Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review

A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed.     

Lack of repair of ultraviolet light damage in Mycoplasma gallisepticum

Molecules with single-stranded tails (rolling circles) were isolated as replicating intermediates in G4 progeny single-stranded DNA synthesis. Lysates from infected cells harvested late in infection during single-stranded DNA synthesis were not deproteinised but analysed directly in caesium chloride and propidium diiodide gradients. The gradient fractionated them on the basis of tail length. If the lysates were first deproteinised however, the tailed replicative intermediates banded as a peak at a density just greater than that of replicative form II DNA (RFII) and did not spread down the gradient. The origin of synthesis of the viral strand tail was mapped by electron microscopy as 55 to 60% away from the single EcoRI cleavage site. Termination molecules finishing a round of viral strand DNA synthesis have been identified as molecules consisting of a closed single-stranded DNA circle attached by a very small region to the parent double-stranded DNA circle.     

The role of DNA polymerase I-associated 5'-exonuclease in replication of coliphage M13 replicative-form DNA.

The conversion of both parental- and progeny-nascent open circular M13 RF DNA into covalently closed RF I is drastically reduced in an $\text{E. coli}$ mutant deficient in the 5′ → 3′ exonuclease associated with DNA polymerase I. The nascent progeny RF DNA also contains a significant proportion of fragments of smaller than unit length.     

Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry

The measurement of fluorescence lifetimes is known to be hindered by the wavelenght-dependent and photocathode area-dependent time response of photomultiplier tubes. A simple and direct method is described to minimize the effects in photomultiplier tubes for phase-modulation fluorometry. Reference fluorophores of known lifetime were used in place of the usual scattering reference. The emission wavelenghts of the reference and sample were matched by either filters or a monochromator, and the use of a fluorophore rather than a scatter decreases the differences in spatial distribution of light emanating from the reference and sample. Thus photomultiplier tube artifacts are minimized. Five reference fluorophores were selected on the basis of availability, ease of solution preparation, and constancy of lifetime with temperature and emission wavelenght. These compounds are p-terphenyl, PPO, PPD, POPOP and dimethyl POPOP. These compounds are dissolved in ethanol to give standard solutions that can be used over the temperature range from ?55 to +55°C. Purging with inert gas is not necessary. The measured phase and modulation of the reference solution is used, in conjunction with the known reference, lifetime, to calculate the actual phase and modulation of the exictation beam. The use of standard fluorophores does not require separate experiments to quantify photomultiplier effects, and does not increase the time required for the measurement of fluorescence lifetimes. Examples are presented which demonstrate the elimination of artifactual photomultiplier effects in measurements of the lifetimes of DADH (0.4 ns) and indole solutions quenched by iodide. In addition, the use of these reference solutions increases the accuracy of fluorescence lifetime measurements ranging ranging to 30 ns. We judge this method to provide more reliable lifetime measurements by the phase and modulation method. The test solutions and procedures we describe may be used by other laboratories to evaluate the performance of their phase fluorometers.     

Precise localization of the origin of replication in a physical map of HeLa cell mitochondrial DNA and isolation of a small fragment that contains it

The precise positions of the origin of replication³ and of the D-loop within the HpaII restriction map of HeLa cell mitochondrial DNA have been investigated. For this purpose, 7 S DNA, which is the heavy-chain initiation sequence, was used as a template for fragment-primed DNA synthesis by Escherichia coli DNA polymerase I. The results indicate clearly that the origin of replication lies in HpaII fragment 8 at about 80 base-pairs from the border with fragment 17, and that the D-loop region extends from this site, through fragment 17, to a position in fragment 10 which is about 365 base-pairs from the border with fragment 17. Sequential digestion of fragment 8 with HaeIII enzyme has allowed the isolation of a subfragment, about 200 base-pairs long, that contains the origin of replication.     

Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III

We have cloned in Escherichia coli and sequenced a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The resistance gene was located by analysis of the initiation and termination codons in an open reading frame (ORF) of 792 bp. The deduced gene product, a 3'5'-aminoglycoside phosphotransferase of type III, has an Mr of 29,200. Comparison of its amino acid sequence with those of type I (Oka et al., 1981) and type II (Beck et al., 1982) 3' phosphotransferase, from transposable elements Tn903 and Tn5, respectively, indicated a statistically significant structural relationship between these enzymes from phylogenetically remote bacterial genera. The degree of homology observed indicate that phosphotransferase type III and type I genes have diverged from a common ancestor and that the phosphotransferase type II gene has emerged more recently from the type I evolutionary pathway.     

Restriction map of Chinese hamster mitochondrial DNA containing replication coordinates: comparison with Syrian hamster mitochondrial genome

A precise physical map, containing the structurally and operationally defined D-loop origin, terminal region, and direction of heavy-strand replication, has been constructed for mitochondrial DNA (mtDNA) from ovary (CHO-KI) and lung cells of Chinese hamster (Cricetulus griseus 2 N = 22), and compared with our previously established genome coordinates for mtDNA from Syrian hamster ( Mesocricetus auratus 2 N = 44). All four HpaI sites in Cricetulus are conserved in Mesocricetus (8 sites). Extensive variation exists for hexanucleotides cleaved by EcoRI HindIII PstI. KpnI and BamHI. Sequence divergence between Chinese and Syrian hamster mtDNAs, as reflected from analysis of the mapped recognition sites for these six endonucleases, is estimated as 5–9% base substitutions. mtDNAs from both hamster and several other mammalian species contain a commonly conserved HpaI site in the region of light strand initiation.     

Effects of lead and cadmium on chemical composition and total water content of the pupal parasitoid, Pimpla turionellae

The parasitoid Pimpla turionellae L. (Hymenoptera, Ichneumonidae) was fed on Cd, Pb and Cd+Pb-contaminated food (33g Cd, 82g Pb and 33g Cd+82g Pb per gram food fresh weight, respectively). Significant decrease in the total lipid and protein content was found along with an increase in the water content particularly in Cd-contaminated parasitoids.     

Why the evolution of resistance to anthropogenic toxins normally involves major gene changes: the limits to natural selection

Standard population genetic theory suggests that adaptation should normally be achieved by the spread of many genes each of small effect (polygenes), and that adaptation by major genes should be unusual. Such models depend on consideration of the rates of acquisition of adaptation. In practice, adaptation to pollutants and anthropogenic toxins has most frequently been achieved by the spread of major genes. A simple model is developed to explain this discrepancy, in which the determining factor is not the rate of spread, but the maximum response achievable under the two contrasting models of polygenic or major gene inheritance. In the short term, for a given mean and genetic variance, characters in which the additive genetic variance is produced by the segregation of many genes of small effect at intermediate gene frequencies are unable to produce as large a response to directional selection as characters in which the variance is caused by genes of large effect at low frequency. If the target for selection is a long way from the mean prior to selection (as it may well be for adaptation to novel anthropogenic stresses) then adaptation can only be achieved by species possessing major genes. The model is discussed with reference to the example of heavy metal tolerance in plants.     

Comparisons of benthic invertebrates between riffles and pools

Benthic species assemblages in upstream and downstream ends of riffles and in pools were investigated seasonally in the first five orders of an alluvial gravel stream with distinct pool and riffle channel form. Riffles comprised < 10% of stream area and were separated by pools with extensive bedrock substrate (ca. 15–85% of total surface area) which was scoured during periodic high flow. Virtually all taxa were more abundant in riffles than in pools, except chironomids which were more equally distributed. Inconsistent results were obtained for upstream-downstream comparisons within riffles. Intermittent headwater reaches (orders 1 & 2) which supported half as many taxa retained this pattern during periods of flow, although riffles at these sites were dry from mid-June to mid-November. Pools which contained more gravel, indicating less disturbance during high flow, had a richer assemblage of benthic species than other pools. Many invertebrates in pools may have been there as a result of drift from their preferred riffle habitats, but the presence of gravel in the pools indicates less intense flow disturbance during floods, provides protection from the mild scouring that does occur during floods, and provides refugia from predators.