 20 2 nucleosomes, each containing 190-200 base pairs of DNA. About 50% of the nucleosomes are not close together, but connected by segments of DNA of irregular lengths which correspond to about 1 5 x of the viral genome, irrespective of the ionic strength. Micrococcal nuclease digestion studies show that there is about 200 base pairs of DNA in the biochemical unit of SV40 chromatin. Therefore, the visible internucleosomal DNA of the SV40 minichromosome does not arise from an unfolding of a fraction of the 190-200 base pairs of DNA initially wound in the nucleosome. These results support the chromatin model which proposes that the same DNA length is contained in the nucleosome and the biochemical unit. Results from extensive micrococcal nuclease digestion suggest that an SV40 nucleosome consists of a 'core' containing a DNA segment'of about 135 base pairs associated to a DNA fragment more susceptible to nuclease attack. The addition of histone H1 results in a striking condensation of the SV40 minichromosome, which supports the assumption that histone HI is involved in the folding of chromatin fibers.