A segmentation gene in tribolium produces a polycistronic mRNA that codes for multiple conserved peptides

Segmentation genes in insects are required for generating the subdivisions of the early embryo. We describe here a new member of the gap family of segmentation genes in the flour beetle Tribolium, mille-pattes (mlpt). mlpt knockdown leads to transformation of the abdominal segments into thoracic segments, providing embryos with up to ten pairs of legs. We show that there are crossregulatory interactions between mlpt and the known gap genes in Tribolium, suggesting that mlpt is itself a gap gene. The mlpt gene reveals an unusual structure, as it encodes a polycistronic mRNA that codes for four peptides. mlpt appears to be the prototype of this previously unknown gene structure in eukaryotes, as we find homologous genes with the same polycistronic arrangement in other insect genomes as well.     

Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 x 10(-8) M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.     

Are ABA,ethylene or their interaction involved in the response of leaf growth to soil water deficit? An analysis using naturally occurring variation or genetic transformation of ABA production in maize

The role of abscisic acid (ABA) and its possible interaction with ethylene in mediating leaf elongation response to soil water deficit are a matter of controversy. To address this question, we used a set of maize genotypes with various levels of ABA either due to natural variability or to genetic transformation targeted on NCED/VP14, a key enzyme of ABA synthesis. The transgenic lines yielded less strong phenotypes than available mutants, making it possible to use them under normal growing conditions. We focused on leaf elongation during night periods in order to avoid the confounding effect of ABA on leaf water status. Our results suggest that over a wide range, internal ABA level (measured in both leaf extracts or xylem sap) has no clear effect on leaf elongation response to soil water deficit, except in the case of an antisense line presenting the strongest reduction in ABA accumulation that showed a slight maintenance of leaf elongation during water deficit. Leaf ethylene production rate was variable and not related to water deficit except in the ABA-deficient transgenic lines where it was increased by water deficit on average but not systematically. Moreover, variability in ethylene production rate was not linked to variability in elongation rate. Our results thus suggest that neither ABA nor ethylene seems to play a major role in the control of leaf elongation response to soil water deficit.     

Increased tolerance of Litopenaeus vannamei to white spot syndrome virus (WSSV) infection after oral application of the viral envelope protein VP28

It has been generally accepted that invertebrates such as shrimp do not have an adaptive immune response system comparable to that of vertebrates. However, in the last few years, several studies have suggested the existence of such a response in invertebrates. In one of these studies, the shrimp Penaeus monodon showed increased protection against white spot syndrome virus (WSSV) using a recombinant VP28 envelope protein of WSSV. In an effort to further investigate whether this increased protection is limited to P. monodon or can be extended to other penaeid shrimp, experiments were performed using the Pacific white shrimp Litopenaeus vannamei. As found with P. monodon, a significantly lower cumulative mortality for VP28-fed shrimp was found compared to the controls. These experiments demonstrate that there is potential to use oral application of specific proteins to protect the 2 most important cultured shrimp species, P. monodon and L. vannamei, against WSSV. Most likely, this increased protection is based on a shared and, therefore, general defence mechanism present in all shrimp species. This makes the design of intervention strategies against pathogens based on defined proteins a viable option for shrimp culture.     

Phase variation and genomic architecture changes in Azospirillum

The plant growth-promoting rhizobacterium Azospirillum lipoferum 4B generates in vitro at high frequency a stable nonswimming phase variant designated 4V(I), which is distinguishable from the wild type by the differential absorption of dyes. The frequency of variants generated by a recA mutant of A. lipoferum 4B was increased up to 10-fold. The pleiotropic modifications characteristic of the phase variant are well documented, but the molecular processes involved are unknown. Here, the objective was to assess whether genomic rearrangements take place during phase variation of strain 4B. The random amplified polymorphic DNA (RAPD) profiles of strains 4B and 4V(I) differed. RAPD fragments observed only with the wild type were cloned, and three cosmids carrying the corresponding fragments were isolated. The three cosmids hybridized with a 750-kb plasmid and pulse-field gel electrophoresis analysis revealed that this replicon was missing in the 4V(I) genome. The same rearrangements took place during phase variation of 4BrecA. Large-scale genomic rearrangements during phase variation were demonstrated for two additional strains. In Azospirillum brasilense WN1, generation of stable variants was correlated with the disappearance of a replicon of 260 kb. For Azospirillum irakense KBC1, the variant was not stable and coincided with the formation of a new replicon, whereas the revertant recovered the parental genomic architecture. This study shows large-scale genomic rearrangements in Azospirillum strains and correlates them with phase variation.     

Experimental verification of the crucial roles of Glu73 in the catalytic activity and structural stability of goose type lysozyme

The roles of Glu(73), which has been proposed to be a catalytic residue of goose type (G-type) lysozyme based on X-ray structural studies, were investigated by means of its replacement with Gln, Asp, and Ala using ostrich egg-white lysozyme (OEL) as a model. No remarkable differences in secondary structure or substrate binding ability were observed between the wild type and Glu(73)-mutated proteins, as evaluated by circular dichroism (CD) spectroscopy and chitin-coated celite chromatography. Substitution of Glu(73) with Gln or Ala abolished the enzymatic activity toward both the bacterial cell substrate and N-acetylglucosamine pentamer, (GlcNAc)(5), while substitution with Asp did not abolish but drastically reduced the activity of OEL. These results demonstrate that the carboxyl group of Glu(73) is directly involved in the catalytic action of G-type lysozyme. Furthermore, the stabilities of all three mutants, which were determined from the thermal and guanidine hydrochloride (GdnHCl) unfolding curves, respectively, were significantly decreased relative to those of the wild type. The results obtained clearly indicate the crucially important roles of Glu(73) in the structural stability as well as in the catalytic activity of G-type lysozyme.     

Consequences of fetal irradiation on follicle histogenesis and early follicle development in rat ovaries

Follicle histogenesis, in which follicles arise from fragmenting ovigerous cords, is a poorly understood mechanism that is strictly dependent upon the presence of germ cells. Our previous studies have shown that severely germ cell-depleted rat ovaries after fetal gamma-irradiation display modifications of follicular endowment and dynamics during the immature period. The primordial follicle stock was absent and the follicles with primary appearance remained quiescent longer than in control ovaries during the neonatal period. The aim of the present work was to analyze the initial steps of follicle histogenesis, and to investigate the etiology of the alterations observed in the development of irradiated ovaries. Just after birth, we observed, in addition to sterile ovigerous cords, the emergence of the first follicles which exhibited several abnormal features as compared to those of control ovaries. Most of the follicles appeared as primary follicles, as they were composed of a layer of cuboidal-shaped granulosa cells surrounding an enlarged oocyte. Interestingly, the granulosa cells of these primary-like follicles did not proliferate and did not express the genes for anti-Müllerian hormone (Amh) or bone morphogenetic protein receptor type II (Bmpr2), both of which are normally expressed from the primary stage onwards. In contrast, the oocytes strongly expressed the gene for growth and differentiation factor 9 (Gdf9), which is normally upregulated from the primary follicle stage onwards, which suggests an uncoupling of granulosa cell development from oocyte development. In addition, irradiated ovaries displayed a higher frequency of follicles that contained 2 or 3 oocytes, which are also referred to as multi-oocyte follicles (MOFs). Examination at the time of follicle histogenesis indicated that MOFs arise from incomplete ovigerous cord breakdown. Taken together, the results of this study indicate that severe perturbations of follicular histogenesis take place following irradiation and massive germ cell depletion during fetal life. In addition to the classically described sterile cords, we have pointed out the differentiation of MOFs and primary-like quiescent follicles, which finally evolve into growing follicles and participate in ovarian function. We propose that these phenotypes are closely correlated to the proportion of granulosa cells to oocytes at the time of neonatal follicle histogenesis.     

Structural characterization of the papaya cysteine proteinases at low pH

Current control of gastrointestinal nematodes relies primarily on the use of synthetic drugs and encounters serious problems of resistance. Oral administration of plant cysteine proteinases, known to be capable of damaging nematode cuticles, has recently been recommended to overcome these problems. This prompted us to examine if plant cysteine proteinases like the four papaya proteinases papain, caricain, chymopapain, and glycine endopeptidase that have been investigated here can survive acidic pH conditions and pepsin degradation. The four papaya proteinases have been found to undergo, at low pH, a conformational transition that instantaneously converts their native forms into molten globules that are quite unstable and rapidly degraded by pepsin. As shown by activity measurements, the denatured state of these proteinases which finally results from acid treatment is completely irreversible. It is concluded that cysteine proteinases from plant origin may require to be protected against both acid denaturation and proteolysis to be effective in the gut after oral administration.     

Structural properties of prion protein protofibrils and fibrils: an experimental assessment of atomic models

Decades after the prion protein was implicated in transmissible spongiform encephalopathies, the structure of its toxic isoform and its mechanism of toxicity remain unknown. By gathering available experimental data, albeit low resolution, a few pieces of the prion puzzle can be put in place. Currently, there are two fundamentally different models of a prion protofibril. One has its building blocks derived from a molecular dynamics simulation of the prion protein under amyloidogenic conditions, termed the spiral model. The other model was constructed by threading a portion of the prion sequence through a beta-helical structure from the Protein Data Bank. Here we compare and contrast these models with respect to all of the available experimental information, including electron micrographs, symmetries, secondary structure, oligomerization interfaces, enzymatic digestion, epitope exposure, and disaggregation profiles. Much of this information was not available when the two models were introduced. Overall, we find that the spiral model is consistent with all of the experimental results. In contrast, it is difficult to reconcile several of the experimental observables with the beta-helix model. While the experimental constraints are of low resolution, in bringing together the previously disconnected experiments, we have developed a clearer picture of prion aggregates. Both the improved characterization of prion aggregates and the existing atomic models can be used to devise further experiments to better elucidate the misfolding pathway and the structure of prion protofibrils.