Chemical-enzymatic synthesis, cloning and expression of a synthetic gene coding for an env protein fragment of the human T-cell leukemia virus type 1.

A synthetic gene for a 88 amino acid long env protein fragment of the human T-cell leukemia virus type 1 (HTLV1) has been assembled by ligation of 35 oligodesoxyribonucleotides, which were chemically synthesized by the phosphotriester segmental support method. After cloning into the pEX vector this HTLV1 env-protein fragment was expressed in E. coli.

Temperature behaviour of human serum albumin.

Structural alterations of albumin, their dependence on concentration and the role of free --SH groups at thermal denaturation, as well as the reversibility of thermally induced structural changes, were studied. Application of various physical methods provides information on a series of structural parameters in a major concentration range. Apart from changes of the helix content, heat treatment gives rise to beta structures which are amplified on cooling and which are correlated with the aggregation of albumin. With rising temperature and concentration the proportion of beta structures and aggregates increases. At degrees of denaturation of up to 20% complete renaturation is possibly in every case. The structure content is concentration-dependent even at room temperature. It may be that intermolecular interactions induce additional alpha-helix structures which are less stable, however, than the ones stabilized by intramolecular interactions. Unfolding of the pocket containing the free --SH group of cysteine-34 enables disulphide bridges to be formed leading to stable aggregates and irreversible structural alterations. Through binding of N-ethymaleimide to free --SH groups, which blocks the formation of disulphide bridges, it is possible to prevent aggregation and irreversible conformational changes. At temperatures below 65--70 degrees C, oligomers are formed mainly via intermolecular beta structures.

Isolation and characterization of salt-soluble, unsheared chromatin.

From rat liver nuclei depending on the extraction time, 10 to 20% of total chromatin has been extracted with a solution containing 0.1 M ammonium sulfate, 2 mM MnCl2, 0.1 M Tris-HCl, pH 7.9. We term this chromatin chromatin S. It has a protein: DNA ratio of 1.3, the full amount of the 5 histones in an undegraded state, and a RNA: DNA ratio of approximately 0.2. Its nonhistone protein pattern, obtained by gel electrophoresis exhibits a rich spectrum of proteins in a broad range of molecular weights. Electrophoretic analysis of the DNA fragments obtained by micrococcus nuclease digestion of chromatin S yields the same digestion pattern as that of nuclei. Thus, chromatin S fulfils an essential criterion of unsheared chromatin. In contrast to other chromatin preparations described so far, this chromatin is soluble at a salt concentration of 0.1 M ammonium sulfate. We have shown previously that it exhibits a compact conformation, low intrinsic viscosity and low radius of gyration obtained by light scattering measurements. Its mean molecular weight was determined to be nearly 10(8).

Characterization of the phenol soluble and insoluble nonhistone proteins of in vivo (32)P-labelled rat liver nuclei by high-resolution gel electrophoresis.

The (32)P-labelling patterns of phenol soluble and insoluble nonhistone proteins of in vivo labelled rat liver nuclei freed of the soluble nuclear proteins have been determined after separation by high-resolution gel electrophoresis. The bulk of the proteins of the nuclear residues was phenol soluble. Seven percent of the proteins of the nuclear residues was obtained with the aqueous phase. As shown in this paper both fractions contain (32)P-labelled proteins but they represent different types of nonhistone proteins.

Infrared spectral studies of carbon monoxide complexes of microsomal cytochromes P-450 and P-448.

The characteristic infrared spectral parameters of the carbonyl stretching vibration (the maximum position nu CO, the band width at half height delta nu 1/2 and the apparent molar extinction coefficient epsilon M) have been determined for the CO complexes of microsomal cytochromes P-450 (PB-induced) and P-448 (3-MC-induced) from rabbits and rats. The cytochromes P-450 and P-448 from the same species as well as the cytochromes prepared by the same inducer but from the two different species yielded nu CO band parameters different from each other and from the bacterial cytochrome P-450cam [1]. These differences are discussed in connection with (1) the presence of different protein moities (multiple forms) of cytochromes, (2) changes in the order of the nearest neighbour environment (accessibility) of the CO binding site and (3) the presence of (endogenous bound substrate. The heme-carbonyl of the microsomal cytochromes senses subtle changes in the nearest heme environment by changing the solvent from H2O to D2O.