Interaction of PRK1 Receptor-like Kinase with a Putative elF2B β-Subunit in Tobacco

PRK1, a receptor-like kinase that is expressed in pollen, pollen tubes, and ovaries, has been shown to play important roles in pollen development and embryo sac development in Petunia inflata. We have used the kinase domain of PRK1 as a bait in the yeast two-hybrid system to identify PRK1-interacting proteins. The screening resulted in isolation of a cDNA encoding a protein highly homologous to the human and yeast beta-subunit of translation initiation factor 2B (eIF2B-beta), which was designated NeIF2Bbeta. eIF2B is a guanine nucleotide exchange protein that functions in the regulation of translation in eukaryotic cells. Deletion mutants of NeIF2Bbeta were analyzed for their interaction with PRK1, and the results suggested that the N-terminal half of NeIF2Bbeta, especially the region between residue 103 and 235, is important for the interaction. This protein association was confirmed by in vitro binding assay of the recombinant NeIF2Bbeta and PRK1 proteins. Despite high sequence homology between NeIF2Bbeta and its yeast counterpart, the NeIF2Bbeta cDNA could not rescue the phenotype of the yeast mutant strain lacking the GCD7 gene encoding eIF2B-beta, when transferred into the mutant strain.     

Ectopic expression of β-lactoglobulin transgenes

Genomic constructs comprising the ovine β-lactoglobulin gene are expressed in a position-independent manner in the mammary gland of transgenic mice. In some lines however, constitutive low-level transgene expression was detected in all other tissues. This ectopic expression presumably represents a position-dependent phenomenon since it was observed in only a proportion (40%) of the lines generated. Different lines of BLG transgenic mice displayed similar temporal patterns of ectopic expression. This pattern differed from that of BLG in the mammary gland. These data imply that the DNA elements that direct position-independent expression of β-lactoglobulin transgenes in the mammary gland do not have the ability to insulate them from position effects in other tissues. Furthermore, the relatively high frequency and constitutive nature of ectopic expression suggests that transgene integration may not be totally random.     

The expression of three desaturase genes of Spirulina platensis in Escherichia coli DH5α – Heterologous expression of Spirulina-desaturase genes

The genes from a cyanobacterium--Spirulina platensis strain C1--that encode the acyl-lipid desaturases (desC, desA and desD) involved in gamma-linolenic (GLA) synthesis have been successfully expressed for the first time in Escherichia coli by employing a pTrcHisA expression system. In this report, the authors describe the expression of the three Spirulina N-terminal 6xHis-desaturases as well as the functional analysis of these recombinant proteins. The gene products of desC, desA and desD have approximate molecular masses of 37, 45, and 47 kDa, respectively. Enzymatic activity measurement of these products was carried out in vivo to demonstrate that (i) the expressed proteins are in functional form, and (ii) the cofactors of the host system can complement the system of Spirulina platensis. The study demonstrated that the gene products of desC and desA catalyzed the reactions in vivo where the enzyme substrates were provided in appropriate concentration. This indicates that the delta9 and delta12 desaturases were expressed in the heterologous host in their active form, and that these two reactions can be carried out in an E. coli host cell using its cofactors system. In contrast, delta6 desaturase activity can be detected only in vitro where electron carriers are provided. This suggests that while this enzyme is expressed in the heterologous host in its active form, its function in vivo is suppressed, as the electron carriers of the host system cannot complement the system of Spirulina platensis.     

Gene expression profiling – Clusters of possibilities

Advances in qPCR technology allow studies of increasingly large systems comprising many genes and samples. The increasing data sizes allow expression profiling both in the gene and the samples dimension while also putting higher demands on sound statistical analysis and expertise to handle and interpret its results. We distinguish between exploratory and confirmatory statistical studies. In this paper we demonstrate several techniques available for exploratory studies on a system of Xenopus laevis development from egg to tadpole. Techniques include hierarchical clustering, heatmap, principal component analysis and self-organizing maps. We stress that even though exploratory studies are excellent for generating hypotheses, results have not been proven statistically significant until an independent confirmatory study has been performed. An exploratory study may certainly be valuable in its own right, and there are often not enough resources to report both an exploratory and a confirmatory study at the same time. However, exploratory and confirmatory studies are intimately connected and we would like to raise that awareness among qPCR practitioners. We suggest that scientific reports should always have a hypothesis focus. Reports are either hypothesis generating, from an exploratory study, or hypothesis validating, from a confirmatory study, or both. In either case, we suggest the generated or validated hypotheses be specifically stated.     

Interaction of PRK1 Receptor-like Kinase with a Putative elF2B β-Subunit in Tobacco

PRK1, a receptor-like kinase that is expressed in pollen, pollen tubes, and ovaries, has been shown to play important roles in pollen development and embryo sac development in Petunia inflata. We have used the kinase domain of PRK1 as a bait in the yeast two-hybrid system to identify PRK1-interacting proteins. The screening resulted in isolation of a cDNA encoding a protein highly homologous to the human and yeast -subunit of translation initiation factor 2B (eIF2B-), which was designated NeIF2B. eIF2B is a guanine nucleotide exchange protein that functions in the regulation of translation in eukaryotic cells. Deletion mutants of NeIF2B were analyzed for their interaction with PRK1, and the results suggested that the N-terminal half of NeIF2B, especially the region between residue 103 and 235, is important for the interaction. This protein association was confirmed by in vitro binding assay of the recombinant NeIF2B and PRK1 proteins. Despite high sequence homology between NeIF2B and its yeast counterpart, the NeIF2B cDNA could not rescue the phenotype of the yeast mutant strain lacking the GCD7 gene encoding eIF2B-, when transferred into the mutant strain.     

Effects of pH on expression and stabilization of β-galactosidase by recombinantE. coli with a thermally-inducible expression system

Summary Effects of pH on -galactosidase expression and stabilization were investigated using recombinantE. coli harboring an expression vector with a thermally-inducible P_L promoter. Expression of -galactosidase was strongly promoted by lowering culture pH when culture temperature was raised to the induction temperature. Optimal pH for induction ranged from 5.4–5.8. The degradation of expressed -galactosidase could be reduced by lowering the culture temperature while at the same time slightly increasing the culture pH in the -gal degradation stage.     

Genetic expression of β-mannosidase activity in different tissues of mice

Beta-Mannosidase activity of liver, kidney, and spleen of two inbred strains of mice and their crosses has been assayed with the synthetic aubstrate p-nitrophenyl-beta-d-mannoside. Activity is low in C57BL/Kl mice and high in DBA/2/Kl mice. Hybrid animals have intermediate levels of beta-mannosidase activity. Segregation of enzyme activities occurs in the F-2 and backcross generations, and there are good correlations between acitities in the three tissuses of F-2 and backcross animals. Some evidence points to a single gene difference in crosses between C57BL and DBA with respect to this mannosidase variation. Curves for enzyme activities at different substrate concentrations and pHs obtained with preparations from DBA and C57BL mice show some differences. These are interpreted as a possible strain variation in a structural gene for this enzyme.     

Two approaches to construct mammalian expression vector of shRNA to reduce expression and replication of HBV in vitro

Two approaches have been developed to construct plasmids that mediate RNA interference to inhibit the replication and expression of HBV in 2.2.15 cell. The overlapping PCR extension and restriction enzyme-digestion were used to generate DNA fragments encoding designed shRNA based on sequences of ORF C of HBV genome. The pU6 derived vectors were constructed to develop plasmid based shRNA delivery systems termed pU6/HBVi. There were significant reductions in the expression of HBsAg and HBeAg between cells transfected with pU6/HBVi and control groups (as to HBsAg: P < 0. 01; and HBeAg: P < 0. 01). Consistently, the HBV DNA copies were reduced from 2.71 x 10(7) to <5 x 10(2) copies with or without pU6/HBVi. These results suggested that shRNA delivery by recombinant plasmids harboring shRNA encoding DNA fragment of interest generated either by overlapping PCR extension or restriction enzyme-digestion, could inhibit expressions of viral proteins and reduce viral replications. The pU6 derived plasmids might be a useful shRNA delivery system in mammalian cells. In addition, we found siRNA based on stealth 2311 was a potent RNAi target of HBV genome.     

Developmental expression of β-glucosidase in olive leaves

Plant β-glucosidases catalyze the hydrolysis of glycosidic linkages and play a vital role in defense against pathogens and stress. The present work investigated the relationship between leaf development and β-glucosidase protein content in Olea europea L. (cv. Picual) leaves. The total chlorophyll content increased with leaf age in current-season leaves. Immunoblot analysis revealed that the content of 61 kD protein of β-glucosidase also increased with leaf age, and that the enzyme existed in three isoforms (pI 5.8–6.2). Statistical analysis indicated a strong correlation between chlorophyll and β-glucosidase protein contents.     

Cloning and expression of a β-galactosidase gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta(?). Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

The expression of β-glucuronidase during preimplantation development of mouse embryos

β-Glucuronidase activity was measured in mouse embryos during the preimplantation period of development by using a microfluorometric assay. A 100-fold increase in activity was observed between 57 (8-cell stage) and 84 hr (morulae) of development. Activity changes between 30 and 60 hr were also significant. Genetic variants of β-glucuronidase occur between the strains of mice $\text{C57BL}\text{6J}$ and $\text{C3H}\text{HeJ}$ which differ in levels of activity and heat denaturation kinetics. Activity changes and heat denaturation kinetics of β-glucuronidase in $\text{C57BL}\text{6}\text{,}\text{C3H}\text{HeJ}$ and F₁ hybrid embryos were compared, and it was demonstrated that paternal genes were expressed during the 100-fold increase in activity and that embryonic genes may be functioning between 30 and 60 hr of development.