Evidence that microRNA precursors, unlike other non-coding RNAs, have lower folding free energies than random sequences

MOTIVATION: Most non-coding RNAs are characterized by a specific secondary and tertiary structure that determines their function. Here, we investigate the folding energy of the secondary structure of non-coding RNA sequences, such as microRNA precursors, transfer RNAs and ribosomal RNAs in several eukaryotic taxa. Statistical biases are assessed by a randomization test, in which the predicted minimum free energy of folding is compared with values obtained for structures inferred from randomly shuffling the original sequences. RESULTS: In contrast with transfer RNAs and ribosomal RNAs, the majority of the microRNA sequences clearly exhibit a folding free energy that is considerably lower than that for shuffled sequences, indicating a high tendency in the sequence towards a stable secondary structure. A possible usage of this statistical test in the framework of the detection of genuine miRNA sequences is discussed.     

mRNAs have greater negative folding free energies than shuffled or codon choice randomized sequences

An examination of 51 mRNA sequences in GenBank has revealed that calculated mRNA folding is more stable than expected by chance. Free energy minimization calculations of native mRNA sequences are more negative than randomized mRNA sequences with the same base composition and length. Randomization of the coding region of genes yields folding free energies of less negative magnitude than the original native mRNA sequence. Randomization of codon choice, while still preserving original base composition, also results in less stable mRNAs. This suggests that a bias in the selection of codons favors the potential formation of mRNA structures which contribute to folding stability.     

Structural RNA has lower folding energy than random RNA of the same dinucleotide frequency

We present results of computer experiments that indicate that several RNAs for which the native state (minimum free energy secondary structure) is functionally important (type III hammerhead ribozymes, signal recognition particle RNAs, U2 small nucleolar spliceosomal RNAs, certain riboswitches, etc.) all have lower folding energy than random RNAs of the same length and dinucleotide frequency. Additionally, we find that whole mRNA as well as 5'-UTR, 3'-UTR, and cds regions of mRNA have folding energies comparable to that of random RNA, although there may be a statistically insignificant trace signal in 3'-UTR and cds regions. Various authors have used nucleotide (approximate) pattern matching and the computation of minimum free energy as filters to detect potential RNAs in ESTs and genomes. We introduce a new concept of the asymptotic Z-score and describe a fast, whole-genome scanning algorithm to compute asymptotic minimum free energy Z-scores of moving-window contents. Asymptotic Z-score computations offer another filter, to be used along with nucleotide pattern matching and minimum free energy computations, to detect potential functional RNAs in ESTs and genomic regions.     

Microsatellites that violate Chargaff's second parity rule have base order-dependent asymmetries in the folding energies of complementary DNA strands and may not drive speciation

Models for meiotic recombination based on Crick's “unpairing postulate” require symmetrical extrusion of stem-loop structures from homologous DNA duplexes. The potential for such extrusion is abundant in many species and, for a given single-strand segment, can be quantitated as the “folding of natural sequence” (FONS) energy value. This, in turn, can be decomposed into base order-dependent and base composition-dependent components. The FONS values of top and bottom strands in most Caenorhabditis elegans segments are close, as are the corresponding base order-dependent and base composition-dependent components; any discrepancies are in the base composition-dependent component. This suggests that the strands would extrude with similar kinetics. However, interspersed among these segments and at the ends of chromosomes (telomeres) are segments containing short tandem repeats (microsatellites) which, by virtue of their high variability, have been postulated to inhibit the pairing of homologous chromosomes and hence drive speciation. In these segments, there are usually wide discrepancies between the FONS values of top and bottom strands, mainly attributable to differences in base order-dependent components. Analyses of artificial microsatellites of different unit sizes and base compositions show that this asymmetrical distribution of folding potential is greatest for microsatellites when the units are short and violate Chargaff's second parity rule. It is proposed that when there is folding asymmetry, recombination proceeds by special, strand-biased, somatic mechanisms analogous to those operating with Chi sequences in Escherichia coli. If meiotic recombination in the germ-line requires extrusion symmetry, then a general inhibitory influence of microsatellite-containing segments could mask the antirecombinational influence of their variability. Thus, microsatellites may not have driven speciation.     

Cytological evidence that both RNA and DNA may form a complex with the same protein

Deoxyribonucleic acid can be added back to protein sites from which the original nucleic acid, ribo- or deoxyribo-, is removed. If sections of frozen-substituted ovarian follicle cells of a leafhopper are first extracted by hot trichloracetic acid to remove nucleic acids and then immersed in a solution of a commerical preparation of deoxyribonucleic acid, the nucleic acid becomes attached to nuclear and cytoplasmic sites and can be rendered visible by the Feulgen reaction. The addition occurs in certain other tissues as well. The results are discussed in relation to biochemical and other cytochemical investigations of the nucleoprotein complex.     

The joint distribution of patterns in random sequences with application to the RC-measure for expressivity

A method previously developed for computation of pattern probabilitiesin random sequences under Markov chain models. We extend thismethod to the calculation of the joint distribution for twopatterns. An application yields the distribution of the rightchoice measure for expressivity and how significance boundsdepend on sequence length. These bounds are used to show thatthe choice of pyrimidine in codon position 3 of Escherichiacoli genes deviates considerably from a general Markov processmodel for coding regions. We also derive some statistical evidencethat this significant deviation is limited to codon position3.     

Quantitation of bovine sperm cytoplasmic calcium with Quin-2 and Fura-2: evidence that external calcium does not have direct access to the sperm cytoplasm

Internal calcium levels of sperm loaded with Quin-2 in the absence or presence of exogenous calcium were 63 +/- 5 and 189 +/- 19 nM, respectively. These values were similar to those determined by Fura-2. Surprisingly, however, dye loaded sperm depleted of internal calcium did not take up calcium from the medium into the cytoplasm upon re-addition of this ion. Uptake was rapid and maximal, however, if these cells were exposed to the calcium ionophores A23187 or ionomycin. Increasing [Quin-2]i progressively lowered [Ca2+]i in spite of the presence of exogenous calcium during dye loading. This anomaly was not due to interference of the fluorophores with calcium uptake, since exogenous 45Ca2+ was taken up at the same rate and to the same extent by control and fluorophore loaded sperm. This 45Ca2+ uptake was mitochondrial and energy dependent. Also, inhibition of mitochondrial calcium accumulation during dye loading lowered [Ca2+]i to values similar to those observed for calcium depleted sperm. These results suggest an extreme impermeability of the sperm plasma membrane to direct calcium entry into the cytoplasm while substantial amounts of calcium entry occurs into the sperm mitochondria.     

A non-equilibrium thermodynamic theory of leakage of complexed Na+ from muscle,with NMR evidence that the non-complexed fraction of muscle Na+ is intra-vacuolar rather than extra-cellular

Recent experimental evidence has provided increasing support for the hypotheses that 60 to 80 per cent of intracellular Na⁺ exists in a complexed state, and that intracellular water exists in a semi-organized, non-liquid state having low solubility for Na⁺. Using these postulates, a previous crude theory of Na⁺ leakage from the cell based on electronion conduction analogies has been redeveloped in a more complete and detailed fashion, following a non-equilibrium thermodynamic approach. The theory, which is based on the postulate of almost 100 per cent complexing of intracellular Na⁺, predicts that Na⁺ leakage from muscle should conform to the Elovich equation, which closely agrees with experiment, despite the fact that experiments indicate that 20 to 40 per cent of muscle Na⁺ isnot complexed. To resolve this apparent paradox, the leakage of complexed and non-complexed Na⁺ from muscle was measured by nuclear magnetic resonance (NMR). The non-complexed Na⁺ leaked much more slowly than the complexed Na⁺, suggesting that the non-complexed Na⁺ may be confined within vacuoles surrounded by an activation energy barrier at the vacuolar membrane. This implies that the measured curves of Na⁺ leakage showing Elovich kinetics are due mostly to leakage of complexed Na⁺ as the theory requires, and that the leakage of 20 to 40 per cent non-complexed Na⁺ is mostly delayed until later times.     

Maternal inheritance of mitochondrial DNA (mtDNA) in the Pacific oyster (Crassostrea gigas): a preliminary study using mtDNA sequence analysis with evidence of random distribution of MitoTracker-stained sperm mitochondria in fertilized eggs

In many bivalve species, paternal and maternal mitochondrial DNA (mtDNA) from sperm and eggs is transmitted to the offspring. This phenomenon is known as doubly uniparental inheritance (DUI). In these species, sperm mtDNA (M type) is inherited by the male gonad of the offspring. Egg mtDNA (F type) is inherited by both male and female somatic cells and female gonadal cells. In Mytilidae, sperm mitochondria are distributed in the cytoplasm of differentiating male germ cells because they are transmitted to the male gonad. In the present study, we investigated maternal inheritance of mtDNA in the Pacific oyster, Crassostrea gigas. Sequence analysis of two mitochondrial non-coding regions revealed an identical sequence pattern in the gametes and adductor muscle samples taken from six males and five females. To observe whether sperm mitochondria were specifically located in the cytoplasm of differentiating germ cells, their distribution was recorded in C. gigas fertilized eggs by vital staining with MitoTracker Green. Although the 1D blastomere was identified in the cytoplasm of differentiating germ cells, sperm mitochondria were located at the 1D blastomere in only 32% of eggs during the 8-cell stage. Thus, in C. gigas, sperm mitochondria do not specifically locate in the germ cell region at the 1D blastomere. We suggest that the distribution of sperm mitochondria is not associated with germ cell formation in C. gigas. Furthermore, as evidenced by the mtDNA sequences of two non-coding regions, we conclude that mitochondrial DNA is maternally inherited in this species.     

Molecular phylogenetic examination of the delphinoidea trichotomy: congruent evidence from three nuclear loci indicates that porpoises (Phocoenidae) share a more recent common ancestry with white whales (Monodontidae) than they do with true dolphins (Delphinidae)

Porpoises (Phocoenidae), dolphins (Delphinidae), and the two species of Monodontidae (beluga and narwhal) together constitute the superfamily Delphinoidea. Although there is extensive evidence supporting the monophyly of this superfamily, previous studies involving morphology, as well as sequence analysis of mitochondrial genes, have failed to yield a clear picture of the relative relationships within the group. Here we present the first examination of this issue from the perspective of single-copy nuclear genes at the DNA sequence level. The data involve three such loci: von Willebrand factor (vWF), interphotoreceptor retinoid binding protein (IRBP), and lactalbumin. The vWF and IRBP data sets consist of protein-coding fragments, whereas the sequenced lactalbumin fragment is predominately intronic. All phylogenetic analyses involving at least one representative from each of the three Delphinoidea families congruently support a beluga/porpoise clade. The levels of sequence divergence for most of these data appear to roughly concur with a paleontological date for the radiation of the Delphinoidea at 11-15 MYA but, in agreement with mitochondrial DNA sequence analyses, suggest that the extant major groups of cetaceans radiated approximately 25 MYA, 10 million years later than inferred from paleontological data.