Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication

Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3D(pol), in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3D(pol)-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3D(pol) was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3D(pol) could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3D(pol) to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu(6) to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3D(pol) in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.     

Cis-acting elements stimulating kinetoplastid guide RNA-directed editing

The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created through the insertion and deletion of specific uridylates. The editing reactions are required to be highly specific in order to ensure that functional open reading frames are created in edited mRNAs and that potentially deleterious modification of normally nonedited sequence does not occur. Selection-amplification and mutagenesis were previously used to identify the optimal sequence requirements for in vitro editing. There is, however, a minority of natural editing sites with suboptimal sequence. Several cis-acting elements, obtained from an in vitro selection, are described here that are able to compensate for a suboptimal editing site. An A + U sequence element within the 5'-untranslated region of cytochrome b mRNA from Leishmania tarentolae is also demonstrated to function as a cis-acting guide RNA and is postulated to compensate for a suboptimal editing site in vivo. Two proteins within an enriched editing extract are UV-cross-linked to two different in vitro selected editing substrates more efficiently than poorly edited RNAs. The results suggest that these proteins contribute to the specificity of the editing reaction.     

Quantitative trait loci for susceptibility to tapeworm infection in the red flour beetle

Parasites have profound effects on host ecology and evolution, and the effects of parasites on host ecology are often influenced by the magnitude of host susceptibility to parasites. Many parasites have complex life cycles that require intermediate hosts for their transmission, but little is known about the genetic basis of the intermediate host's susceptibility to these parasites. This study examined the genetic basis of susceptibility to a tapeworm (Hymenolepis diminuta) in the red flour beetle (Tribolium castaneum) that serves as an intermediate host in its transmission. Quantitative trait loci (QTL) mapping experiments were conducted with two independent segregating populations using amplified fragment length polymorphism (AFLP) markers and randomly amplified polymorphic DNA (RAPD) markers. A total of five QTL that significantly affected beetle susceptibility were identified in the two reciprocal crosses. Two common QTL on linkage groups 3 and 6 were identified in both crosses with similar effects on the phenotype, and three QTL were unique to each cross. In one cross, the three main QTL accounted for 29% of the total phenotypic variance and digenic epistasis explained 39% of the variance. In the second cross, the four main QTL explained 62% of the variance and digenic epistasis accounted for only 5% of the variance. The actions of these QTL were either overdominance or underdominance. Our results suggest that the polygenic nature of beetle susceptibility to the parasites and epistasis are important genetic mechanisms for the maintenance of variation within or among beetle strains in susceptibility to tapeworm infection.     

Production of glucose oxidase using Aspergillus niger and corn steep liquor

Glucose oxidase production was optimized using an isolated strain of Aspergillus niger and an economical nutrient source, corn steep liquor (CSL). The culture produced 580 +/- 30 units/ml of the enzyme using 70 g/l sucrose as the carbon source. Using CSL as the sole nutrient source enzyme synthesis was increased to 640 +/- 36 units/ml. None of the nitrogen sources (nitrates of calcium, sodium, ammonium, potassium and yeast extract, malt extract, and peptone) was beneficial to the enzyme synthesis. Aeration and agitation enhanced enzyme synthesis to 850 +/- 45 units/ml. Glucose oxidase has numerous applications in food industry and clinical fields.     

Immobilisation of cyclodextrin glucanotransferase from <Emphasis Type="Italic">Bacillus circulans</Emphasis> ATCC 21783 on purified seasand

Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans (ATCC 21783) was immobilised on a silica-based support: purified seasand. Although adsorption of 98% was achieved, considerable desorption was encountered. This problem was minimised by crosslinking the adsorbed enzyme with glutaraldehyde. The immobilised enzyme after crosslinking could be used repeatedly for cyclodextrin (CD) production in a batch process. The activity retention was 80% at the end of the eighth cycle. The immobilised enzyme showed a shift in the pH optimum towards the alkaline side and also an improvement in the pH stability compared to the free enzyme. It catalysed the formation of β-CD as a major product. A significant amount of α-CD production was also observed on prolonged incubation. Electronic Publication     

D-cbl, a negative regulator of the Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis

During Drosophila oogenesis, asymmetrically localized Gurken activates the EGF receptor (Egfr) and determines dorsal follicle cell fates. Using a mosaic follicle cell system we have identified a mutation in the D-cbl gene which causes hyperactivation of the Egfr pathway. Cbl proteins are known to downregulate activated receptors. We find that the abnormal Egfr activation is ligand dependent. Our results show that the precise regulation of Egfr activity necessary to establish different follicle cell fates requires two levels of control. The localized ligand Gurken activates Egfr to different levels in different follicle cells. In addition, Egfr activity has to be repressed through the activity of D-cbl to ensure the absence of signaling in the ventral most follicle cells.     

Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat)

The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin-JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P2' of substrates, are similar to those of penicillopepsin-JT1, endothiapepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate-enhancing effect was due to a hydrogen bond between the -NH- of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C-terminal to Asp215. Thr219 of penicillopepsin-JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in k(cat) when a P3 residue was present in the substrate, which was similar to that of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the -NH- of P3 to the enzyme, the scissile peptide bond between P1 and P1' becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.