Studies on deoxyribonucleoproteins. IX. Subfractions of deoxyribonucleoproteins from rat and mouse liver.

By carrier-free continuous electrophoresis, deoxyribonucleoprotein from rat and mouse liver could be separated into two subfractions. The more anodic fraction (DNP I), comprising 5 - 8 per cent of the total, contains fewer proteins (two types of histones only). [3H]Cyclophosphamide caused in vivo a 2.5 times higher alkylation of the DNA in in DNP I than of the DNA in DNP II. These and additional results led to the suggestion of a structural model with DNP I as a spacer in the deoxyribonucleoprotein fiber.     

Fractionation of chromosomal deoxyribonucleoproteins by partition in two-phase aqueous polymer systems

Deoxyribonucleoprotein (DNP)¹ prepared by shearing chromatin of mouse cells may be fractionated in 2-phase aqueous Dextran-polyethyleneglycol mixtures. A partial separation of DNPs with different non-histone protein/DNA ratios may be obtained in a single-step partition. Separation of a spectrum of fractions of DNP has been obtained by countercurrent distribution using the same 2-phase polymer system. DNP fractions which bear nascent RNA (representing approximately $\text{1}\text{3}$ of the total DNA) may be separated from the major fraction of DNP; they are found in the same region of the distribution pattern as DNP fractions with the highest non-histone protein/DNA ratio.     

Fractionation of liver proteins by preparative electrophoresis

Summary. Proteomics offers unique possibilities to investigate changes in the levels and modifications of proteins involved in the pathomechanisms of diseases and toxic events. However, search for potential drug targets and disease or toxicity markers is limited by the fact that mainly the high-abundance, hydrophilic proteins are visualized in two-dimensional gels. Here we studied the enrichment of rat liver cytosolic proteins by preparative electrophoresis. Preparative electrophoresis was performed with the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Lithium dodecyl sulfate was exchanged against agents compatible with isoelectric focusing prior to the two-dimensional gel electrophoresis. Proteins were identified from two-dimensional gels by matrix-assisted laser desorption ionization time-of-flight mass specrometry. Low- and middle-size proteins and low-abundance proteins, which had not been found before, were enriched by preparative electrophoresis. The present study represents a contribution of proteomics in the quantification of differences in the levels of low-abundance liver proteins in toxicity studies.     

Fractionation and characterization of DNA at sites of replication from rat liver nuclei

Regenerating rat liver nuclei when sonicated and centrifuged in a Cs2SO4 gradient were fractionated into three distinct bands. These bands were designated as light band (LB), middle band (MB), and heavy band (HB) according to their density. LB and MB were shown to consist of large granular particles with varying electron densities, but LB also contained remnants of nuclear membrane. When analysed by gel electrophoresis, LB and MB displayed more than 35 bands of proteins. The third fraction, HB, consisted largely of small chromatin fibers and its proteins were predominantly the four histones of the nucleosomal core particle. Following short pulses with [3H]thymidine in vivo, the specific activity of DNA in LB and MB was significantly higher than that of bulk DNA contained in HB. DNA in all three fractions became equally labelled as the duration of the labelling interval increased beyond 30 min. Newly synthesized DNA was characterized by electrophoresis on analytical 1.7% acrylamide -0.5% agarose composite gels. After a 1-min labelling interval in vivo, 17% of the rapidly labelled DNA from LB and MB was stationary at the gel origin like replication forks from E. coli, but only 3% of HB DNA had zero mobility. Electron microscopy confirmed the presence of DNA replication forks in LB, MB, and HB. With increasing time of synthesis the proportion of labelled DNA exhibiting zero mobility decreased in all three fractions. Denaturation of DNA or digestion of single-stranded DNA with S1 nuclease released newly synthesized DNA from the gel origin. Ribonuclease was without effect. DNA recovered from LB and MB also had a higher molecular weight than the HB DNA. Together these results indicate (1) that LB and MB are enriched in newly replicated DNA; (2) that an increased proportion of newly replicated DNA in LB and MB is associated with DNA replication forks; and (3) that the replicating DNA recovered in LB and MB may be associated with other nuclear constituents in situ because this DNA appears to be protected from the more frequent chain breaks introduced into the bulk of chromatin (HB) by sonication.     

Fractionation of the nucleotides of the acid-soluble liver fraction of rats

A method for separation of rat liver acid-soluble nucleotides was developed including ion exchange polyethyleneimine cellulose chromatography, followed by rechromatography of the separate fractions on Dowex 1 and Aminex MS resins. It is simple, reproducible and does not require expensive reagents and devices. The sensitivity of the method in respect to orotate is 100 pM in a sample. Data on the content of liver basic 5'-ribonucleotides and their derivatives were obtained by the proposed method.