Manfred Renz

An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels

A procedure for quick and simple elution of DNA from agarose gels is presented. After electrophoresis, bands of interest are cut out of the gel and the slices are equilibrated in a neutral salt buffer. The slices are then frozen and centrifuged through a filtration assembly whereby the DNA-containing buffer is squeezed out. The method is simple, quick, and suitable for the safe handling of small amounts of DNA (less than 1 microgram). The isolated DNA is susceptible to any enzymatic reaction and also to chemical sequencing. The method is most useful for rapid preparation of specifically end-labeled DNA fragments (e.g., for sequencing), but may also be utilized for any other preparative applications.

The chromosome fiber: evidence for an ordered superstructure of nucleosomes.

Chromosome fibers isolated from lymphocyte nuclei and prepared for electron microscopy by techniques designed to preserve their native structure have a distinctly knobby appearance, suggesting that DNA and protein are not distributed evenly along the fiber axis. Individual knobs (superbeads) are arranged in tandem and have an average diameter of about 200 Å. Mild nuclease digestion of isolated nuclei releases apparent monomer superbeads that are composed of nucleohistone particles with the properties of nucleosomes. The kinetics of digestion indicate that the superbead is a discrete structural unit containing, on the average, about eight nucleosomes.

Chromatin association and DNA binding properties of the c-fos proto-oncogene product

As a first step in the analysis of the molecular function of the nuclear c-fos proto-oncogene product we have studied its subnuclear localization in serum-stimulated mouse fibroblasts where it forms a non-covalent, apparently monodisperse complex with another nuclear protein, p39. The c-fos/p39 complex is almost quantitatively released from intact nuclei by DNasel or micrococcus nuclease treatment under conditions where only a minor fraction of DNA and nuclear proteins is released. In gel filtration experiments, c-fos/p39 comigrates with chromatin and seems to be associated with regions of increased DNasel accessibility. c-fos/p39 is bound to chromatin by electrostatic forces of moderate strength since greater than 90% of the complex can be eluted from nuclei at 0.4 M NaCl. In vitro, the c-fos/p39 complex in nuclear extracts binds to double- and single-stranded calf thymus DNA, suggesting that the association of c-fos/p39 with chromatin is at least in part due to its interaction with DNA. In agreement with this conclusion, c-fos/p39 is released from nuclei by incubation with tRNA, presumably due to competition for binding sites. Our observations are compatible with the hypothesis that c-fos may play a role in the regulation of gene expression.

Regulation of c-fos transcription in mouse fibroblasts: identification of DNase I-hypersensitive sites and regulatory upstream sequences.

In quiescent mouse fibroblasts, the c‐fos gene is expressed at very low levels, but is rapidly and transiently inducible by peptide growth factors. In this study, we have identified in quiescent cells five DNase I‐hypersensitive sites located ‐1700, ‐290, +10, +240 and +700 bp relative to the 5′ cap site. After serum stimulation, the distinct nuclease hypersensitive site at position +10 rapidly disappeared, and instead a broad region of DNase I accessibility between positions 0 and +250 occurred. Nucleotide sequence analysis of the 5′‐flanking region of the mouse c‐fos gene showed that the hypersensitive site around position ‐290 is located in a region that is highly conserved between mouse and human, and that contains an enhancer‐like structure. When the mouse c‐fos promoter and 351 bp of 5′‐flanking sequences were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into NIH3T3 cells efficient, constitutive expression of CAT activity was observed, even in unstimulated, quiescent cells. However, removal of a 256‐bp stretch upstream from position ‐95 completely abolished CAT expression, indicating that sequences within a region of approximately 350 bp upstream from the cap site are indispensable for c‐fos transcription. In addition, our findings point to the existence of other sequence elements that exert negative regulation in the absence of growth factor stimulation. Such sites may be found around the growth factor‐responsive nuclease hypersensitive sites in the vicinity of the cap site.

Simple DNA sequences of Drosophila virilis isolated by screening with RNA

We isolated clones from different types of genomic Drosophila virilis libraries by screening with mRNA labelled in vitro. The DNA regions of the clones showing cross-hybridization with the RNA were sequenced. All of them contained different types of simple sequences, which most probably were solely responsible for the hybridization. We show that simple sequences and their transcription are not a phenomenon that is restricted to Drosophila. Simple sequences can be detected in all eucaryotes and at least three types (poly[d(G--T)] X poly[d(C--A)], poly[d(G--A)] X poly-[d(C--T)] and poly(dA) X poly(dT] are also widely transcribed.

Native and reconstituted chromosome fiber fragments.

The structure of hen erythrocyte chromatin fibers was studied with the electron microscope. Chromatin fiber fragments with a length of about 5,000 Å and an average diameter of 320 Å are composed of 13 globular subunits (superbeads) which contain different numbers of nucleosomes. Their number average corresponds to 17 nucleosomes. — The interaction of lysine-rich histones with nucleosome chains was investigated by reconstitution experiments and was found to be semi-cooperative.

Study on the somatic pairing of polytene chromosomes

Plasmids containing Drosophila virilis DNA (pDv118, pDv719, pDv714 and pDv117), characterized and localized on D. virilis chromosomes in Riede et al. (1983) were localized by in situ hybridization with polytene chromosomes of the hybrids D. virilis × D. lummei, D. virilis × D. novamexicana, and D. virilis × D. lacicola. The degree of somatic pairing was determined by comparing the four plasmids in the three hybrids. We found that somatic pairing in the polytene chromosomes decreased with decreasing DNA homology of the bands. — Additional cytological studies indicated that (1) each band can pair independently of its neighboring bands, (2) visible structural differences between bands have no influence on pairing of the surrounding chromosome region, (3) heteromorphic bands can pair by themselves, and (4) inversions of chromosome regions disturb the somatic pairing process but are not the primary cause for nonpairing in hybrids.

DNA sequence divergence in the Drosophila virilis group

DNA sequence divergence was analyzed in some sibling species of the Drosophila virilis group. Clones comprising about 0.1% of the genome DNA were selected at random from a D. virilis library for a comparative study on DNA from D. lummei, D. novamexicana, D. borealis, and D. lacicola. Blot hybridization experiments indicated that about 70% of DNA from D. lummei and D. novamexicana and less than 50% of DNA from D. borealis and D. lacicola share sequences that are homologous to DNA in D. virilis. This finding is in excellent agreement with the genealogical tree based on cytological studies (Throckmorton 1982). — Four plasmids with inserts which are present in one or a few copies per genome were hybridized in situ to polytene chromosomes. These experiments demonstrate that (1) homologous “unique” DNA sequences are localized exclusively in homologous bands and (2) homologous bands that appear to be identical in different species may contain different DNA sequences.

Affinity chromatography of chromatin on single-stranded DNA-agarose columns.

Abstract Passing chromatin fragments of rat liver nuclei through DNA-agarose columns results in the removal of all histones of the H1 class and almost all (≥95%) nonhistone proteins from the chromatin and thus leads to the separation of DNA molecules containing nucleosomal histones only. Elution of the porteins bound to DNA-agarose by salt gradients leads to a fractionation of chromosomal proteins indicating that they bind with different affinities to single-stranded DNA. This simple and fast procedure is suitable for both the isolation of histone H1-depleted chromatin and the fractionation of nonhistone proteins.