Recently duplicated maize R2R3 Myb genes provide evidence for distinct mechanisms of evolutionary divergence after duplication

R2R3 Myb genes are widely distributed in the higher plants and comprise one of the largest known families of regulatory proteins. Here, we provide an evolutionary framework that helps explain the origin of the plant-specific R2R3 Myb genes from widely distributed R1R2R3 Myb genes, through a series of well-established steps. To understand the routes of sequence divergence that followed Myb gene duplication, we supplemented the information available on recently duplicated maize (Zea mays) R2R3 Myb genes (C1/Pl1 and P1/P2) by cloning and characterizing ZmMyb-IF35 and ZmMyb-IF25. These two genes correspond to the recently expanded P-to-A group of maize R2R3 Myb genes. Although the origins of C1/Pl1 and ZmMyb-IF35/ZmMyb-IF25 are associated with the segmental allotetraploid origin of the maize genome, other gene duplication events also shaped the P-to-A clade. Our analyses indicate that some recently duplicated Myb gene pairs display substantial differences in the numbers of synonymous substitutions that have accumulated in the conserved MYB domain and the divergent C-terminal regions. Thus, differences in the accumulation of substitutions during evolution can explain in part the rapid divergence of C-terminal regions for these proteins in some cases. Contrary to previous studies, we show that the divergent C termini of these R2R3 MYB proteins are subject to purifying selection. Our results provide an in-depth analysis of the sequence divergence for some recently duplicated R2R3 Myb genes, yielding important information on general patterns of evolution for this large family of plant regulatory genes.     

Hepatitis B and C in the hemodialysis unit of Tocantins,Brazil: serological and molecular profiles

A survey was conducted in the hemodialysis population of the state of Tocantins, Brazil, aiming to assess the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, to analyze associated risk factors, and also to investigate these viruses genotypes distribution. During January and March 2001, all patients (n = 100) were interviewed at the unique dialysis unit in Tocantins. Blood samples were collected and serum samples were screened for HBV serological markers. Hepatitis B surface antigen positive samples were tested for HBV DNA. All samples were also tested for anti-HCV antibodies and HCV RNA. An overall prevalence of 45% was found for HBV infection (4% were HBsAg/anti-HBc positive, 2% were anti-HBc only and 39% had anti-HBc/anti-HBs markers). Concerning HCV infection, anti-HCV and HCV RNA were detected in 13% and 14% of the subjects, respectively. Three patients were HCV RNA positive and anti-HCV negative, resulting in an overall HCV prevalence of 16%. Univariate analysis of risk factors showed that only shift and length of tile on hemodialysis were associated with HBV and HCV positivity respectively. Among the four HBsAg-positive samples, HBV DNA was detected in three of them, which were identified as genotype A by restriction fragment length polymorphism (RFLP) analysis. All 14HCV RNA-positive samples were genotyped by INNO-LiPA. Genotypes la and 3a were found in 85% and 15%, respectively. The present data show low HBsAg and HCV prevalence rates. The risk factors associated with HBV and HCV positivity suggest that nosocomial transmission may influence in spreading these viruses in the dialysis unit studied.     

Growth and hepatic acetyl coenzyme-A carboxylase activity are affected by dietary protein level in European seabass (Dicentrarchus labrax)

Two trials were undertaken with European seabass (Dicentrarchus labrax) to estimate the protein requirements for maintenance and growth as well as the effect of dietary protein level on the activity of hepatic acetyl coenzyme-A carboxylase (ACoAC). Six diets were formulated to contain graded levels of protein (from 5 to 55% crude protein (CP)) at a constant (12%) lipid level. Three other diets were also formulated to contain 35, 45 and 55% CP, but with a higher lipid level (19%). Groups of 10 individually marked fish (IBW: 100 g) and groups of 8 fish (IBW: 160 g) were used in trial I and II, respectively. Fish were fed to visual satiety and intake was recorded. At the end of both studies, whole body, liver and plasma samples were withdrawn for analyses. Growth rate was improved with increasing dietary CP level. Despite not being the object of a statistical analysis, feed efficiency tended to be enhanced at higher dietary CP level and protein efficiency ratio tended to decrease with increased protein intake. The reduction of the dietary protein/energy ratio, due to the increase of dietary lipids further improved growth and feed utilisation. Data from both experiments indicate 4.5+/-0.5 g kg(-1) d(-1) as the daily protein intake for maximum N gain and 520+/-50 mg kg(-1) d(-1) as the maintenance needs for nitrogen balance. An increase of dietary CP level, up to 25%, increased ACoAC activity. A further increase in dietary CP level (35 to 55%) did not affect liver ACoAC activity. The increase in dietary lipid level depressed significantly liver ACoAC specific activity.     

Automated image analysis to improve bead ingestion toxicity test counts in the protozoan Tetrahymena pyriformis

AIMS: To improve bead ingestion counts in Tetrahymena pyriformis by automated image analysis as an alternative to direct-counts. METHODS AND RESULTS: Fluorescent latex beads were added to T. pyriformis cultures for ingestion tests. The number of beads ingested by 25 cells was counted directly by epifluorescence microscopy and compared with similar data from image analysis. anova indicated that counts were not significantly different (P < 0.05). The image analysis particularly provided advantages in terms of speed. CONCLUSIONS: The image analysis is superior to direct beads counting in T. pyriformis particularly in terms of speed of analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The image analysis method is very rapid and will allow many more toxicological analyses to be undertaken with less operator error.     

Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain

The genetic program that underlies the generation of visceral motoneurons in the developing hindbrain remains poorly defined. We have examined the role of Nkx6 and Nkx2 class homeodomain proteins in this process, and provide evidence that these proteins mediate complementary roles in the specification of visceral motoneuron fate. The expression of Nkx2.2 in hindbrain progenitor cells is sufficient to mediate the activation of Phox2b, a homeodomain protein required for the generation of hindbrain visceral motoneurons. The redundant activities of Nkx6.1 and Nkx6.2, in turn, are dispensable for visceral motoneuron generation but are necessary to prevent these cells from adopting a parallel program of interneuron differentiation. The expression of Nkx6.1 and Nkx6.2 is further maintained in differentiating visceral motoneurons, and consistent with this the migration and axonal projection properties of visceral motoneurons are impaired in mice lacking Nkx6.1 and/or Nkx6.2 function. Our analysis provides insight also into the role of Nkx6 proteins in the generation of somatic motoneurons. Studies in the spinal cord have shown that Nkx6.1 and Nkx6.2 are required for the generation of somatic motoneurons, and that the loss of motoneurons at this level correlates with the extinguished expression of the motoneuron determinant Olig2. Unexpectedly, we find that the initial expression of Olig2 is left intact in the caudal hindbrain of Nkx6.1/Nkx6.2 compound mutants, and despite this, all somatic motoneurons are missing. These data argue against models in which Nkx6 proteins and Olig2 operate in a linear pathway, and instead indicate a parallel requirement for these proteins in the progression of somatic motoneuron differentiation. Thus, both visceral and somatic motoneuron differentiation appear to rely on the combined activity of cell intrinsic determinants, rather than on a single key determinant of neuronal cell fate.     

Bioconversion of nitriles by Candida guilliermondii CCT 7207 cells immobilized in barium alginate

Nitrile degradation by Candida guilliermondii CCT 7207 using free and immobilized cell systems was compared. Different specific growth rates were observed for immobilized (mumax=0.021 h(-1)) and the free cells (mumax=0.029 h(-1)). The maximum specific rate of acetic acid formation was 0.387 h(-1) and 0.266 h(-1) for free and immobilized cells, respectively. Cell adhesion to the support materials was confirmed by scanning electron microscopy. When immobilized, the yeast was able to use high nitrile and amide concentrations (aliphatic and aromatic) as nitrogen sources. The results suggest that C. guilliermondii CCT 7207 presents a physiological pattern potentially useful for the bioremediation of polluted environments or for the bioproduction of amides and organic acid of high commercial value.     

Screening and characterization of Bacillus thuringiensis isolates from Brazil for the presence of coleoptera-specific cry genes

Forty-three Bacillus thuringiensis isolates from Brazil and 3 from Argentina were screened, using the polymerase chain reaction (PCR), for various coleoptera-specific cry genes. Seven isolates produced specific and/or nonspecific DNA fragments in a PCR reaction with primers specific for two coleopteran cry genes and 4 of these produced DNA fragments with primers specific for 7 known coleopteran cry genes. These isolates showed, by electron microscopy, the presence of spherical crystals. They also showed proteins of around 70 kDa which were immunologically similar to the Cry3Aa protein from B. thuringiensis subsp. tenebrionis. The 3 isolates from Argentina were toxic to T. molitor, and although no isolate from Brazil showed toxicity, they might show toxicity to another insect species.     

Deletion of follicle-stimulating hormone (FSH) receptor residues encoded by exon one decreases FSH binding and signaling in the rat

The rat FSH receptor (rFSHR) shares considerable homology with the rat LH receptor (rLHR), yet binds human FSH (hFSH) with high fidelity, suggesting that the binding determinant encoded by the rFSHR gene shares no homology with the analogous rLHR primary sequence, thereby affording specificity of ligand binding. Two such regions of primary sequence have been previously identified and studied by peptide challenge tests and immunoneutralization studies. We therefore implemented site-directed mutagenesis to delete the regions S9-N30 and D300-F315 of the mature rFSHR sequence. The mutant receptor (DeltarFSHR) cDNAs were expressed in insect cells. The large deletion DeltarFSHRS9-N30 and a smaller deletion, DeltarFSHRS9-S18, did not bind (125)I-hFSH. However, DeltarFSHRK19-R29 and DeltarFSHRD300-F315 bound (125)I-hFSH with an affinity indistinguishable from wild-type rFSHR. The deletion mutants DeltarFSHR S9-N30 or DeltarFSHRS9-S18 were not detectable on the cell surface by flow cytometry unless cells were sheared. Although (125)I-hFSH binding to DeltarFSHRK19-R29 was normal, this form of the receptor was defective for signal transduction whereas DeltarFSHRD300-F315 was not. Furthermore, neither region seems to be a specificity determinant, since their removal did not result in high-affinity binding of hCG to DeltarFSHR.     

Definition of minimal domains of interaction within the recombination-activating genes 1 and 2 recombinase complex

During V(D)J recombination, recognition and cleavage of the recombination signal sequences (RSSs) requires the coordinated action of the recombination-activating genes 1 and 2 (RAG1/RAG2) recombinase complex. In this report, we use deletion mapping and site-directed mutagenesis to determine the minimal domains critical for interaction between RAG1 and RAG2. We define the active core of RAG2 required for RSS cleavage as aa 1-371 and demonstrate that the C-terminal 57 aa of this core provide a dominant surface for RAG1 interaction. This region corresponds to the last of six predicted kelch repeat motifs that have been proposed by sequence analysis to fold RAG2 into a six-bladed beta-propeller structure. Residue W317 within this sixth repeat is shown to be critical for mediating contact with RAG1 and concurrently for stabilizing binding and directing cleavage of the RSS. We also show that zinc finger B (aa 727-750) of RAG1 provides a dominant interaction domain for recruiting RAG2. In all, the data support a model of RAG2 as a multimodular protein that utilizes one of its six faces for establishing productive contacts with RAG1.     

The effect of stirring and seeding on the AcPheLeuNH(2) synthesis and crystallization in a reversed micellar system

The present work describes the enzymatic synthesis and simultaneous crystallization of the dipeptide AcPheLeuNH(2) by alpha-chymotrypsin in a reversed micellar system of tetradecyltrimethylammonium bromide (TTAB)/heptane/octanol/carbonate buffer. The low solubility of the dipeptide in the micellar solution led to the formation and growth of needle-like crystals during the synthesis as soon as supersaturation was achieved. The crystallization process then followed a typical pattern, proceeding in three phases: nucleation, de-supersaturation, and re-equilibrium of saturation. Crystallization was followed by visual observation with an optical microscope, and the increase of crystal number and size was confirmed. Experiments showed that the supersaturation concentration decreases with the addition of AcPheLeuNH(2) seeds before the reaction, and also with a decrease of the stirring speed. It was also observed that the increase of both seed concentration and stirring advances the start of crystallization, so that the dipeptide is more quickly removed from solution. The consequent decrease in its loss through hydrolysis causes an increase in its yield. Both stirring and seeding could constitute important generic strategies for promoting crystallization of more soluble dipeptides during their synthesis in similar reversed micellar systems.