Native rat liver NHC protein-DNA interactions have been investigated by use of a nitrocellulose filter assay sensitive in detection of protein-DNA complexes. Optimal conditions for DNA-protein interactions occurs at low ionic strength conditions (110 mM phosphate buffer). A fraction of NHC proteins was enriched 25-fold by their affinity for rat DNA immobilized on cellulose columns under these conditions. At higher ionic strength (260 mM-0.04M phosphate buffer and 0.15 M sodium chloride), this fraction binds approximately sevenfold less to rat DNA but with a substantial increase in stability of the complexes. Equilibrium competition experiments indicate that at the higher ionic strength there is a considerable DNA sequence specificity of the rat DNA binding NHC protein. Since rat DNA contains three components as defined by their reassociation kinetics: single copy DNA (C0t1/2pure = 1.6 times 103); middle repetitive DNA (C0t1?1PURE = 1.1); and highly repetitive (C0t1/2pure smaller than 0.02). The two former were isolated and employed in the DNA binding assays. At the high ionic strength criterion, the rat DNA binding NHC proteins showed a substantial preference for a subset of middle repetitive DNA sequences. This suggests a preferential interaction between a class of NHC proteins and a class of middle repetitive DNA sequences.

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We describe a mild method for the separation and fractionation of nonhistone chromosomal proteins (NHC proteins) on a preparative scale. Rat liver chromatin is dissociated in 3 M NaCl-10 mM Tris-HCl (pH 8), and is resolved into DNA and protein by gel filtration on Bio-Gel A-50m. The resulting mixture of chromosomal proteins is concentrated by ammonium sulfate precipitation and histones are then removed by cation exchange chromatography on Bio-Rex T J. he histone components of chromatin are now relatively well understood, separated, and even to a considerable degree sequenced (Elgin et al., 1971;Wilhelm et al., 1971). It remains of interest to characterize the nonhistone protein components of chromatin in a similar way. Many approaches to the study of this class of proteins have already been reported (Wang,

Cloned DNA containing the first nine exons of the rat albumin gene was digested with EcoRI and HindIII, and the resulting fragments were used to screen for regions with relatively high affinity for protein. Of three restriction fragments preferentially bound, the fragment containing the first two exons of the albumin gene was consistently bound over others by heat-stable protein extracted from liver nuclei with 0.35-1.0 M NaCl. Proteins extracted with lower and higher ionic strength buffers bound the DNA fragments, but with little specificity. The DNA fragment that was preferentially bound consistently by the 1.0 M nuclear extract was subcloned into pBR325 and was used to isolate the specific DNA-binding activity. After purification, histone H1 was the polypeptide with preferential DNA-binding activity. Histone H1 has a high-affinity binding site in the 5' end of the rat albumin gene within 440 5'-flanking base pairs and the first two exons of the gene.

The sensitivity and application of the polymerase chain reaction (PCR) for the diagnosis of parvovirus B19 (B19) infection was investigated by simultaneously assaying a collection of 279 consecutively received samples for presence of anti-B19 IgM and IgG antibodies by Western blot and for B19 DNA by PCR and dot-blot hybridization (dot-blot); samples were sera from patients with suspected B19 infection. PCR and dot-blot detected B19 DNA in 9% (16/179) and 1% (2/179), respectively of Ab-positive samples (IgM+/IgG-, IgM+IgG+, IgM-IgG+), and in 28% (15/54) and 2% (1/54), respectively, of IgM+ samples. PCR also detected B19 DNA in 2% (2/100) of IgM-/IgG- samples, both of which had normal total IgG and IgM levels. PCR is of unique value because it permits diagnosis of B19 infection even in the absence of specific acute phase (IgM) and in the presence or absence of convalescent-phase (IgG) Ab.