Fractionation, isolation and characterization of nonhistone chromosomal protein from calf thymus.

1) A method is described for the separation and fractionation of nonhistone chromosomal proteins from salt-urea dissociated calf thymus chromatin. After precipitating DNA in the dissociated chromatin solution with LaCl3, the chromosomal proteins in the supernatant were fractionated by SP-Sephadex C-25 column chromatography using a combination of NaCl stepwise and linear gradient elutions. Much care was taken to prevent proteolytic degradation of the chromosomal proteins during the preparation. 2) Among the protein fractions separated by this chromatography, twenty subfractions were found to be homogeneous on SDS-polyacrylamide gel electrophoresis. These purified proteins account for about 18% of the whole chromosomal protein. Eleven subfractions of these purified nonhistone proteins had ratios of acidic to basic amino acids above 1.0 and the nine remaining subfractions had ratios below 1.0, corresponding to nonhistone proteins of basic character. 3) The molecular weights of the purified nonhistone proteins ranged from 7,400 to 19,000.     

Stabilization of double-stranded DNA molecule by nonhistone peptidic effector from calf thymus

A peptidic effector from calf thymus causes a strong stabilization of DNA doublestranded molecule in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (<5000). The biological activity of the thymic factor cannot be attributed to a histone fragment. Melting data of the control DNA and of the DNA-active factor complex in various conditions of ionic strength and dielectric constant of the solution medium are recorded.     

Conformational changes in subfractions of calf thymus histone H1.

This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.     

Conformational study of calf brain tubulin

The conformation of calf brain tubulin has been monitored by circular dichroism, optical rotatory dispersion, and spectrophotometric titration as a function of pH, temperature, ligand concentrations, and denaturants. At pH 7, calf brain tubulin maintains its structural integrity between 5 and 37 °C as determined by circular dichroism. Furthermore, the presence of MgCl ₂ up to 1.6 × 10^?2m does not induce any observable changes in the circular dichroism spectra, nor does 10^?4m CaCl₂. With increasing pH, the spectral data can best be described as a gradual loosening of the secondary structure between pH 7 and 9. Both spectral and titrimetric data suggest a major unfolding of tubulin between pH 9 and 10. The apparent pK of tyrosine shifts from 10.85 to 9.98 upon transferring from buffer to 6 m guanidine hydrochloride, indicating that at least 14 of the 15 tyrosine groups are not fully accessible to protons in the native protein. The single disulfide bridge in calf brain tubulin helps to maintain a domain which is highly resistant to unfolding by denaturants.     

The quantitative protein composition of calf thymus chromatin.

The proteins of calf thymus chromatin were analysed quantitatively using a combination of polyacrylamide and cellogel electrophoreses. Quantification was achieved by determining the staining values of each purified histone with amido-black. The results indicate that, on average, 700 molecules histone F1, 850 of F2al, 1440 of F2a2, 1 890 of F2b, 2380 of F3 and 500-1000 non-histone molecules are bound per 10(5) base pairs of DNA. This suggests a moderately dense protein covering of the DNA.     

Conformational study of α1-acid glycoprotein

The secondary structure of the protein moiety of the α₁-acid glycoprotein (orosomucoid) was evaluated from its primary structure by using the Lim and Chou and Fasman predictions, and the corresponding dichroic spectrum was calculated. The experimental dichroic spectrum of the whole glycoprotein was compared with the summation of (i) the calculated dichroic spectrum due to the protein moiety and (ii) the experimental dichroic spectrum of the carbohydrate moiety. The results are in good agreement with the fact that the carbohydrate moities do not produce any pertubation of the protein conformation. In addition, we observed that four out of five glycan chains are linked to Asn residues which are situated either in a reverse β-turn or in regions where charged and polar residues are numerous, that is, on the outside of the protein.     

Xeroderma pigmentosum group A correcting protein from calf thymus.

A proteinous factor was purified from calf thymus and HeLa cells, which specifically corrects the excision repair defect of xeroderma pigmentosum complementation group A (XP-A) cells. Recovery of UV-induced unscheduled DNA synthesis after microinjection of XP-A cells was used as a quantitative assay for the correcting activity of protein preparations. XP-A correcting protein appears to be very stable as it withstands heating to 100 degrees C and treatment with SDS or 6 M urea. A molecular weight of 40-45 kD was found both under native (gel filtration) and denaturing (SDS-PAGE) conditions. Calf XP-A protein binds to single-stranded DNA more strongly than to double-stranded DNA, but shows no clear preference for UV-irradiated DNA. Polyclonal antibodies raised against human recombinant XP-A protein, which strongly inhibit UV-induced unscheduled DNA synthesis of normal human cells, completely abolished XP-A correcting activity when mixed with calf thymus preparations. This indicates a close relationship between human gene product and the calf protein. In the final preparation two main protein bands were present. Only one band at approx. 41 kD showed both DNA binding activity in Southwestern blots and immune reaction with human XP-A antibody, suggesting that this is the active calf XP-A correcting factor.     

An auxiliary protein for DNA polymerase-delta from fetal calf thymus

An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.     

Fractionation of calf thymus DNA based on its interaction with homologeous f1 histone. Melting curves of the obtained fractions

Calf thymus DNA fractions were obtained by precipitation with the homologeous f₁ histone and their melting curves were investigated. An increase of the melting temperatures of DNA remaining in the supernatants was observed. Within the range of 5–50 % of the DNA precipitated the melting temperatures and the melting intervals of DNA in the sediment remained constant. The obtained values /38 mole % GC, ΔT_m = 7.0°C/ suggest that DNA found in the precipitates corresponds to the main calf thymus DNA. Despite its heterogeneity this group of DNA molecules does not undergo fractionation using f₁ histone. We assume that the molecules of the main DNA all contain specific areas to which the f₁ histone attaches in our experimental conditions. The main DNA molecules, regardless of their base composition, seem to contain these specific areas in amounts causing equal precipitation probability. They seem to differ in this respect from some GC rich fractions, possibly those of satellite DNA.     

HMG (high-mobility-group)-14/17-like proteins in calf thyroid. Thyrotropin-dependent phosphorylation and comparison with calf thymus proteins.

Two-dimensional polyacrylamide-gel electrophoresis of acid extracts of thyroid and thymus tissue, and of thyroid nuclei, revealed the presence of three HClO4-soluble nuclear proteins, PS.1, PS.2 and PS.3, whose electrophoretic mobilities closely resembled those of HMG (high-mobility-group) proteins 14 and 17. PS.1 co-migrated with HMG 14 on CM-Sephadex column chromatography. Like HMG 14, PS.2 and PS.3 were phosphorylated in calf thyroid slices; 32P-labelling of PS.3 was stimulated by thyrotropin. Thyrotropin also induced a rapid increase in the labelling of A5, an HMG-14/17-like protein found in whole calf thyroid and thymus tissue, but not in thyroid nuclei.     

Partial purification of "omega" protein from calf thymus.

Proteins which relax supercoiled DNA, called "omega" proteins, are thought to be involved in DNA replication. Calf thymus is a plentiful source of "omega" protein activity. It has been extensively purified and has been characterized as behaving similarly to other eukaryotic "omega" proteins in completely relaxing either positively or negatively supercoiled DNA, requiring a salt concentration of about 0.2 M NaCl or KCl, and not requiring Mg2+. A transient nick must occur but could not be detected. A new assay for "omega" activity is described which is rapid and sensitive, and depends on the fluorescence enhancement of ethidium intercalating duplex DNA.