A phylogenetic classification of gastropod aquaporins.

Successful responses to the multifarious challenges of controlling water balance are critical for snails' survival in the great diversity of habitats they occupy. Advances are being made in understanding how such challenges are approached at the molecular level, including through the study of aquaporins, which are proteins functioning to facilitate the passage of water and other small molecules across cellular membranes. Deduced aquaporin amino acid sequences from partial genomic assemblies of three neritimorph species were added to available gastropod data and sequences from other taxa to make a phylogenetic classification of these proteins using maximum likelihood and Bayesian analyses. We identified ten groups, designated as G1 to G10, containing sequences from multiple major gastropod lineages. At least six of the groups appear to be encoded by multiple genes within at least some species. Five weakly-associated sequences from Neritimorpha were not allocated to a group. The designated groups G1, G2, G3, G4, G5 and G7 (previously defined as Malacoglyceroporins) formed clades containing only gastropod sequences and were strongly supported by Bayesian inference. G1, G2, G3 and G5 were also strongly supported by maximum likelihood analyses. Group G6 (previously defined as Malacoaquaporins)was included with sequences from the oyster, Crassostrea gigas in a strongly supported clade. Groups G8 and G9 included only gastropod sequences but were not strongly supported. Groups G8 and G10 were designated to include all the gastropod sequences belonging respectively to strongly-supported clades including human aquaglyceroporins and aquaammoniaporins. Most groups have been found in a wide range of gastropod lineages but all identified representatives of group G7 belong to Apogastropoda whereas G2 is known only from Patellogastropoda and Neritimorpha.

Pulmonate phylogeny based on 28S rRNA gene sequences: a framework for discussing habitat transitions and character transformation.

Pulmonate snails occupy a wide range of marine, estuarine, freshwater and terrestrial environments. Non-terrestrial forms are supposed to be basal in pulmonate evolution but the group's phylogeny is not well resolved either morphologically or on the basis of available DNA sequence data. The lack of a robust phylogeny makes it difficult to understand character polarization and habitat transformation in pulmonates. We have investigated pulmonate relationships using 27 new sequences of 28S rRNA from pulmonates and outgroups, augmented with data from GenBank. The complete alignments comprised about 3.8kb. Maximum parsimony, maximum likelihood and Bayesian analyses of alignments generated under different assumptions are reported. Complete alignments appear to have a degree of substitution saturation so where there is conflict between hypothesised relationships more weight is given to analyses where regions of random similarity are excluded and which are not affected by this complication. Monophyly of the five main pulmonate groups was robustly supported in almost all analyses. The marine group Amphiboloidea and the freshwater Glacidorbidae are the most basal. The remaining pulmonates (Siphonariidae, Hygrophila and Eupulmonata) form a moderately-supported monophyletic group in all analyses bar one probably affected by saturation of substitutions. Siphonariidae, a predominantly marine and intertidal family, and Eupulmonata (mainly terrestrial with marine, estuarine and freshwater species) form a strongly supported clade that is the sister group to Hygrophila (freshwater). Multiple colonizations of freshwater and terrestrial habitats by pulmonate snails are suggested. No analyses strongly support the possibility of habitat reversions. The colonizations of freshwater by Hygrophila and of land by Stylommatophora were apparently phylogenetically independent although it cannot yet be excluded that there were transient terrestrial phases in the history of the former group or freshwater phases in the latter.

The phylogenetic position of Neritimorpha based on the mitochondrial genome of Nerita melanotragus (Mollusca: Gastropoda).

This is the first report of the mitochondrial gene order and almost-complete DNA sequence of a representative of the Neritimorpha, the highest-ranking gastropod clade lacking such data. Mitochondrial gene order in Nerita is largely plesiomorphic. Its only difference from the cephalopod Octopus vulgaris is a tRNA transposition shared by Vetigastropoda and Caenogastropoda. Genome arrangements were not informative enough to resolve the evolutionary relationships of Neritimorpha, Vetigastropoda and Caenogastropoda. The sister-group taxon of Neritimorpha varied in sequence-based analyses. Some suggested that Neritimorpha is the sister group of Caenogastropoda plus Heterobranchia and some that Neritimorpha and Caenogastropoda are sister groups. No analysis significantly supported the hypothesis that Vetigastroda is more closely related to Caenogastropoda than is Neritimorpha.

Molecular phylogenetics of Caenogastropoda (Gastropoda: Mollusca).

Caenogastropoda is the dominant group of marine gastropods in terms of species numbers, diversity of habit and habitat and ecological importance. This paper reports the first comprehensive multi-gene phylogenetic study of the group. Data were collected from up to six genes comprising parts of 18S rRNA, 28S rRNA (five segments), 12S rRNA, cytochrome c oxidase subunit I, histone H3 and elongation factor 1alpha. The alignment has a combined length of 3995 base positions for 36 taxa, comprising 29 Caenogastropoda representing all of its major lineages and seven outgroups. Maximum parsimony, maximum likelihood and Bayesian analyses were conducted. The results generally support monophyly of Caenogastropoda and Hypsogastropoda (Caenogastropoda excepting Architaenioglossa, Cerithioidea and Campanilioidea). Within Hypsogastropoda, maximum likelihood and Bayesian analyses identified a near basal clade of nine or 10 families lacking an anterior inhalant siphon, and Cerithiopsidae s.l. (representing Triphoroidea), where the siphon is probably derived independently from other Hypsogastropoda. The asiphonate family Eatoniellidae was usually included in the clade but was removed in one Bayesian analysis. Of the two other studied families lacking a siphon, the limpet-shaped Calyptraeidae was associated with this group in some analyses, but the tent-shaped Xenophoridae was generally associated with the siphonate Strombidae. The other studied hypsogastropods with an anterior inhalant siphon include nine families, six of which are Neogastropoda, the only traditional caenogastropod group above the superfamily-level with strong morphological support. The hypotheses that Neogastropoda are monophyletic and that the group occupies a derived position within Hypsogastropoda are both contradicted, but weakly, by the molecular analyses. Despite the addition of large amounts of new molecular data, many caenogastropod lineages remain poorly resolved or unresolved in the present analyses, possibly due to a rapid radiation of the Hypsogastropoda following the Permian-Triassic extinction during the early Mesozoic.

Marsupial MHC class II beta genes are not orthologous to the eutherian beta gene families.

The major histocompatibility complex (MHC) class II DRB, DQB, DPB, and DOB gene clusters are shared by different eutherian orders. Such an orthologous relationship is not seen between the beta genes of birds and eutherians. A high degree of uncertainty surrounds the evolutionary relationship of marsupial class II beta sequences with eutherian beta gene families. In particular, it has been suggested that marsupials utilize the DRB gene cluster. A cDNA encoding an MHC class II beta molecule was isolated from a brushtail possum mesenteric lymph node cDNA library. This clone is most similar to Macropus rufogriseus DBB. Our analysis suggests that all known marsupial beta-chain genes, excluding DMB, fall into two separate clades, which are distinct from the eutherian DRB, DQB, DPB, or DOB gene clusters. We recommend that the DAB and DBB nomenclature be reinstated. DAB and DBB orthologs are not present in eutherians. It appears that the marsupial and eutherian lineages have retained different gene clusters following gene duplication events early in mammalian evolution.

Population genetics of wolf spiders of fragmented habitat in the wheat belt of New South Wales.

Possible effects of habitat fragmentation on the population genetics of a species of wolf spider (Lycosidae) from remnant Callitris woodland in the wheat belt of central western New South Wales in Australia are examined. Single-strand conformational analysis of mitochondrial cytochrome oxidase (subunit 1) was used to characterize the haplotypes of 295 spiders in six blocks each of four woodland sites. DNA sequences were collected from 119 of these spiders to confirm haplotype scoring, allow phylogeny estimation and permit calculation of sequence-based statistics. Intra-block tests do not suggest widespread effects of fragmentation. Genetic diversity is high in all blocks, with 25 haplotypes being identified. Nucleotide diversity is relatively low, as all of the haplotypes are closely related. One block had a significantly low value for the Ewens/Watterson test of neutrality and one block's value was nearly significantly high. Two blocks had nearly significant values of the Harpending Raggedness Index testing for recent population bottlenecks. No other intrablock tests approach significance. Interpopulation comparisons show significant nonhomogeneity of haplotype frequencies globally and in all pairwise comparisons. Relationships between woodland blocks based on haplotype frequencies are discordant with geographical proximity. Haplotype distribution patterns suggest that population structuring existed prior to fragmentation. We develop two measures of genetic distinctiveness to identify subpopulations of interest for conserving evolutionary processes in a species' regional population. One is based on the sum of pairwise FST values and one on the spatial distribution of genetic variation. High values of the measure suggest a subpopulation might have been recently perturbed and low values that it is relatively undisturbed. The two measures identify different blocks as being of particular interest.

Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development.

Auxin plays a key role in lateral root formation, but the signaling pathway for this process is poorly understood. We show here that NAC1, a new member of the NAC family, is induced by auxin and mediates auxin signaling to promote lateral root development. NAC1 is a transcription activator consisting of an N-terminal conserved NAC-domain that binds to DNA and a C-terminal activation domain. This factor activates the expression of two downstream auxin-responsive genes, DBP and AIR3. Transgenic plants expressing sense or antisense NAC1 cDNA show an increase or reduction of lateral roots, respectively. Finally, TIR1-induced lateral root development is blocked by expression of antisense NAC1 cDNA, and NAC1 overexpression can restore lateral root formation in the auxin-response mutant tir1, indicating that NAC1 acts downstream of TIR1.

Phylogenetic investigations of the stephanoberyciformes and beryciformes, particularly whalefishes (Euteleostei: Cetomimidae), based on partial 12S rDNA and 16S rDNA sequences.

DNA data were collected from a number of acanthomorph fishes for 12S rDNA (30 sequences) and 16S rDNA (39 sequences) to investigate the phylogenetic relationships of genera within Cetomimidae (whalefishes) and of this family within the Stephanoberyciformes/Beryciformes assemblage. The Cetomimidae are apparently monophyletic. Within the family, species of Gyrinomimus and Cetomimus form a clade but the former genus is paraphyletic with respect to the latter. Cetostoma is sister to Ditropichthys rather than to Gyrinomimus plus Cetomimus as suggested by morphological analyses. Rondeletiidae + Cetomimidae + Barbourisiidae are shown, as expected from morphological analyses, as a monophyletic group in the 12S rDNA analyses, but not in the 16S rDNA or combined analyses, although the shortest trees showing the group require only one extra step in each case. These three families plus Melamphaidae (our sample of Stephanoberyciformes) are not shown as a group in any analysis, with Melamphaidae being sister to Berycidae in the 16S and combined analyses, but dispersed in the 12S analyses. Maximum-parsimony trees without imposed constraints are notably shorter than trees constrained to show ordinal groupings or either of the two main current hypotheses of Stephanoberyciformes/Beryciformes relationships. The length difference is highly significant for most comparisons using either 12S or 16S rDNA sets or their combination, and significant or nearly so for all comparisons. In particular, the Beryciformes is unlikely to be monophyletic. The Holocentridae are included, with high bootstrap and Bremer support, in a clade of non-beryciforms comprising the Gempylidae, Zeidae, and Atheriniformes (the only higher acanthomorphs sampled) and not with other Beryciform families. In these data, the Berycidae are the sister to the Melamphaidae, a stephanoberyciform family.

Phylogenetic studies of marsupials based on phosphoglycerate kinase DNA sequences.

Phosphoglycerate kinase sequences were obtained for 313 aligned bases of 41 individuals from 39 marsupial species. In contrast to previous molecular analyses, the relationships suggested by these data show a high level of congruence with morphologically defined orders and families. Four main monophyletic lineages are recognizable. These are the monogeneric orders Microbiotheria (Dromiciops australis) and Notoryctemorphia (Notoryctes typhlops), a grouping of the American orders Didelphimorphia and Paucituberculata, and the Australasian species other than N. typhlops. Within the Australasian lineage, there are again four main monophyletic groups; the Dasyuridae, two peramelemorph (bandicoot) lineages (one comprised of pseudogene sequences) and the Diprotodontia. This topology is not greatly affected by the exclusion of pseudogenes except that a clade of syndactylous species (Peramelemorphia plus Diprotodontia) is recovered. Two other peramelemorph pseudogenes have inserts of about 1 kb with high levels of similarity to LINE 1 elements. The Diprotodontia is notable for its relative lack of intersequence variation in comparison to the Dasyuromorphia.

Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus sites.

We showed previously that p34(cdc2)/cyclin B (MPF) hyperphosphorylates poly(A) polymerase (PAP) during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevented complete phosphorylation and MPF-mediated repression. Here we show that PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites was required for repression of PAP activity, and partial phosphorylation had no detectable effect. The consensus sites were phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites were phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remained so throughout maturation, while the non-consensus sites did not become fully phosphorylated until after 12 h of metaphase II arrest. We propose that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. We discuss the possibility that this reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle.

Cell-cycle related regulation of poly(A) polymerase by phosphorylation.

The poly(A) tail found on almost all eukaryotic messenger RNAs is important in enhancing translation initiation and determining mRNA stability. Control of poly(A)-tail synthesis thus has the potential to be a key regulatory step in gene expression and is indeed known to be important during early development in many organisms. To study a possible basis for such regulation, we examined phosphorylation of poly(A) polymerase (PAP) by p34(cdc2)/cyclin B (maturation/mitosis-promoting factor, MPF). We show here that PAP can be phosphorylated in vivo and in vitro by MPF. Consistent with this, PAP becomes hyperphosphorylated both during meiotic maturation of Xenopus laevis oocytes and in HeLa cells arrested at M phase, times in the cell-cycle when MPF is known to be active. We show further that hyperphosphorylation by MPF dramatically reduces the activity of purified PAP, and that PAP isolated from mitotic HeLa cells is similarly inhibited by hyperphosphorylation. This repression probably contributes to the well established reductions in poly(A)+ RNA and/or protein synthesis known to occur in M-phase cells.

DNA regeneration in the polymerase chain reaction.

This paper is an analysis of a model of the regeneration of damaged DNA during the polymerase chain reaction (PCR) made to estimate the degree to which recombination between similar but not identical sequences ("jumping PCR") compromises the results obtained with the technique. The analysis is applicable to museum or field-weathered specimens and also to the damage, such as single-strand nicks, which can occur during storage. Two main results are derived: (i) oligonucleotide priming competes extremely effectively against regeneration derived from priming by overlaps of damaged DNA strands. (ii) Extending result (i), it is shown that jumping PCR is not likely to cause inaccurate sequence determination in standard PCR protocols. One exception arises where it is required to determine the sequence of one specified variant of a piece of DNA which is present in many thousand copies. But, generally, experiments can be performed with confidence that the results are very rarely distorted by jumping PCR. The basic model is extended to investigate how to increase the average length of DNA fragments input to PCR experiments. Increased input of less-damaged material to the PCR can theoretically be achieved by performing several cycles of denaturation, re-association and replication on the sample DNA in the absence of exogenous primers. Experimental work will be necessary to confirm this. However, jumping PCR may occur at substantial frequencies during such pretreatment. This problem cannot be avoided but can be reduced in pretreatment protocols by studying sequences (such as mtDNA or rDNA) where multiple copies are subjected to evolutionary homogenizing processes.

Spectrophotometric quantitation of DNA on blots after ethanol-solubilization of the MTT-formazan from anti-digoxigenin-based detection of nucleic acids.

The tetrazolium salt, 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) has recently been established as a substitute for Nitro-blue tetrazolium (NBT) in stain mixtures using antibody-conjugated alkaline phosphatase for the location of proteins on Western blots (Heegaard, 1990). Experiments reported here show that MTT is as sensitive as NBT in digoxigenin-labeled probe localization (on nucleic acid blots) utilizing alkaline-phosphatase-labelled, anti-digoxigenin antibodies. Moreover, as the formazan from MTT is soluble in ethanol, it is shown that spectrophotometric quantitation can be used to estimate the amount of target DNA on dot and Southern blots. For dot blotting, pBR328 was used as the probe and pBR322 as target. For Southern blots, human rDNA was used as the probe and total genomic calf DNA as the target. Staining response was linear over at least six twofold DNA dilutions in both types of blot.

Developmental changes in the isoenzymes controlling glycolysis in the acridine grasshopper,Caledia captiva.

In this paper is reported an example of extensive developmental changes in the isoenzymes controlling a biochemical pathway: more than half of the glycolytic enzymes of the grasshopper,Caledia captiva differ in electrophoretic phenotype between embryonic and adult stages. A similar pattern of changes is found in each of the taxa ofC. captiva, which is actually a species complex. The present example of developmental variation differs from that described for glycolytic enzymes in vertebrates in two main points. Firstly, the changes between the phenotypes of the embryos and adults are co-incident in time, occurring near hatching. Secondly, in contrast to vertebrates where embryospecific isoenzymes are rare, there exist inC. captiva isoenzymes of trehalase, glucosephosphate isomerase, aldolase, pyruvate kinase, lactate dehydrogenase and 6-phosphogluconate dehydrogenase which are found in the embryo but not in the adult. Some of the variable enzymes also exhibit tissue specificity in the adult. The existence of the changes, whatever their basis, shows that the theory that the expression of housekeeping genes is developmentally invariant is not generally correct.

The effects of linked loci on estimates of relative viability derived from pedigree analysis.

The effect of viability selection at linked loci on segregation ratios at a specified studied locus is analysed in this paper for the crosses of a heterozygote to a homozygote and of two heterozygotes. Both autosomal and sex-linked loci are treated. The analysis indicates that it would be unusual for the change in frequency of a given allele at the studied locus to be distorted by as much as one per cent by the combined effects (assumed to be additive) of selection at all linked loci. This result encourages the use of pedigree analysis in tests of the null hypothesis of selective neutrality of the genotypes at a particular locus since the inevitable cases where selection at linked loci does lead to incorrect rejection of the null hypothesis should be relatively few.

The relative viabilities of alternative human genotypes.

The relative viability of heterozygotes is investigated for two groups of loci. Group A includes serum antigen systems and polymorphisms demonstrated after electrophoresis by general stains or histochemical methods. Group B includes dominantly inherited traits and enzyme deficiencies. The two types of offspring of the mating of a heterozygote and a homozygote are expected to occur in the segregation ratio of 1:1. For loci where there is complete penetrance and complete ascertainment of sibs, significant deviations from this ratio can be ascribed to selection at the locus or at a locus in linkage disequilibrium. The complication of linkage disequilibrium is eliminated by analysis of a large number of traits and the tentative conclusion is reached that there is a prevalence of loci with heterozygote advantage. The estimates of viability differentials would be improved by the analysis of more pedigrees from a disparate sample of traits.