In Paramecium caudatum, whether synkaryon division products will become germinal micronuclei or somatic macronuclei is associated with their brief anteroposterior localization in the cell at the end of the third synkaryon division. The four posterior nuclei all differentiate into macronuclear anlagen. Only one of the anterior four is selected as the germinal nucleus of the next generation as the other three become degenerates. In starving exconjugant cells, more than two presumptive micronuclei were observed by staining with carbol fuchsin. In contrast, when exconjugants were supplied with the culture medium, only one of the presumptive micronuclei was observed as it divided in the exconjugant cell divisions, while the other three became pycnotic. The induction of the germinal nuclear selection process in exconjugant cells seems to be closely associated with the nutrient supply.
The protein biosynthetic system has been extended to incorporate nonnatural amino acids in addition to the 20 naturally occurring ones. Transfer RNAs were chemically aminoacylated with nonnatural amino acids and the aminoacylated tRNAs were added to the cell lysate of E. coli, together with the target mRNA. The positions of the nonnatural amino acids were directed by the 4-base codon/anticodon pairs that are orthogonal to the existing 3-base codon/anticodon pairs. Here, the position-specific incorporation of nonnatural amino acids was applied to incorporate an electron-accepting amino acid, 2-anthraquinonylalanine (anqAla) into a DNA-binding protein, λ-Cro repressor. The position of anqAla was directed by a CGGG 4-base colon introduced at several different positions on the mRNA. Among the several mutant proteins, the 64angAla Cro showed a strong binding to the consensus double-stranded DNA.
A native protein in aqueous salt solution usually unfolds reversibly upon heating at a transition temperature (Tm) with unfolding enthalpy (ΔHm). The method to predict Tm and ΔHm from the 3D structure assuming simple two-states transition has been developed using experimental values of Tm, ΔHm and heat capacity change (ΔCp, u) compiled in the database“ProTherm”. In order to account for dependence of thermostability on pH and salt concentration, the difference in electrostatic interactions between native and denatured states (Δhu, ele) was computed applying Debye-Huckel type potential between partial charges located on ionizable side chain atoms. Standard unfolding free energy (ΔG0u), enthalpy (ΔH0u), and ΔCp, u at 25°C after subtracting electrostatic energies were obtained using a data set of 63 experimental data on 32 uncharged proteins. Each of these quantities was assumed to be a sum of products of the difference in accessible surface area (ΔASA) of an i-th constituent atomic group between the native and unfolded extended structure times a corresponding adequate constant. Proportional constants for six (aliphatic, aromatic, hydroxyl, amide, carbonyl, and thiol) atomic groups were determined using the data set. Thus, ΔG0u, ΔH0u and ΔCp, u for a protein at uncharged form of known 3D structure can be computed using its ΔASA value of every group, and ΔGu(T) at T is determined according to thermodynamic relation, adding Δhu, ele(T). Values of Tm and ΔHm for the protein may, therefore, be evaluated as values at ΔGu(T)=0. The agreement of calculated and experimental values for the data set used for the determination of the constants was practically the same as that for another data set of 33 experimental data on Tm and ΔHm for 19 proteins with standard deviations of about 12°C and 38kcal/mol, respectively. The stability and instability of correctly and misfolded proteins (hemerythrin, and Ig-variable domain fragment) were successfully explained in terms of ΔGu.