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.      

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.     

Induction of capsular polysaccharide synthesis by rho-fluorophenylalanine in Escherichia coli wild type and strains with altered phenylalanyl soluble ribonucleic acid synthetase

Escherichia coli K-12 strain AB259 can be induced to form capsular polysaccharide (mucoid clones) by dl-p-fluorophenylalanine (FPA; 5 x 10(-6)m on agar plates at 37 C or 8 x 10(-5)m in liquid medium at 30 C). The change was shown to be phenotypic. An increase in enzymes probably involved in capsular polysaccharide synthesis [phosphomannose isomerase (3.3-fold), uridine diphosphate-d-galactose-4-epimerase (2.5-fold), and guanine diphosphate-l-fucose synthetase] was demonstrated as a result of growth in FPA. These increases appear sufficient to account for the increased synthesis of capsular polysaccharide due to growth in FPA. FPA-resistant derivatives of strain AB259 were obtained by selecting mutants on FPA-containing agar or by transducing in an altered phenylalanyl soluble ribonucleic acid synthetase that activates FPA poorly. Mucoid clones were formed by these strains only in the presence of 30 to 1,000 times as much FPA. Among these strains, there was a close correlation between incorporation of FPA-C(14) and induction of capsular polysaccharide synthesis. The results are thus consistent with the following model: FPA is incorporated into the protein product of the R(1) gene (repressor) and alters it sufficiently to allow derepression of several enzymes.     

Derepression of Phosphomannose Isomerase by Regulator Gene Mutations Involved in Capsular Polysaccharide Synthesis in Escherichia coli K-12

A regulator gene mutation (capR) that causes increased synthesis of capsular polysaccharide and derepressed synthesis of several enzymes involved in polysaccharide synthesis also derepresses phosphomannose isomerase (PMI) synthesis. In contrast, a second mutation (capS, which maps separately from capR) that causes increased production of the same polysaccharide does not lead to increased synthesis of PMI (nor of several of the other enzymes involved in polysaccharide synthesis). Introduction of the capR9 allele by transduction or mutation of capR(+) to capR can change the phenotype of a mannose-negative nonmucoid strain to a mannose-positive mucoid phenotype. Thus, genotype capR(+)man-2 is mannose-negative and nonmucoid, but genotype capR9 man-2 is mannose positive and mucoid. Other interactions between these alleles in the synthesis of capsular polysaccharide are recorded.     

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.      

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.     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

Cloning of gene lon (capR) of Escherichia coli K-12 and identification of polypeptides specified by the cloned deoxyribonucleic acid fragment

A mutation in the lon (capR) gene of Escherichia coli K-12 results in overproduction of capsular polysaccharide and increased sensitivity to ultraviolet and ionizing radiations. The lon (capR) gene deoxyribonucleic acid was cloned from a new F′ factor. The new plasmids, designated pBZ201 and pBZ203, (i) contained an additional 8.2-megadalton (Md) EcoRI fragment that had the same mobility as one of the EcoRI fragments of the F′, and (ii) conferred repression of capsular polysaccharide synthesis and repression of sensitivity to ultraviolet radiation in a bacterial transformation experiment with capR mutant recipient strains. A capR9 mutant plasmid, pBZ201M9, was also isolated and conferred expression of mucoidy and ultraviolet sensitivity to a capR⁺ (lon⁺) strain, indicating that the capR9 allele was dominant. Plasmids pBZ201M80, pBZ201M9-INSA, and pBZ201M9-INSB were characterized by transformation as containing recessive capR mutant alleles. Heteroduplex analyses and agarose gel electrophoresis of restriction endonuclease digests of plasmid DNA preparations revealed that (i) pBZ201M9-INSA and pBZ201M9-INSB each contains a 0.5-Md insertion (probably IS1) in the cloned DNA fragment at the same site, and (ii) pBZ201 and pBZ203, both capR⁺ plasmids, contain the same 8.2-Md fragment cloned in opposite orientations with respect to the cloning vehicle, pSC101. Plasmid-specified polypeptides were determined by using strain CSR603 maxicells containing each plasmid. Two new polypeptides were coded by the lon⁺ (capR⁺) 8.2-Md DNA fragment: Z1, 94 kilodaltons (94K), and Z2, 67K. The maxicells containing recessive capR mutant plasmids were deficient only in synthesis of the 94K polypeptide, and the dominant (capR9) mutant plasmid specified 5 to 10 times more of the 94K polypeptide than the maxicells containing the capR⁺ plasmid. Other data indicated that the capR9-specified “94K polypeptide” was not identical to the capR⁺-specified “94K polypeptide.” Thus the altered mutant polypeptide was synthesized in increased quantities, suggesting a defective mode of autogenous regulation for the capR9 polypeptide and effective autogenous regulation of the capR⁺ polypeptide.     

Second-site mutations in capR (lon) strains of Escherichia coli K-12 that prevent radiation sensitivity and allow bacteriophage lambda to lysogenize.

capR (lon) mutants of Escherichia coli K-12 are mucoid and sensitive to ultraviolet (UV) and X-ray radiation as well as to nitrofurantoin. The mutants form filaments after exposure to these agents. capR mutants are also conditionally lethal since they die when plated on complex medium even without UV treatment; this phenomenon is designated "complex medium-induced killing". Furthermore, capR mutants are poorly lysogenized by bacteriophage lambda. Second-site revertants were isolated by plating on media containing nitrofurantoin. All 17 of the independent revertants studied were still mucoid but resistant to UV radiation. Sixteen of the 17 revertants contained a mutation, sulA, that cotransduced with pyrD (21 min). A second locus, sulB, was also found that cotransduced with leu (2 min). Studies with partial diploids (F'pyrD+ sulA+/pyrD36 sulA17 capR9 (lon) demonstrated that sulA+ is dominant to sulA; thus the indicated partial diploid is UV sensitive, whereas the haploid parent is UV resistant. Furthermore, two other phenotypic traits of capR (lon) mutants were reversed by the sul mutation:complex medium-induced killing and the inability of lambda phage to efficiently lysogenize capR strains. On the basis of these and other results, the following model is suggested to explain capR (lon) and sul gene interactions. capR (lon) is a regulator gene for the structural genes sulA+ and sulB+. Depression of both sul operons results in UV sensitivity and decreased ability of lambda to lysogenize, whereas inactivation of either sul+ protein by mutation to sul prevents these phenomena.