Characterizing the effects of the protein environment on the reduction potentials of metalloproteins

The reduction potentials of electron transfer proteins are critically determined by the degree of burial of the redox site within the protein and the degree of permanent polarization of the polypeptide around the redox site. Although continuum electrostatics calculations of protein structures can predict the net effect of these factors, quantifying each individual contribution is a difficult task. Here, the burial of the redox site is characterized by a dielectric radius R _p (a Born-type radius for the protein), the polarization of the polypeptide is characterized by an electret potential ? _p (the average electrostatic potential at the metal atoms), and an electret-dielectric spheres (EDS) model of the entire protein is then defined in terms of R _p and ? _p. The EDS model shows that for a protein with a redox site of charge Q, the dielectric response free energy is a function of Q ², while the electret energy is a function of Q. In addition, R _p and ? _p are shown to be characteristics of the fold of a protein and are predictive of the most likely redox couple for redox sites that undergo different redox couples.     

Molecular evidence for the origins of Antarctic fishes: paraphyly of the Bovichtidae and no indication for the monophyly of the Notothenioidei (Teleostei)

The notothenioids are an Antarctic suborder of perciform fishes to which increasing interest is being devoted. To investigate their origin, one must address two questions. First, are Bovichtidae (Bovichtus, Cottoperca, Pseudaphritis), the sister-group of the rest of the suborder, monophyletic ? Secondly, what is the sister-group of the Notothenioidei ? These questions were addressed by determining the complete nucleotide sequence of the D2 and D8 domains of 28S rDNA (759 sites, among which 158 informative for parsimony), for 6 notothenioids and a collection of 6 outgroup taxa including the Trachinoidei and Zoarcoidei. Different outgroups (or combinations of outgroups) and different weighting schemes support the inference that Pseudaphritis is closer to the rest of the Notothenioidei than Cottoperca and Bovichtus are. Relationships of Cottoperca and Bovichtus remain unclear with respect to outgroups. Our molecular data therefore clearly show that the Bovichtidae are paraphyletic, but their relationships are not those suggested by Balushkin in 1992. Our data provide no indication of the monophyly of the Notothenioidei in its classical sense. Most of the homoplasy is due to outgroup sequences and interrelationships of outgroups are unresolved. Some morphological synapomorphies shared by Pseudaphritis and the rest of the non-bovichtid Notothenioidei are proposed, including some that were identified by Voskoboynikova in 1993.     

Posttranscriptional repression of the cel gene of the ColE7 operon by the RNA-binding protein CsrA of Escherichia coli

Carbon storage regulator (CsrA) is a eubacterial RNA-binding protein that acts as a global regulator of many functionally diverse chromosomal genes. Here, we reveal that CsrA represses expression from an extrachromosomal element of Escherichia coli, the lysis gene (cel) of the ColE7 operon (cea-cei-cel). This operon and colicin expression are activated upon SOS response. Disruption of csrA caused ∼5-fold increase of the lysis protein. Gel mobility shift assays established that both the single-stranded loop of the T1 stem–loop distal to cei, and the putative CsrA binding site overlapping the Shine–Dalgarno sequence (SD) of the cel gene are important for CsrA binding. Substitution mutations at SD relieved CsrA-dependent repression of the cel gene in vivo. Steady-state levels and half-life of the cel mRNA were not affected by CsrA, implying that regulation is mediated at the translational level. Levels of CsrB and CsrC sRNAs, which bind to and antagonize CsrA, were drastically reduced upon induction of the SOS response, while the CsrA protein itself remained unaffected. Thus, CsrA is a trans-acting modulator that downregulates the expression of lysis protein, which may confer a survival advantage on colicinogenic E. coli under environment stress conditions.     

Drosophila Dyrk2 Plays a Role in the Development of the Visual System

The DYRKs (dual-specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that are associated with a number of neurological disorders, but whose biological targets are poorly understood. Drosophila encodes three Dyrks: minibrain/Dyrk1A, DmDyrk2, and DmDyrk3. Here we describe the creation and characterization of a DmDyrk2 null allele, DmDyrk2^1w17. We provide evidence that the smell impaired allele smi35A¹, is likely to encode DmDyrk2. We also demonstrate that DmDyrk2 is expressed late in the developing third antennal segment, an anatomical structure associated with smell. In addition, we find that DmDyrk2 is expressed in the morphogenetic furrow of the developing eye, that loss of DmDyrk2 in the eye produced a subtle but measurable defect, and that ectopic DmDyrk2 expression in the eye produced a strong rough eye phenotype characterized by increased secondary, tertiary and bristle interommatidial cells. This phenotype was dependent on DmDyrk2 kinase activity and was only manifest when expressed in post-mitotic non-neuronal progenitors. Together, these data indicate that DmDyrk2 is expressed in developing sensory systems, that it is required for the development of the visual system, and that the eye is a good model to identify DmDyrk2 targets.     

Identification of the yellow skin gene reveals a hybrid origin of the domestic chicken

Yellow skin is an abundant phenotype among domestic chickens and is caused by a recessive allele (W*Y) that allows deposition of yellow carotenoids in the skin. Here we show that yellow skin is caused by one or more cis-acting and tissue-specific regulatory mutation(s) that inhibit expression of BCDO2 (beta-carotene dioxygenase 2) in skin. Our data imply that carotenoids are taken up from the circulation in both genotypes but are degraded by BCDO2 in skin from animals carrying the white skin allele (W*W). Surprisingly, our results demonstrate that yellow skin does not originate from the red junglefowl (Gallus gallus), the presumed sole wild ancestor of the domestic chicken, but most likely from the closely related grey junglefowl (Gallus sonneratii). This is the first conclusive evidence for a hybrid origin of the domestic chicken, and it has important implications for our views of the domestication process.     

Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein

We have recently discovered that the Masculinizer (Masc) gene encodes a CCCH tandem zinc finger protein, which controls both masculinization and dosage compensation in the silkworm Bombyx mori. In this study, we attempted to identify functional regions or residues that are required for the masculinizing activity of the Masc protein. We constructed a series of plasmids that expressed the Masc derivatives and transfected them into a B. mori ovary-derived cell line, BmN-4. To assess the masculinizing activity of the Masc derivatives, we investigated the splicing patterns of B. mori doublesex (Bmdsx) and the expression levels of B. mori IGF-II mRNA-binding protein, a splicing regulator of Bmdsx, in Masc cDNA-transfected BmN-4 cells. We found that two zinc finger domains are not required for the masculinizing activity. We also identified that the C-terminal 288 amino acid residues are sufficient for the masculinizing activity of the Masc protein. Further detailed analyses revealed that two cysteine residues, Cys-301 and Cys-304, in the highly conserved region among lepidopteran Masc proteins are essential for the masculinizing activity in BmN-4 cells. Finally, we showed that Masc is a nuclear protein, but its nuclear localization is not tightly associated with the masculinizing activity.     

Molecular handcuffing of the relaxosome at the origin of conjugative transfer of the plasmid R1162

The assembly of plasmid-encoded proteins at a unique site (oriT) on the plasmid R1162, to form a complex called the relaxosome, is required for conjugative transfer of the plasmid and for negative regulation of neighboring promoters. Two-dimensional chloroquine gel electrophoresis was used to show that oriTs are physically coupled at the relaxosome. This interaction requires all the relaxosome proteins, which are assembled into a structure resulting in a decrease in the average linking number of the plasmid DNA in the cell. Molecules with higher superhelical densities are preferentially selected for assembly of the relaxosome. Genetic data obtained earlier indicate that the molecular coupling reported here is a ‘handcuffing’ reaction that contributes to the regulation of adjacent plasmid promoters. However, although these promoters affect the expression of the genes for replication, plasmid copy-control is regulated independently. This is the first time ‘handcuffing’ has been observed at an oriT, and its possible significance for transfer is discussed.     

New occurrences of heteromorph ammonites in the Berriasian-Valanginian of the Crimean mountains

A new species, Bochianites (?) ambiguus sp. nov., is described from the Lower Berriasian of the Eastern Crimea. It is proposed that the name B. laevis Liu, 1988 be used instead of B. levis Arkadiev, 2008, as the former is a senior synonym and homonym. For the first time a species of the genus Leptoceras is described from the Upper Berriasian-Lower Valanginian of the former Soviet Union. Problems in the identification of heteromorph ammonites when they are partially preserved are discussed, and the geographical distribution and facial affinities of the heteromorph ammonite family Bochianitidae are considered.     

Characterization of the Bacterial Community of the Chemically Defended Hawaiian Sacoglossan Elysia rufescens

Sacoglossans are characterized by the ability to sequester functional chloroplasts from their algal diet through a process called kleptoplasty, enabling them to photosynthesize. The bacterial diversity associated with sacoglossans is not well understood. In this study, we coupled traditional cultivation-based methods with 454 pyrosequencing to examine the bacterial communities of the chemically defended Hawaiian sacoglossan Elysia rufescens and its secreted mucus. E. rufescens contains a defense molecule, kahalalide F, that is possibly of bacterial origin and is of interest because of its antifungal and anticancer properties. Our results showed that there is a diverse bacterial assemblage associated with E. rufescens and its mucus, with secreted mucus harboring higher bacterial richness than entire-E. rufescens samples. The most-abundant bacterial groups affiliated with E. rufescens and its mucus are Mycoplasma spp. and Vibrio spp., respectively. Our analyses revealed that the Vibrio spp. that were highly represented in the cultivable assemblage were also abundant in the culture-independent community. Epifluorescence microscopy and matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS) were utilized to detect the chemical defense molecule kahalalide F on a longitudinal section of the sacoglossan.     

Mutants of the bithorax complex and determinative states in the thorax of Drosophila melanogaster.

Homoeotic mutations of the bithorax complex cause segmental transformations. The genes in which these mutations occur are good candidates for genes that are involved in determination. The determination system in imaginal discs must have at least two functions. One is a cell heredity function that is responsible for maintaining the determined state during growth and development. A second is the expression of the determined state (e.g., different imaginal discs have different morphologies). The homoeotic mutations of the bithorax complex could be affecting either of these two functions. I have found that when posterior haltere disc cells, that are transformed by the mutation postbithorax so that they form wing cuticle in situ, regenerate anterior structures, these structures are anterior wing. This is the same result as that seen when wild-type posterior-wing disc cells regenerate anterior structures. On the other hand, when anterior haltere disc cells transformed by the mutation bithorax³, so that they produce wing cuticle in situ, regenerate, they produce posterior haltere structures. This is unlike wild-type anterior-wing disc cells, which regenerate posterior-wing structures. From these results, I conclude that bithorax³ affects the expression of the determined state and postbithorax affects the cell heredity of determination.     

The retinal pigment epithelium of the eye regulates the development of scleral cartilage

The majority of vertebrate species have a layer of hyaline cartilage within the fibrous sclera giving an extra degree of support to the eyeball. In chicks, this is seen as a cuplike structure throughout the scleral layer. However, the mechanisms that control the development of scleral cartilage are largely unknown.Here we have studied the phases of scleral cartilage development and characterised expression profiles of genes activated during the cartilage differentiation programme. CART1 and SOX9, the earliest markers of pre-committed cartilage, are expressed in the mesenchyme surrounding the optic cup. Later AGGRECAN, a matrix protein expressed during chondrocyte differentiation, is also expressed. The expression of these genes is lost following early removal of the optic cup, suggesting a role for this tissue in inducing scleral cartilage. By grafting young retinal pigment epithelium (RPE) and retina into cranial mesenchyme in vivo, it was found that RPE alone has the ability to induce cartilage formation.There are some exceptions within the vertebrates where scleral cartilage is not present; one such example is the placental mammals. However, we found that the cartilage differentiation pathway is initiated in mice as seen by the expression of Cart1 and Sox9, but expression of the later cartilage marker Aggrecan is weak. Furthermore, cartilage forms in mouse peri-ocular mesenchyme micromass culture. This suggests that the process halts in vivo before full differentiation into cartilage, but that murine scleral mesenchyme has retained the potential to make cartilage in vitro.RA, Wnts and Bmps have been linked to the cartilage development process and are expressed within the developing RPE. We find that RA may have a role in early scleral cartilage development but is not likely to be the main factor involved.These data reveal the course of scleral cartilage formation and highlight the key role that the optic cup plays in this process. The driving element within the optic cup is almost certainly the retinal pigmented epithelium.     

A systematic analysis of the gap gene system in the moth midge Clogmia albipunctata

The segmentation gene hierarchy of Drosophila melanogaster represents one of the best understood of the gene networks that generate pattern during embryogenesis. Some components of this network are ancient, while other parts of the network have evolved within the higher Diptera. To further understand the evolution of this gene network, we are studying the role of gap genes in a representative of a basally diverging dipteran lineage, the moth midge Clogmia albipunctata. We have isolated orthologues of all of the Drosophila trunk gap genes from Clogmia, and determined their domains of expression during the blastoderm stage of development, in relation to one another, and in relation to the expression of even-skipped (Calb-eve), a component of the pair-rule system that is directly regulated by the gap genes in Drosophila. We find that hunchback (Calb-hb), Krüppel (Calb-Kr), knirps (Calb-knl), giant (Calb-gt) and tailless (Calb-tll) are all expressed in patterns consistent with a gap segmentation role during blastoderm formation, but huckebein (Calb-hkb) is not. In the anterior half of the embryo, the relative positions of the gap gene expression domains in relation to one another, and in relation to the eve stripes, are rather well conserved. In the posterior half of the embryo, there are significant differences. Posteriorly, Calb-gt is expressed only transiently and very weakly, in a domain that overlaps entirely with that of Calb-knl. At late blastoderm stages, none of the candidate genes we have tested is expressed in the region between the posterior Calb-knl domain and Calb-tll. It is likely that the regulation of Calb-eve expression in this posterior region depends on combinations of gap gene factors that differ from those utilised for the same stripes in Drosophila. Both the gap and the pair-rule patterns of gene expression are dynamic in Clogmia, as they are in Drosophila, shifting anteriorly as blastoderm development proceeds.