Expression of helix-loop-helix type negative regulators of differentiation during limb regeneration in urodeles and anurans.

The urodele is capable of regenerating its limb by forming a blastema even in the adult. By contrast, the anuran, which is phylogenetically close to the urodele, loses this ability during metamorphosis and forms blastema-like tissues that develop only into a spike-like structure in the adult. In order to compare the molecular mechanism of the formation and maintenance of the blastema between the urodele and anuran, the genes encoding helix-loop-helix (HLH) type negative regulators of differentiation were characterized for both the Japanese newt, Cynops pyrrhogaster, and African clawed frog, Xenopus laevis. Cynops homologs of Id2, Id3, and HES1 and Xenopus Id2 were identified. To learn the roles of these genes in regeneration, their expression was examined. The expression of Id2 and Id3 was low in unamputated limbs, but was up-regulated in blastemas of both adult newt and Xenopus. Interestingly, transcripts of the two Id genes showed specific localizations in the blastema and the expression patterns were very similar in both species through the early to medium bud stage. Id2 was expressed predominantly in the blastemal epidermis, and Id3 was expressed equally in the blastemal epidermis and mesenchyme including cells in precartilage condensations. HES1 expression was up-regulated in the newt blastemal epidermis. It was thought that the up-regulation of these genes in the epidermis was related to the proliferation of the cells and that increased expression of these genes in the mesenchyme was related to the undifferentiated state of the blastemal cells. These results and considerations strongly suggested that the state of differentiation is similar in the early to medium bud blastema of both urodeles and anurans. The expression of Id3 remained high through to the digits stage in newts. In contrast, its expression in Xenopus decreased in spike-like regenerates, which correspond to palette-digits stage of newt regenerates. From these results, it was suggested that the blastema redifferentiates earlier in the frog than in the newt, and therefore the timing of redifferentiation of the cartilage is crucial for complete regeneration.

Structural diversity in the alpha 2-->8-linked polysialic acid chains in salmonid fish egg glycoproteins. Occurrence of poly(Neu5Ac), poly(Neu5Gc), poly(Neu5Ac, Neu5Gc), poly(KDN), and their partially acetylated forms.

alpha 2-->8-Linked polysialic acid (polySia) chains terminate O-linked oligosaccharide chains on Salmonidae fish egg polysialoglycoproteins (PSGPs). Expression of these surface PSGPs are developmentally regulated and the polySia epitope is functionally implicated in a number of distinct species-specific cell-cell recognition events during fertilization and early embryogenesis. To better understand the functional diversity of these PSGPs, structural studies of the polySia chains isolated from three genera and eight species of Salmonidae fish eggs were carried out by chemical, immunochemical, enzymatic, and 1H NMR methods. A remarkable degree of structural diversity was found, including differences in the N-acyl groups, i.e. N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), and in the presence of either O-acetyl substitution at C4, C7, or C9 or O-lactyl substitution at C9. The presence of heteropolymers containing both Neu5Ac and Neu5Gc residues was also an unexpected finding. Accordingly, the different forms of alpha 2-->8-linked homo- and heteropolymers of these polySia structures include: poly(Neu5Ac), poly(Neu5Gc), poly(Neu5,chi Ac2), poly(Neu5Gc chi Ac), poly(Neu5Ac, Neu5Gc), poly-(Neu5Ac, Neu5,chi Ac2), poly(Neu5Ac, Neu5Gc chi Ac), poly(Neu5Gc,Neu5,chi Ac2), and poly(Neu5Gc, Neu5-Gc chi Ac), where chi represents the site of acetylation at carbon atom 4, 7, or 9. The significance of this new structural information, together with our recent finding of alpha 2-->8-linked polydeaminoneuraminic acid, poly(KDN), in the rainbow trout egg vitelline envelope, is that it demonstrates the natural occurrence of multiple forms of alpha 2-->8-linked polySia chains in Salmonidae fish glycoproteins that have not been previously described. The results also predict that a remarkable array of polysialylated glycoconjugates is yet to be discovered in animals other than teleost fishes.

Intramolecular stacking association and conformation properties of a 'cap' structure, m7G5'pppUm, and the related model compounds.

The stacking equilibrium quotient of the m7G5'pppUm unit, which occurs as the 5'-terminal "cap" of certain eukaryotic mRNA's, was determined by temperature-dependent difference spectrophotometry as Kstack = 1.82 at 25 degrees and pH 5. In order to evaluate the contribution of different structural modifications to the net stabilization of the cap structures of mRNA, a variety of compounds related to m7G5'pppUm were synthesized and their stacking properties were studied by the same method and compared. The results are summarized as: (1) Introduction of a methyl group into N-7 of G residue results in an increase in base stacking. (2) Methylation at 2'-OH of U residue also stabilizes the stacked structure of G-containing dimers, but it does not influence stacking interaction in m7G-containing dimers. (3) The effect of different types of internucleotide linkages on the order of stacking tendencies is: N5'ppN' greater than N5'pppN' greater than NpN'. UV hypochromicity and CD spectral measurements of the relevant dimers were also conducted, and the hypochromicity values and CD spectra of dimers in their stacked conformation were estimated by making use of the determined Kstack values. The results indicate that, while 2'-O-methylation exerts very little effect on the stacked conformation of the dimers, methylation at N-7 and the nature of the internucleotide linkage strongly influence the stacked conformation, thereby forming unusual left-handed conformations in m7G5'pppU(m), m7G5'ppU(m), and G5'ppU(m).

Simple model to account for the deoxy-versus ribodimer stacking quotient data. Estimation of apparent and intrinsic equilibrium quotients for intramolecular stacking association of purine deoxy- and ribodinucleoside monophosphates.

Stacking equilibrium quotients were evaluated for three purine deoxyribo-dimers, d-ApA, d-m6Apm6A and d-GpG, using procedures developed in earlier studies, and compared with those for the corresponding ribo-dimers. It was shown that the deoxyribo-dimers were apparently more strongly stacked than the corresponding ribo-dimers at 25 degrees C. To explain the observed difference in stability (as measured by the apparent stacking quotient) in the two series of dimers a new extended two-state model is advanced, that the differences in the apparent stacking quotients are due to a limited availability of the number of the sugar-ring puckering modes in the stacked ribo-dimers as compared with the case in the stacked deoxyribo-counterparts. The intrinsic stacking quotients, which are interrelated with the apparent stacking quotients by the microscopic sugar puckering equilibrium constants, were also evaluated for the purine deoxy- and ribo-dimers, and were found to be the same in any pair of these dimers.

Secondary ion mass spectrometry: the scope and limitations in structural analysis of oligoribonucleotides.

Applicability of secondary ion mass spectrometry (SIMS) for structural analysis of oligoribonucleotides was studied. For this purpose a number of oligoribonucleotides having various base sequences, chain lengths, and internucleotide linkages were prepared, and their positive ion SIMS spectra were recorded without derivatization. The soft ionization produced ions possessing relatively low internal energies which underwent little fragmentation, so that both pseudo-molecular and sequence (fragment) ions were observed, the former being more abundant than the latter. Nevertheless, sufficient information can be obtained to elucidate the structure of oligoribonucleotides. Thus SIMS can be used as a useful method for structural analysis of a wide variety of oligoribonucleotides.

Determination of microscopic ionization constants and microscopic stacking equilibrium quotients of adenylyl(3' --> 5')adenosine.

Using the basic ionization constants for a pair of isomers, m1ApA and Apm1A, and the measured values for the overlapping pK values of ApA, the microscopic ionization constants and intramolecular stacking quotients for the monoprotonated ApA were estimated. The results indicate that, in contrast to the case of GpG, ApA did not exhibit preferential protonation on either site of 3'- and 5'-linked nucleoside bases and neither enhanced nor diminished stacking was observed for ApA and ApA as compared to ApA.

Hydrogen exchange kinetics of nucleic acids. Double and triple helices with Hoogsteen-type basepairs.

The kinetics of hydrogen-tritium exchange reaction have been followed by a Sephadex technique of a double-helical poly(ribo-2-methylthio-adenylic acid) . poly(ribouridylic acid) complex with the Hoogsteen-type basepair. Only one hydrogen in every 2-methylthio-adenine . uracil basepair has been found to exchange at a measurably slow rate, 0.023 s-1 (at 0 degrees C), which is, however, much greater than that for a double-helix with the Watson-Crick type A . U pair. The kinetics of hydrogen-tritium exchange were also examined by triple-helical poly(rU) . poly(rA) . poly(rU) which involves both the Watson-Crick and Hoogsteen basepairings. Here, three hydrogens in every U . A. U base triplet have been found to exchange at a relatively slow rate, 0.0116 s-1 (at 0 degrees C). The kinetics of hydrogen-deuterium exchange reactions of these polynucleotide helices have also been followed by a stopped-flow ultraviolet absorption spectrophotometry at various temperatures. On the basis of these experimental results, the mechanism of the hydrogen exchange reactions in these helical polynucleotides was discussed. In the triple helix, the rate-determining process of the slow exchange of the three (one uracil-imide and two adenine-amino) hydrogens is considered to be the opening of the Watson-Crick part of the U. A. U triplet. This opening is considered to take place only after the opening of the Hoogsteen part of the triplet.

Stacking properties of a highly hydrophobic dinucleotide sequence, N6, N6-dimethyladenylyl(3' leads to 5')N6, N6-dimethyladenosine, occurring in 16--18-S ribosomal RNA.

The thermal denaturation ultraviolet absorption spectra of N6,N6-dimethyladenylyl(3' leads to 5')-N6,N6-dimethyladenosine (m2(6)Apm2(6)A), which is a common sequence in 16--18-S ribosomal RNA, in aqueous buffer at pH 7 have been measured over the temperature range 3-90 degrees C. These data have been used to determine the thermodynamic quantitites associated with the intramolecular stacking equilibria. At 25 degrees C in neutral aqueous solution m2(6)Apmw(6)A exists mainly (about 81%) as a stacked form, so that the stacking interactions are stronger than those in the parent unmethylated adenylyl-(3'-5')adenosine (ApA), where about 52% is stacked. From the parameters of delta H and delta S, it is concluded that 'hidden' hydrophobic inteactions are of prime importance in the enhanced stability of m2(6)Apm2(6)A. Transphosphorylation reaction of ApA and m2(6)Apm2(6)A to form the corresponding cyclic 2',3'-phosphates has been studied. First-order rate constants at 25 degrees C for the reactions, which are base-catalyzed, have been obtained. Insertion of two methyl groups at N-6 of ApA reduces the rate of transphosphorylation. Effects of stacking on rates are discussed in the light of reaction mechanisms.

Interferon induction and its dependence on the primary and secondary structure of poly(inosinic acid).poly(cytidylic acid).

The synthetic interferon (IF) inducer rIn.rCn was modified by substituting the ribosyl residues with either their corresponding deoxy(dC or dI) or 2'-O-methyl analogues (mC or mI). The polynucleotide duplexes of these analogues are inactive as IF inducers. Circular dichroism (CD) studies revealed that, while the deoxy substitution produces significant changes in the conformation of rIn.rCn, the 2'-O-methyl substitution produces no detectable change. Biological competition experiments indicated that the methylated duplexes mIn.rCn, rIn.mCn, and mIn.mCn all compete with rIn.rCn for IF induction, while the deoxy duplexes dIn.rCn and rIn.dCn do not. These results are consistent with those predicted from the CD data. Copolymer duplexes (mI,rI)n.(mC,rC)n of varying degrees of methylation and residue clustering were also evaluated for IF induction in human fibroblasts. The IF-inducing capabilities of these duplexes correlated highly with the presence of clusters containing six or more consecutive ribosyl residues. These combined observations suggest that interaction of rIn.rCn with the cell in the induction process may occur in a biphasic manner involving first the topological recognition of a large segment of the RNA to allow for proper binding to the putative cellular receptor, followed by recognition of a much smaller region of the RNA corresponding to 6--12 consecutive ribosyl residues (0.5 to 1 helical turn) which is responsible for the triggering of the induction process.