Mitsuo Zama

Specifie codon usage pattern and its implications on the secondary structure of silk fibroin mRNA

We have identified two distinctive regions of the repetitive unit nucleotide sequence of fibroin mRNA of Bombyx mori. The codon usage for the major amino acids, glycine, alanine and serine is distinctly different in these two regions, indicating that it is determined by the fibroin mRNA or gene structure but not by the tRNA population. Comparative computer analyses of nucleotide substitutions in the unit sequence suggest that selection has operated on the codon usage to optimize the secondary structure characteristic of the fibroin mRNA.

Quantitative determination of single-stranded sections in DNA using the fluorescent probe acridine orange

Abstract A method for the quantitative determination of single-stranded sections in DNA was developed. Fractions of denatured sections can be estimated using a suitable calibration curve which is obtained on the basis that acridine orange bound to denatured DNA emits a weaker fluorescence at 530 nm and a stronger one at 640 nm than does the dye bound to native DNA. The most suitable conditions, where dye is bound to both types of DNA with the same efficiency, were determined to be an ionic strength of 0.01 and a DNA phosphate to bound dye ratio of 7.5. Under these conditions, the fluorescence intensity at 530 nm of the dye added to the mixture of native and heat-denatured DNA decreased linearly as the fraction of denatured DNA increased. In relation to making a calibration curve, the conformation of heat-denatured calf thymus DNA at an ionic strength of 0.01 was studied. When heated DNA was chilled, only a few percent of total nucleotides reformed the double helical structure in spite of the considerable decrease in hyperchromicity.

The nature of strong binding between acridine orange and deoxyribonucleic acid as revealed by equilibrium dialysis and thermal renaturation.

Abstract The strong binding of acridine orange with DNA was studied by means of equilibrium dialysis and thermal renaturation of the complex. Thermodynamic parameters for the strong binding with native and denatured DNA were calculated from the data of equilibrium dialysis. The association constant and the change of free energy were nearly equal for both types of DNA, but the changes of enthalpy and entropy were quite different. Binding to both types of DNA may be strong binding in nature, but the accompanying effects on the secondary structures of DNA would be different. It was found that the presence of acridine orange inhibits the completion of renaturation, presumably due to a stabilizing effect of the bound dye on the “wrong” base pairs as they are formed. The results support the notion that binding of acridine orange with the double helix is an intercalation into adjacent base pairs, while the binding with single strand is a partial intercalation into adjacent bases.

Difference between polylysine and polyarginine in changing DNA structure upon complex formation

Abstract Soluble complexes of DNA with poly-L-lysine and poly-L-arginine were prepared by dialysis or mixing method, and their circular dichroism spectra and viscosities were measured. Both complexes formed by dialysis method had different circular dichroism spectra from that of free DNA respectively, i.e., the spectrum of the DNA-poly-L-lysine complex was similar to that of DNA at high salt concentration and the spectrum of the DNA-poly-L-arginine complex resembled that of DNA under melting at elevated temperature. The viscosity of the both complexes decreased pronouncedly with the increase in peptide to DNA ratio, although the viscosity of the DNA-poly-L-lysine complex was more rapidly decreased than that of the DNA-poly-L-arginine complex. The difference between the two polypeptides in changing DNA structure was observed also in the complexes formed by mixing method.

Isolation of the giant ramified nuclei of the posterior silk glands of Bombyx mori

Abstract The posterior silk glands of Bombyx mori are highly differentiated to produce fibroin, the specific cocoon protein. We have now succeeded in isolating the nuclei from the posterior silk gland cells, and a giant arborescent nucleus with a strikingly complicated morphology was observed. Biochemical analysis of the composition of the nuclei confirmed the existence of the four core histones in equimolar quantities and H1 histone. The isolated nuclei obtained here might be prerequisite for the study of the structure of the giant polyploid nucleus and its relation to the tissue-specific fibroin gene expression.

Structure and circular dichroism of DNA—Polylysine—Polyarginine complex

Abstract Distinct differences were found between DNA—polylysine and DNA—polyarginine complexes formed under identical conditions of salt-gradient dialysis in the change of the CD spectrum as a function of salt concentrations. Based on the findings, the CD and the induced optical activities of the bound dye of the DNA—polylysine—polyarginine complex, formed by the step-gradient dialysis procedure from 5 M urea—2 M NaCl, were investigated in order to evaluate the relative contributions of polylysine and polyarginine to the structural changes of DNA. It was shown that the two polypeptides do not act additively in changing the CD spectrum of DNA in the DNA—polylysine—polyarginine complex, but a small amount of polyarginine is effectively able to block large spectral changes induced by polylysine throughout the NaCl concentration range examined (1–0.01 M). It appears that the most likely effect of polyarginine binding on DNA—polylysine complex is to break down a regularity of the aggregated structure characterized by its large and anomalous CD. Acridine orange, bound to the peptide-free portions in the DNA—polylysine—polyarginine complex, exhibited characteristic extrinsic Cotton effects differing from either of the DNA—polylysine—dye or the DNA—polyarginine—dye system.

Quantitative study of dye binding to DNA-polylysine and DNA-polyarginine complexes

Abstract Soluble complexes of poly-L-lysine and poly-L-arginine with DNA were prepared by a salt gradient dialysis and were investigated on stoichiometric aspect using a dye binding technique. It was found that only partial cationic residues on polyarginine are effective for occupying DNA phosphate groups, while all residues in polylysine can occupy.

The study of the DNA structure in DNA-polylysine and DNA-polyarginine complexes: Induced optical activities of bound dyes

Abstract Induced optical activities of acridine orange and actinomycin D bound to DNA-polylysine and DNA-polyarginine complexes were measured in order to obtain some information about the DNA conformation in the complexes. In the case of acridine orange, the anomalous circular dichroism (CD) spectra obtained at high molar ratios of the dye to DNA phosphate in the visible and ultraviolet regions, differed greatly from the spectrum of the dye bound to free DNA. The ultraviolet CD spectra of actinomycin D bound to the complexes also differed markedly from that of actinomycin D bound to free DNA. The CD spectra of DNA-polylysine-dye and DNA-polyarginine-dye systems were qualitatively different from each other for both dyes. From these findings, the possibility of specific conformational alterations of the free DNA portions in each DNA-polypeptide complex with the two basic polypeptides were suggested.

Codon usage pattern in α2(I) chain domain of chicken type I collagen and its implications for the secondary structure of the mRNA and the synthesis pauses of the collagen

A stability map of local secondary structure of the mRNA of the triple-helical alpha 2(I) chain domain of chicken type I collagen was obtained by plotting the free energy of the optimal secondary structure of a local segment in mRNA against the segment position along a base sequence of the mRNA. It was found that the positions of the minima of free energy in the plot coincide with the positions where synthesis pauses of the alpha-chain polypeptides of the corresponding sizes translated from the mRNA have been reported to occur (1). The codon usage pattern of each of the three major amino acids of the alpha-chain domain of the collagen, Gly, Pro and Ala, fluctuates considerably along the base sequence segments of the mRNA and a deviation of the pattern from that of the average of the whole alpha 2(I) chain domain mRNA, particularly for Gly codons, leads to a loss of the stability of the local secondary structure of the mRNA. The results suggest that selection has operated on the codon usage to optimize the secondary structure characteristic of the mRNA of the chicken collagen alpha 2(I) chain domain which leads to a nonuniform polypeptide elongation pattern.

Involvement of the histidine residues in the pH-induced conformational change of histone H5.

Comparative studies on the conformational stability of histones H1 and H5 have been carried out by monitoring the pH-induced conformational transitions of the proteins by CD and 1H NMR spectroscopies. The transition point of H1 agrees with the pKa of the carboxyl groups of the acidic residues. In contrast, the transition of H5 is associated with the ionization of the histidine residues which exist exclusively in H5, as well as the deionization of the acidic residues. These observations, combined with the result of the deuterium exchange rates of the histidine C-2 protons, led us to conclude that His-25 and His-62, which are buried in the globular domain, play an important role in the conformational stability of histone H5.