Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Nuclear magnetic resonance studies of isolated structural domains of yeast phosphoglycerate kinase

The structural integrity and substrate binding properties of the two genetically engineered domains of yeast phosphoglycerate kinase were investigated using one- and two-dimensional nuclear magnetic resonance techniques. Both domains were found to fold with regions of native-like structure, with the N-domain showing greater conformational flexibility than the C-domain. The 'basic patch' region of the N-domain is, however, clearly perturbed by removal of the C-domain. This is most likely due to the absence of stabilizing interactions between the C-terminal peptide (including alpha-helices XIII and XIV) and the N-domain. The C-domain is able to bind nucleotide with an affinity only three times less than that of the native protein.     

Flexibility and folding of phosphoglycerate kinase

Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches. Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK. The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These regions are also parts of the molecule which unfold first. Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft. Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle. The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates. These intermediates are more stable in the horse muscle than in the yeast enzyme. In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme. A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis. These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

The slow-refolding step of phosphoglycerate kinase as monitored by pulse proteolysis.

The kinetics of refolding of yeast phosphoglycerate kinase were studied by following the variation in circular dichroism at 218 nm, the recovery of enzyme activity, and the susceptibility to proteolysis by trypsin and V8-protease. A very rapid phase followed by a slower one was detected by circular dichroism, which revealed the formation of secondary structures. The slower phase, with a macroscopic rate constant of 0.35 min-1, was also detected by the susceptibility of the enzyme to both proteases. It was shown that cleavage sites located in the hinge region, in a part of the C-domain and, to a lesser extent, in a region of the N-domain, which are accessible in the intermediate state, became inaccessible during the slow-refolding step of the molecule. These results demonstrate, on the one hand, the role of domains as folding intermediates, and, on the other hand, the locking of the domain structure and the domain pairing that occurs during the slow-refolding step with a rate constant of 0.35 min-1. The return of the enzyme activity occurred in a slower last step upon conformational readjustments induced by domain interactions.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Identification and characterization of a new disulfide isomerase-like protein (DsbD) in Escherichia coli.

Previous studies have established that DsbA and DsbC, periplasmic proteins of Escherichia coli, are two key players involved in disulfide bond formation. A search for extragenic mutations able to compensate for the lack of dsbA function in vivo led us to the identification of a new gene, designated dsbD. Lack of DsbD protein leads to some, but not all, of the phenotypic defects observed with other dsb mutations, such as hypersensitivity to dithiothreitol and to benzylpenicillin. In addition, unlike the rest of the dsb genes, dsbD is essential for bacterial growth at temperatures above 42 degrees C. Cloning of the wild-type gene and sequencing and overexpression of the protein show that dsbD is part of an operon and encodes an inner membrane protein. A 138 amino acid subdomain of the protein was purified and shown to possess an oxido-reductase activity in vitro. Expressing this subdomain in the periplasmic space helped restore the phenotypic defects associated with a dsbD null mutation. Interestingly, this domain shares 45% identity with the portion of the eukaryotic protein disulfide isomerase carrying the active site. We further show that in dsbD mutant bacteria the dithiol active sites of DsbA and DsbC proteins are mostly oxidized, as compared with wild-type bacteria. Our results argue that DsbD generates a reducing source in the periplasm, which is required for maintaining proper redox conditions. The finding that overexpression of DsbD leads to a Dsb- phenotype, very similar to that exhibited by dsbA null mutants, is in good agreement with such a model.     

Evidence for "twisted plane" undulations in golden hamster sperm tails

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.     

Unfolding-refolding of the domains in yeast phosphoglycerate kinase: comparison with the isolated engineered domains

The role of domains as folding units was investigated with a two-domain protein, yeast phosphoglycerate kinase. Each of the domains was produced independently by site-directed mutagenesis. It has been previously demonstrated by several criteria that these domains are able to fold in vivo into a quasi-native structure [Minard et al. (1989a) Protein Eng. 3, 55-60; Fairbrother et al. (1989) Protein Eng. 3, 5-11]. In the present study, the reversibility of the unfolding-refolding process induced by guanidine hydrochloride was investigated for the intact protein and the isolated domains. The transitions were followed by circular dichroism for both domains and the intact protein and by the variations in enzyme activity for the intact protein. Tryptophan residues were used as intrinsic conformational probes of the C-domain. An extrinsic fluorescent probe, N-[[(iodoacetyl)amino]ethyl]-8-naphthylamine-1-sulfonic acid (IAEDANS), was bound to the unique cysteinyl residue Cys97 to observe the conformational events in the N-domain. The unfolding-refolding transitions of each domain in the intact protein and in the isolated domains prepared by site-directed mutagenesis were compared. It was shown that the two domains are able to refold in a fully reversible process. A hyperfluorescent intermediate was detected during the folding of both the isolated C-domain and the intact yeast phosphoglycerate kinase. The stability of each isolated domain was found to be similar, the free energy of unfolding being approximately half that of the intact molecule.