Primary Chemical Structure Research Articles

Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene

Bacteriophage MS2 RNA is 3,569 nucleotides long. The nucleotide sequence has been established for the third and last gene, which codes for the replicase protein. A secondary structure model has also been proposed. Biological properties, such as ribosome binding and codon interactions can now be discussed on a molecular basis. As the sequences for the other regions of this RNA have been published already, the complete, primary chemical structure of a viral genome has now been established.

The Interaction of Apolipoprotein-Serine with Phosphatidylcholine

Abstract Apolipoprotein-serine (apoLP-Ser) is one of the protein constituents of human plasma very low density lipoproteins (VLDL). The protein has 57 amino acids with 1 residue each of methionine and tryptophan. Cleavage of apoLP-Ser with cyanogen bromide yields the NH2-terminal fragment (CNBr I) and COOH-terminal fragment (CNBr II) having 38 and 19 residues, respectively. CNBr II contains the single residue of tryptophan. We have studied the interaction of egg phosphatidylcholine with apoLP-Ser and its cyanogen bromide fragments. The binding of phospholipid was studied in the following ways: (a) by inhibition of the reactivation of defatted β-hydroxybutyrate dehydrogenase from beef heart mitochondria; (b) by changes in circular dichroic and fluorescence spectra; (c) by isolation of complexes of the protein and peptide fragments with phospholipid, using density gradients of KBr; and (d) by rouleaux formation by phospholipid vesicles as determined by electron microscopy after negative staining with phosphotungstic acid. ApoLP-Ser strongly inhibited the reactivation of defatted β-hydroxybutyrate dehydrogenase; 10 µg of the apoprotein inhibited reactivation by 85%. CNBr I and CNBr II inhibited reactivation to a lesser extent than did the intact apoprotein. The α helical content of apoLP-Ser was about 56%, as determined by circular dichroism. Mixing the apoprotein with bilamellar vesicles of phosphatidylcholine resulted in an estimated increase in the α helical content to about 73%. The apparent helicity of CNBr I was increased from 34 to 65% and of CNBr II from 15 to 25% in the presence of phosphatidylcholine. The addition of phospholipid vesicles to apoLP-Ser produced a blue shift (6.0 nm) in the fluorescence maximum, indicating a shift of the tryptophan to a more hydrophobic environment. A similar finding was made with CNBr II in the presence of phospholipid. In ultracentrifugal flotation experiments, complexes of the phospholipid with apoLP-Ser, CNBr I, and CNBr II, each floated at a density less than 1.08 g per ml, whereas apoLP-Ser in the absence of phospholipid sedimented to a density greater than 1.18 g per ml. Finally, when the phosphatidylcholine vesicles were titrated with increasing amounts of the protein or cyanogen bromide fragments and negatively stained with phosphotungstic acid, the vesicles exhibited a series of morphological changes from free vesicles to linear arrays of stacked discs as determined by electron microscopy. These findings are interpreted in light of a theory of phospholipid-binding involving amphipathic helical regions that contain positively and negatively charged amino acids and are consistent with the localization of such regions in the primary chemical structure of the molecule.

The role of antigenic conformation in antigen-antibody complex formation

Iron binding imparts a conformational rigidity and compactness to the ovotransferrin molecule. This appears to cause the inaccessibility of certain antigenic sites to antibody in the formation of antigen-antibody complexes. Iron ovotranseferrin had a lower apparent antigenic valence and formed preferably smaller soluble antigen-antibody complexes that ovotransferrin, as measured by fluorescence polarization, saturated ammonium sulfate precipitation, and sedimentation velocity ultracentrifugation experiments. In primary Ab binding tests and in quantitative precipitin tests, however, both antigens gave similar reactivity. These results and the known identity of the primary chemical structure of ovotransferrin and iron ovotransferrin indicated that conformation differences are responsible for the observed immunochemical behavior. Molecular models, based on hydrodynamic measurements on ovotransferrin, iron ovotransferrin, and antibody molecules, also support this view. The antigenic differences between ovotransferrin and iron ovotransferrin were manifested only in the secondary phase of the antigen-antibody complex formation, emphasizing the need for appropriate secondary phase immunochemical techniques in such studies.

Studies of a growth factor: Fractionation studies and amino acid composition of derived fractions

A protein growth factor for the nematode, Caenorhabditis briggsae, has been shown, by means of electrophoretic and chromatographic separations, to exist in multiple active forms. The amino acid composition of nine of these active fractions has been shown to be essentially identical, although they were of differing specific activities. It is proposed that these active forms have basically the same primary chemical structure and that the major differences between them is their molecular size. Biological specific activity appears to increase as molecular size increases. Maximal biological activity of most of these fractions could be demonstrated only after the protein was subjected to an activation process. Activation is believed to result from a reversible conformational change within the protein molecule.

Allelomorphism and the chemical differences of the human haemoglobins A, S and C.

IN previous communications on this subject, one of us1,2 reported that normal human haemoglobin (A) and sickle-cell haemoglobin (S) differ in only one out of nearly 300 amino-acids in the half-molecule, and that this is the only demonstrable difference in primary chemical structure. We now wish to report on a similar investigation into the structure of another abnormal human haemoglobin—that of haemoglobin C disease.