Scrape loading and sonication loading are two recently described methods of introducing macromolecules into living cells. We have tested the efficacy of these methods for transfection ofmammalian cells with exogenous DNA, using selection systems based either on resistance to the drug G418

A rat liver DNA helix-destabilizing protein (HDP) that has previously been proposed to play a role in transcription has been identified as M chain lactate dehydrogenase (LDH-5; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27). Tryptic peptides accounting for 157 amino acids in the rat liver HDP have been characterized and then matched to the published sequence for the M chain of porcine LDH. Based on amino acid compositions and direct solid-phase protein sequencing, at least 148 of the 157 residues that were compared are identical in both proteins. In addition, both porcine LDH and the rat liver HDP have blocked amino termini and similar amino acid compositions and molecular weights. Rat liver HDP and LDH-5 that were purified to molecular homogeneity had similar specific activities in both single-stranded DNA (ss DNA) binding and LDH assays. HPLC tryptic peptide maps were also identical for both the rat liver HDP and LDH proteins. Since preincubation of HDP in NADH prevents its binding to ss DNA, both NADH and ss DNA may be binding at the same site. Further support for this latter idea derives from chemicalmodification studies which demonstrate that tyrosine-238, which is located near the coenzyme binding site of LDH, seems to be essential for the ability of HDP to bind ss DNA. These results indicate caution in ascribing in vivo roles solely on the basis of binding to ss DNA. Alternatively, they suggest that a single protein may play more than one biological role.

A procedure was developed for isolating nuclei from either the conidial or germinated conidial growth phase of Neurospora crassa. A frozen conidial suspension was lysed by passage through a French pressure cell, and the nuclei were freed from the broken cells by repeated homogenization in an Omni-Mixer. Pure nuclei were obtained from the crude nuclear fraction by density banding in a Ludox gradient. The final nuclear yield was 20 to 30%. The nuclei had a deoxyribonucleic acid (DNA):ribonucleic acid (RNA):protein ratio of 1:3.5:7 and were active in RNA synthesis. The nuclei, stained with the DNA stain 4,6diamidino-2-phenylindole, appeared under fluorescence microscopy as bright blue spheres, 1 ,um in diameter, essentially free from cytoplasmic attachments. Chromatin extracted from the nuclei in a 70 to 75% yield by dissociation with 2 M sodium chloride and 5 M urea had a DNA:RNA:protein ratio of 1:1.05:1.7. Chromatin reconstituted from this preparation exhibited a level of RNA polymerase template activity lower than that of pure Neurospora DNA, but the maximum level of reconstitution obtained was only 10%. Fractionation of Neurospora chromatin on hydroxylapatite separated the histones from the chromatin acidic proteins. The normal complement of histone proteins was present in both the reconstituted and dissociated chromatin preparations. The acidic protein fraction exhibited a variety of bands on sodium dodecyl sulfate gel electrophoresis ranging in molecular weight from 15,000 to 70,000. The gel pattern was much more complex for total dissociated chromatin than for reconstituted chromatin.

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