Evolution of the secondary structures and compensatory mutations of the ribosomal RNAs of Drosophila melanogaster

This paper examines the effects of DNA sequence evolution on RNA secondary structures and compensatory mutations. Models of the secondary structures of Drosophila melanogaster 18S ribosomal RNA (rRNA) and of the complex between 2S, 5.8S, and 28S rRNAs have been drawn on the basis of comparative and energetic criteria. The overall AU richness of the D. melanogaster rRNAs allows the resolution of some ambiguities in the structures of both large rRNAs. Comparison of the sequence of expansion segment V2 in D. melanogaster 18S rRNA with the same region in three other Drosophila species and the tsetse fly (Glossina morsitans morsitans) allows us to distinguish between two models for the secondary structure of this region. The secondary structures of the expansion segments of D. melanogaster 28S rRNA conform to a general pattern for all eukaryotes, despite having highly divergent sequences between D. melanogaster and vertebrates. The 70 novel compensatory mutations identified in the 28S rRNA show a strong (70%) bias toward A-U base pairs, suggesting that a process of biased mutation and/or biased fixation of A and T point mutations or AT-rich slippage-generated motifs has occurred during the evolution of D. melanogaster rDNA. This process has not occurred throughout the D. melanogaster genome. The processes by which compensatory pairs of mutations are generated and spread are discussed, and a model is suggested by which a second mutation is more likely to occur in a unit with a first mutation as such a unit begins to spread through the family and concomitantly through the population. Alternatively, mechanisms of proofreading in stem-loop structures at the DNA level, or between RNA and DNA, might be involved. The apparent tolerance of noncompensatory mutations in some stems which are otherwise strongly supported by comparative criteria within D. melanogaster 28S rRNA must be borne in mind when compensatory mutations are used as a criterion in secondary-structure modeling. Noncompensatory mutation may extend to the production of unstable structures where a stem is stabilized by RNA- protein or additional RNA-RNA interactions in the mature ribosome. Of motifs suggested to be involved in rRNA processing, one (CGAAAG) is strongly overrepresented in the 28S rRNA sequence. The data are discussed both in the context of the forces involved with the evolution of multigene families and in the context of molecular coevolution in the rDNA family in particular.      

Small-subunit ribosomal RNA sequence from Naegleria gruberi supports the polyphyletic origin of amoebas

We have sequenced the small-subunit ribosomal RNA gene of the amoebo- flagellate protozoan Naegleria gruberi. Comparison of this sequence with the rRNA sequences of other eukaryotes resulted in a phylogenetic tree that supports the suggested polyphyletic origin of amoebas and suggests a flagellate ancestry for Naegleria.      

Cross-hybridizing snake satellite, Drosophila, and mouse DNA sequences may have arisen independently

Previous reports have interpreted hybridization between snake satellite DNA and DNA clones from a variety of distant taxonomic groups as evidence for evolutionary conservation, which implies common ancestry (homology) and/or convergence (analogy) to produce the cross- hybridizing sequences. We have isolated 11 clones from a genomic library of Drosophila melanogaster, using a cloned 2.5-kb snake satellite probe of known nucleotide sequence. We have also analysed published sequence data from snakes, mice, and Drosophila. These data show that (1) all of the cross-hybridization between the snake, fly, and mouse clones can be accounted for by the presence of either of two tandem repeats, [GATA]n and [GACA]n and (2) these tandem repeats are organized differently among the different species. We find no evidence that these sequences are homologous apart from the existence of the simple repeat itself, although their divergence from a common ancestral sequence cannot be ruled out. The sequences contain a variety of homogeneous clusters of tandem repeats of CATA, GA, TA, and CA, as well as GATA and GACA. We suggest that these motifs may have arisen by a self-accelerating process involving slipped-strand mispairing of DNA. Homogeneity of the clusters might simply be the result of a rate of accumulation of tandem repeats that exceeds that of other mutations.      

Studies on the fate of homologous DNA applied to seedlings of Matthiola incana.

Seedlings of Matthiola incana (crucifer) are able to take up exogenous homologous DNA by the roots. DNA homogenously labelled with [3H]adenine and 5-bromodeoxyuridine is incorporated into the plants in a macromolecular form. Intact donor DNA and a fraction with a buoyant density intermediate between that of the donor and the recipient DNA can be recovered. Analysis of this intermediate fraction by ultrasonication and alkali treatment allows the suggestion that homologous DNA is integrated as a double-stranded DNA which becomes covalently linked to the recipient DNA. Control experiments in which seedlings were incubated in a mixture simulating donor DNA degradation products in the presence and absence of unlabelled competitors suggest that these results are not due to the breakdown of donor DNA and reincorporation of the products during DNA synthesis in the recipient plants. When ultrasonicated or thermally denatured DNA is applied to the plants it may be degraded and reused for recipient DNA synthesis but it is not recovered in a macromolecular form. The possibility that the intermediate DNA fraction arises by bacterial contamination of the plants can be excluded by several arguments. Autoradiographic studies show that at least part of the radioactivity of the donor DNA taken up by the plants is associated with the cell nucleus.     

A Na+-independent,phloretin-sensitive monosaccharide transport system in isolated intestinal epithelial cells

A monosaccharide transport system in addition to the active Na+-dependent system characteristic of the brush border surface of vertebrate intestinal tissue has been identified in isolated chick intestinal epithelial cells. The newly described system differs in several characteristics from the Na+-dependent process, including function in the absence of Na+; a high sensitivity to phloretin, relative insensitivity to phlorizin; different substrate specificity; and a very high KT and Vmax. The system apparently functions only in a facilitated diffusion manner so that it serves to move monosaccharide across the cell membrane down its chemical gradient. An appreciable fraction of total sugar efflux occurs via the Na+-independent carrier from cells which have accumulated sugar to a steady state. Phloretin selectively blocks this efflux so that a normal steady-state sugar gradient of seven- to eightfold is transformed to a new steady-state gradient which is greater than 14-fold. Locus of the new system is tentatively ascribed to the serosal cell surface where it would serve for monosaccharide transfer between enterocyte and lamina propria of the villus.     

Assay of picomole amounts of ATP, ADP, and AMP using the luciferase enzyme system.

Procedural modifications of the luciferase method for ATP assay in conjunction with enzymatic conversion of AMP and ADP allow the assay of all three adenine nucleotides in quantities ranging from 4 to 20 pmoles. An unmodified Beckman scintillation detector at ambient temperature and in a coincidence mode of operation serves as a suitable instrument for quantitating light emitted by the enzyme preparation. The most significant modifications include use of Ca₃(PO₄) activated crude arsenate extracts of desiccated firefly lanterns, low arsenate concentrations during the assay, and an assay pH of 8.0. Extracts handled in this manner exhibit approximately fivefold higher activity than nonactivated extracts employed at pH 7.4 and 50 mm arsenate. Stability of activated extracts is also somewhat greater than for nonactivated preparations. ADP can be 95% enzymatically converted to ATP by treatment with phosphoenolpyruvate and pyruvate kinase under the conditions described. If myokinase is included, approximately 90% of sample AMP can be converted to ATP. Follwing the appropriate enzymatic treatment, the nucleotides are assayed as ATP and amounts calculated by comparison to curves established for known nucleotide standards. The method is appropriate for perchloric acid extracts of biological tissue and certain considerations necessary for application to experimental situations are described.     

Evidence for an intestinal Na+: Sugar transport coupling stoichiometry of 2.0

Membrane potentials maintained by normally-energized intestinal epithelium interfere with an accurate determination of the Na⁺: sugar coupling stoichiometry associated with Na⁺-dependent transport systems. The interference is due to the fact that basal Na⁺ influx is itself a potential-dependent event, and sugar transport induces a membrane depolarization which therefore modifies basal Na⁺ entry. New information obtained under circumstances in which the membrane potential is maintained near 0 indicates that the true coupling stoichiometry is 2:1 rather than the commonly-accepted value of 1:1. A 2:1 stoichiometry means that cellular electrochemical Na⁺ gradients are adequate to account for recently observed 70-fold sugar gradients maintained by these cells under certain conditions.     

Electron transfer between cytochrome P450cin and its FMN-containing redox partner, cindoxin

Cytochrome P450 reductase, which delivers electrons from NADPH to microsomal P450s, consists of a single polypeptide that contains both FAD and FMN. The bacterial P450cin utilizes a similar electron transport system except the FAD/FMN reductase consists of two separate polypeptides where the FMN protein, cindoxin, shuttles electrons between the FAD-containing cindoxin reductase and P450cin. Here we characterize the kinetics and specificity of electron transfer between cindoxin and P450cin as well as discuss the influence of possible binding surface interactions using homology models.