Relationships among msx gene structure and function in zebrafish and other vertebrates

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.      

Is endothelial-nitric-oxide-synthase-derived nitric oxide involved in cardiac hypoxia/reoxygenation-related damage?

Nitric oxide (NO) has been reported to act both as a destructive and a protective agent in the pathogenesis of the injuries that occur during hypoxia/reoxygenation (H/R). It has been suggested that this dual role of NO depends directly on the isoform of NO synthase (NOS) involved. In this work, we investigate the role that NO derived from endothelial NOS (eNOS) plays in cardiac H/R-induced injury. Wistar rats were submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 12 h and 5 days), with or without prior treatment using the selective eNOS inhibitor l-NIO (20 mg/kg). Lipid peroxidation, apoptosis and protein nitration, as well as NO production (NOx), were analysed. The results showed that l-NIO administration lowered NOx levels in all the experimental groups. However, no change was found in the lipid peroxidation level, the percentage of apoptotic cells or nitrated protein expression, implying that eNOS-derived NO may not be involved in the injuries occurring during H/R in the heart. We conclude that l-NIO would not be useful in alleviating the adverse effects of cardiac H/R.     

Molecular population genetics of ref(2)P, a locus which confers viral resistance in Drosophila

The ref(2)P locus (2-54.2) is polymorphic for two allelic forms in natural populations of Drosophila melanogaster, ref(2)Po and ref(2)Pp. The latter allele confers resistance to the rhabdovirus sigma infecting wild populations. Previous work, based on a small sample of prescreened restrictive (resistant) and permissive (susceptible) alleles, identified a large number of amino acid replacement changes (7) relative to synonymous changes (1). Such protein variability could be the result of variation-enhancing selection. To further test the selection hypothesis, we have examined the DNA sequences of ten randomly chosen lines of D. melanogaster and one line of D. simulans. Nine of the ten lines are permissive; D. simulans does not harbor the virus. The melanogaster alleles contain 4 synonymous changes, 19 noncoding changes, and 13 amino acid replacement changes, indicating a relatively high level of polymorphism. Three sequenced restrictive alleles have nearly identical sequences, indicating that they are relatively young. Compared to the permissive alleles, they share only a complex deletion at codon 34, CAG-AAT to GGA, which our analysis indicates to be the site conferring the restrictive phenotype. Patterns of polymorphism and divergence differ from neutral predictions by several criteria for the amino terminal region, which contains the complex deletion (codons 1-91), but not the remainder of the protein (codons 92-599). We find a higher rate of evolution on the D. melanogaster lineage than on the D. simulans lineage. The relatively large amount of both replacement and silent polymorphism in the permissive alleles and the lack of divergence between permissive and restrictive alleles suggests that the sigma virus and ref(2)P may be engaged in an evolutionary race in which new restrictive alleles are continually arising but are relatively short-lived.      

Sedentary life style of Neotropical sedge wrens promotes song imitation

To what extent has the style of song development among songbirds coevolved with other life history strategies? Among Cistothorus wrens in North America, it seems that sedentary or site-faithful habits of marsh wrens, C. palustris, favour song imitation, but seminomadic habits of sedge wrens, C. platensis, favour song improvisation, whereby each male generates a large but unique song repertoire. In this study, we tested whether more sedentary populations of sedge wrens in the Neotropics would imitate songs. At our primary study site near Cartago, Costa Rica, breeding birds were colour-banded during 1995 and 1996, and follow-up surveys revealed that the birds remained at this site the year round. Extensive tape recording and analysis of songs showed that males had large song repertoires (200-300+ songs), and that many songs were shared among neighbouring males. In addition, males only 27 km distant, at La Pastora, used different songs. Furthermore, matched countersinging, in which two males answer each other with identical song types, was recorded near Brasilia, in Brazil. The sharing of songs among permanent neighbours, microgeographical variation in song, and matched countersinging can be achieved only through song imitation, thus revealing a striking difference in the style of song development among different populations of the sedge wren. In the Neotropics, having predictable neighbours throughout life appears to have favoured song imitation, so that individuals can interact using a common, learned code typical of the local population; among more mobile populations in North America, however, individuals improvise large repertoires of species-typical songs, thereby enabling singing males to communicate with any individual, no matter what the population of origin. Strategies of song development must correlate with life history features, and further surveys are needed to make sense of the great diversity of singing behaviours among songbirds. Copyright 1999 The Association for the Study of Animal Behaviour.     

Pollination ecology and breeding system of the very narrow coastal endemic Seseli farrenyi (Apiaceae). Effects of population fragmentation

The reproductive biology of Seseli farrenyi (Apiaceae), a very narrow endemic to Cape Creus (Catalonia, Spain), including flowering timing patterns, quantity and quality of pollination services (type and frequency of pollinators, pollen carryover, pollen deposition on stigmas and reproductive success measured as fruit set), and breeding system was studied. Given the decline of population size detected in the last twenty years, we also analyzed the effects of fragmentation on pollination mechanisms. Protandry along with strong synchrony of floral development within umbels and sequential inflorescence emission within individual stalks, produces sexual phase alternation that promotes a strong outcrossing despite its non-specific pollination system and its (at least partial) self-compatibility. This pronounced xenogamy is supported by results of the insect exclusion test, hand-pollination experiments, and high P/O ratio. S. farrenyi flowers received visits from at least 28 species of insects, including wasps, small bees, ants, flies, syrphid flies, beetles and stink bugs, with different pollen carry-overs. Heterospecific pollen on stigmas decreased notably during the season (50% to 2.5%), averaging 12%. In the small population the stigmatic pollen loads and seed set decreased, but there was no effect of pollinator visitation rates. It was more affected by the composition of pollinators and their efficiency. The wind had a considerable effect on the plant. Some conservation measures are proposed.     

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma- eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.      

How formalin affects the outcome of routine and special stains

The discovery of formaldehyde for preserving tissue structures produced a new dimension in microscopy. Preserving structure and morphology became important; therefore, identifying a proper fixing agent for particular structures, chemical entities, and tissues, also became important. The methods for demonstrating tissue structures evolved and were implemented with careful observation and documentation of the results and outcomes. Formalin was incorporated into many techniques, and provided helpful results in many cases and hindrances in others. The effects of formalin on the outcomes of routine and special staining techniques are reported here.     

The MMS22L–TONSL heterodimer directly promotes RAD51‐dependent recombination upon replication stress

Homologous recombination (HR) is a key pathway that repairs DNA double‐strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L–TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L–TONSL associates with replication protein A (RPA)‐coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR‐mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L–TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51‐ssDNA nucleoprotein filament formation and RAD51‐dependent strand exchange activity in vitro. Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L–TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR‐mediated restart in vivo.