Making the body plan: precision in the genetic hierarchy of Drosophila embryo segmentation

We quantify fluctuations in protein expression for three of the segmentation genes in the fruit fly, Drosophila melanogaster. These proteins are representative members of the first three levels of a signalling hierarchy which determines the segmented body plan: maternal (Bicoid protein); gap (Hunchback protein); and pair-rule (Even-skipped protein). We quantify both inter-embryo and inter-nucleus (within a single embryo) variability in expression, especially with respect to positional specification by concentration gradient reading. Errors are quantified both early and late in cleavage cycle 14, during which the protein patterns develop, to study the dynamics of error transmission. We find that Bicoid displays very large positional errors, while expression of the downstream genes, Hunchback and Even-skipped, displays far more precise positioning. This is evidence that the pattern formation of the downstream proteins is at least partially independent of maternal signal, i. e. evidence against simple concentration gradient reading. We also find that fractional errors in concentration increase during cleavage cycle 14.     

Structural instability of IncP-1 plasmids in Pseudomonas aeruginosa PAT involves interaction with plasmid pVS1

The structural instability exhibited by IncP-1 plasmids in Pseudomonas aeruginosa strain PAT was shown to be Rec+ dependent and involved interaction with the resident plasmid pVS1. Structural instability resulted from deletion of plasmid deoxyribonucleic acid at a frequency of ca. 10(-2)/cell per generation. Deletants could be stabilized by transduction into P. aeruginosa strain PAO, but in strain PAT deletants had only a transient existence, as continued deletion led eventually to the loss of the entire plasmid. The patterns of markers lost in PAT were used to demonstrate a marker order for R68 similar to that published elsewhere for RP4 (Barth and Grinter, J. Mol. Biol. 113:455-474, 1977), except that only one Tra region was found. R68 also exhibited Rec+-dependent structural instability in PAO(pVS1) derivatives but, unlike the case in PAT, instability was not accompanied by chromosome mobilization. We isolated deletants of pVS1 which were unable to promote structural instability.     

Fluorescence of membrane-bound tryptophan octyl ester: a model for studying intrinsic fluorescence of protein-membrane interactions.

The fluorescence of a membrane-bound tryptophan derivative (tryptophan octyl ester, TOE) has been examined as a model for tryptophan fluorescence from proteins in membrane environments. The depth-dependent fluorescence quenching of TOE by brominated lipids was found to proceed via a dynamic mechanism with vertical fluctuations playing a central role in the process. The activation energy for the quenching was estimated to be 1.3 kcal/mole. The data were analyzed using the distribution analysis (DA) method, which extends the conventional parallax method to account more realistically for the transbilayer distributions of both probe and quencher and for possible variations in the probe's accessibility. DA provides a better fit than the parallax method to data collected with TOE in membranes formed of lipids brominated at either the 4,5, the 6,7, the 9,10, or the 11,12 positions of the sn-2 acyl chain. DA yields information on the fluorophore's most probable depth in the membrane, its conformational heterogeneity, and its accessibility to the lipid phase. Previously reported data on cytochrome b5 and melittin were reanalyzed together with data obtained with TOE. This new analysis demonstrates conformational heterogeneity in melittin and provides estimates of the freedom of motion and exposure to the lipid phase of membrane-embedded tryptophans of cytochrome b5.     

High sero-prevalence of caseous lymphadenitis identified in slaughterhouse samples as a consequence of deficiencies in sheep farm management in the state of Minas Gerais, Brazil

Background

Multiple congenital ocular anomalies (MCOA) syndrome is a hereditary congenital eye defect that was first described in Silver colored Rocky Mountain horses. The mutation causing this disease is located within a defined chromosomal interval, which also contains the gene and mutation that is associated with the Silver coat color (PMEL17, exon 11). Horses that are homozygous for the disease-causing allele have multiple defects (MCOA-phenotype), whilst the heterozygous horses predominantly have cysts of the iris, ciliary body or retina (Cyst-phenotype). It has been argued that these ocular defects are caused by a recent mutation that is restricted to horses that are related to the Rocky Mountain Horse breed. For that reason we have examined another horse breed, the Icelandic horse, which is historically quite divergent from Rocky Mountain horses.

Results

We examined 24 Icelandic horses and established that the MCOA syndrome is present in this breed. Four of these horses were categorised as having the MCOA-phenotype and were genotyped as being homozygous for the PMEL17 mutation. The most common clinical signs included megaloglobus, iris stromal hypoplasia, abnormal pectinate ligaments, iridociliary cysts occasionally extending into the peripheral retina and cataracts. The cysts and pectinate ligament abnormalities were observed in the temporal quadrant of the eyes. Fourteen horses were heterozygous for the PMEL17 mutation and were characterized as having the Cyst-phenotype with cysts and occasionally curvilinear streaks in the peripheral retina. Three additional horses were genotyped as PMEL17 heterozygotes, but in these horses we were unable to detect cysts or other forms of anomalies. One eye of a severely vision-impaired 18 month-old stallion, homozygous for the PMEL17 mutation was examined by light microscopy. Redundant duplication of non-pigmented ciliary body epithelium, sometimes forming cysts bulging into the posterior chamber and localized areas of atrophy in the peripheral retina were seen.

Conclusions

The MCOA syndrome is segregating with the PMEL17 mutation in the Icelandic Horse population. This needs to be taken into consideration in breeding decisions and highlights the fact that MCOA syndrome is present in a breed that are more ancient and not closely related to the Rocky Mountain Horse breed.     

Nuclear genes that promote splicing of group I introns in the chloroplast 23S rRNA and psbA genes in Chlamydomonas reinhardtii

Defence against pathogens in Arabidopsis is orchestrated by at least three signalling molecules: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). The hrl1 (hypersensitive response-like lesions 1) mutant of Arabidopsis is characterized by spontaneous necrotic lesions, accumulation of reactive oxygen species, constitutive expression of SA- and ET/JA-responsive defence genes, and enhanced resistance to virulent bacterial and oomycete pathogens. Epistasis analyses of hrl1 with npr1, etr1, coi1 and SA-depleted nahG plants revealed novel interactions between SA and ET/JA signalling pathways in regulating defence gene expression and cell death. RNA gel-blot analysis of RNA isolated separately from the lesion+ and the lesion- leaves of double mutants of hrl1 revealed different signalling requirements for the expression of defence genes in these tissues. Expression of the ET/JA-responsive PDF1.2 gene was markedly reduced in hrl1 npr1 and in SA-depleted hrl1 nahG plants. In hrl1 nahG plants, expression of PDF1.2 was regulated by benzathiadiazole in a concentration-dependent manner: induced at low concentration and suppressed at high concentration. The hrl1 etr1 plants lacked systemic PR-1 expression, and exhibited compromised resistance to virulent Pseudomonas syringae and Peronospora parasitica. Inhibiting JA responses in hrl1 coi1 plants lead to exaggerated cell death and severe stunting of plants. Finally, the hrl1 mutation lead to elevated expression of AtrbohD, which encodes a major subunit of the NADPH oxidase complex. Our results indicate that defence gene expression and resistance against pathogens in hrl1 is regulated synergistically by SA and ET/JA defence pathways.     

Polyploids with different origins and ancestors form a single sexual polyploid species

Polyploidization is one of the few mechanisms that can produce instantaneous speciation. Multiple origins of tetraploid lineages from the same two diploid progenitors are common, but here we report the first known instance of a single tetraploid species that originated repeatedly from at least three diploid ancestors. Parallel evolution of advertisement calls in tetraploid lineages of gray tree frogs has allowed these lineages to interbreed, resulting in a single sexually interacting polyploid species despite the separate origins of polyploids from different diploids. Speciation by polyploidization in these frogs has been the source of considerable debate, but the various published hypotheses have assumed that polyploids arose through either autopolyploidy or allopolyploidy of extant diploid species. We utilized molecular markers and advertisement calls to infer the origins of tetraploid gray tree frogs. Previous hypotheses did not sufficiently account for the observed data. Instead, we found that tetraploids originated multiple times from extant diploid gray tree frogs and two other, apparently extinct, lineages of tree frogs. Tetraploid lineages then merged through interbreeding to result in a single species. Thus, polyploid species may have complex origins, especially in systems in which isolating mechanisms (such as advertisement calls) are affected directly through hybridization and polyploidy.     

Molecular recognition of human eosinophil-derived neurotoxin (RNase 2) by placental ribonuclease inhibitor

Placental ribonuclease inhibitor (RI) binds diverse mammalian RNases with dissociation constants that are in the femtomolar range. Previous studies on the complexes of RI with RNase A and angiogenin revealed that RI utilises largely distinctive interactions to achieve high affinity for these two ligands. Here we report a 2.0 angstroms resolution crystal structure of RI in complex with a third ligand, eosinophil-derived neurotoxin (EDN), and a mutational analysis based on this structure. The RI-EDN interface is more extensive than those of the other two complexes and contains a considerably larger set of interactions. Few of the contacts present in the RI-angiogenin complex are replicated; the correspondence to the RI-RNase A complex is somewhat greater, but still modest. The energetic contributions of various interface regions differ strikingly from those in the earlier complexes. These findings provide insight into the structural basis for the unusual combination of high avidity and relaxed stringency that RI displays.     

The effects of dietary phytoestrogens on aromatase activity in human endometrial stromal cells

Dietary phytoestrogens have been reported to inhibit aromatase activity in placental microsomes, but the effects in the human endometrium are unknown. Aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, has recently been shown to be expressed in the endometrium of women with endometriosis and is thought to play a role in the pathophysiology of this disease. Therefore, the objective of this study was to screen dietary phytoestrogens for their ability to inhibit aromatase activity in human endometrial stromal cells (ESC) and identify potential novel therapeutic agents for the treatment of endometriosis. The inhibition of aromatase activity by direct interaction with the dietary phytoestrogens genistein, daidzein, chrysin, and naringenin was tested in a cell free assay. Furthermore, test compound effects on aromatase activity in ESC cultures were also examined. Genistein and daidzein were inactive in the human recombinant aromatase assay whereas naringenin and chrysin inhibited aromatase activity. However, genistein (1 nM to 1 mM) stimulated aromatase activity in ESC whereas other phytoestrogens had no effect. Immunopositive aromatase cells were demonstrated in genistein-treated ESC but not in untreated control cultures. Taken together, our data suggest that genistein can increase aromatase activity in ESC likely via increased enzyme expression.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.