Specific sequences within arginine–glycine-rich domains affect mRNA-binding protein function

The discovery of roles for arginine methylation in intracellular transport and mRNA splicing has focused attention on the methylated arginine–glycine (RG)-rich domains found in many eukaryotic RNA-binding proteins. Sequence similarity among these highly repetitive RG domains, combined with interactions between RG-rich proteins, raises the question of whether these regions are general interaction motifs or whether there is specificity within these domains. Using the essential Saccharomyces cerevisiae mRNA-binding protein Npl3 (ScNpl3) as a model system, we first tested the importance of the RG domain for protein function. While Npl3 lacking the RG domain could not support growth of cells lacking Npl3, surprisingly, expression of the RG domain alone supported partial growth of these cells. To address the specificity of this domain, we created chimeric forms of ScNpl3 with RG-rich domains of S. cerevisiae nucleolar proteins, Gar1 and Nop1 (ScGar1, ScNop1), or of the Candida albicans Npl3 ortholog (CaNpl3). Whereas the CaNpl3 RG chimeric protein retained nearly wild-type function in S. cerevisiae, the ScGar1 and ScNop1 RG domains significantly reduced Npl3 function and self-association, indicating RG domain specificity. Nuclear localization of Npl3 also requires specific RG sequences, yet heterologous RG domains allow similar modulation of Npl3 transport by arginine methylation.     

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background  

The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes.     

Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR

Background

Linear regression of efficiency or LRE introduced a new paradigm for conducting absolute quantification, which does not require standard curves, can generate absolute accuracies of ±25% and has single molecule sensitivity. Derived from adapting the classic Boltzmann sigmoidal function to PCR, target quantity is calculated directly from the fluorescence readings within the central region of an amplification profile, generating 4–8 determinations from each amplification reaction.

Findings

Based on generating a linear representation of PCR amplification, the highly visual nature of LRE analysis is illustrated by varying reaction volume and amplification efficiency, which also demonstrates how LRE can be used to model PCR. Examining the dynamic range of LRE further demonstrates that quantitative accuracy can be maintained down to a single target molecule, and that target quantification below ten molecules conforms to that predicted by Poisson distribution. Essential to the universality of optical calibration, the fluorescence intensity generated by SYBR Green I (FU/bp) is shown to be independent of GC content and amplicon size, further verifying that absolute scale can be established using a single quantitative standard. Two high-performance lambda amplicons are also introduced that in addition to producing highly precise optical calibrations, can be used as benchmarks for performance testing. The utility of limiting dilution assay for conducting platform-independent absolute quantification is also discussed, along with the utility of defining assay performance in terms of absolute accuracy.

Conclusions

Founded on the ability to exploit lambda gDNA as a universal quantitative standard, LRE provides the ability to conduct absolute quantification using few resources beyond those needed for sample preparation and amplification. Combined with the quantitative and quality control capabilities of LRE, this kinetic-based approach has the potential to fundamentally transform how real-time qPCR is conducted.     

Microbial production host selection for converting second-generation feedstocks into bioproducts

Although many secondary metabolites with diverse biological activities have been isolated from myxobacteria, most strains of these biotechnologically important gliding prokaryotes remain difficult to handle genetically. In this study we describe the new fast growing myxobacterial thermophilic isolate GT-2 as a heterologous host for the expression of natural product biosynthetic pathways isolated from other myxobacteria. According to the results of sequence analysis of the 16S rDNA, this moderately thermophilic isolate is closely related to Corallococcus macrosporus and was therefore named C. macrosporus GT-2. Fast growth of moderately thermophilic strains results in shorter fermentation and generation times, aspects which are of significant interest for molecular biological work as well as production of secondary metabolites. Development of a genetic manipulation system allowed the introduction of the complete myxochromide biosynthetic gene cluster, located on a transposable fragment, into the chromosome of GT-2. Genetic engineering of the biosynthetic gene cluster by promoter exchange leads to much higher production of myxochromides in the heterologous host C. macrosporus GT-2 in comparison to the original producer Stigmatella aurantiaca and to the previously described heterologous host Pseudomonas putida (600 mg/L versus 8 mg/L and 40 mg/L, respectively).     

Mathematics of quantitative kinetic PCR and the application of standard curves

Fluorescent monitoring of DNA amplification is the basis of real-time PCR, from which target DNA concentration can be determined from the fractional cycle at which a threshold amount of amplicon DNA is produced. Absolute quantification can be achieved using a standard curve constructed by amplifying known amounts of target DNA. In this study, the mathematics of quantitative PCR are examined in detail, from which several fundamental aspects of the threshold method and the application of standard curves are illustrated. The construction of five replicate standard curves for two pairs of nested primers was used to examine the reproducibility and degree of quantitative variation using SYBER® Green I fluorescence. Based upon this analysis the application of a single, well- constructed standard curve could provide an estimated precision of ±6–21%, depending on the number of cycles required to reach threshold. A simplified method for absolute quantification is also proposed, in which quantitative scale is determined by DNA mass at threshold.     

Adeno-associated virus vector integration junctions.

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.     

Effect of osmotic stress on the developmental competence of germinal vesicle and metaphase II stage bovine cumulus oocyte complexes and its relevance to cryopreservation

The effects of osmotic stress on germinal vesicle (GV) and metaphase II (MII) stage bovine cumulus oocyte complexes (COCs) were evaluated by first exposing them to various anisotonic NaCl solutions (75, 150, 600, 1200, 2400, and 4800 +/- 5 mOsm/kg) for 10 min and then returning them to isotonic TL-Hepes solution (270 +/- 5 mOsm/kg) at 20 +/- 2 degrees C. Percentages of oocyte maturation, fertilization, polyspermy, cleavage, and blastocyst formation were measured as endpoints. Exposure to anisotonic conditions had a significant (P < 0.05) effect on the developmental competence of both GV and bovine MII COCs. Oocytes at the GV stage were more sensitive to anisotonic stress than MII oocytes (P < 0.05). None of the GV oocytes developed to the blastocyst stage after exposure to hypertonic conditions (2400 or 4800 mOsm solutions), while exposure to hypotonic conditions (75 or 150 mOsm solutions) resulted in significantly lower (P < 0.05) blastocyst formation (9% and 13%, respectively) compared to the isotonic control (25%). A dramatic decrease to 4% development to blastocyst was observed for MII oocytes following exposure to a 4800 mOsm solution. Blastocyst formation of MII oocytes which were exposed to 75, 150, 600, 1200, or 2400 mOsm solutions were similar (15%, 20%, 18%, 14%, and 13%, respectively; P > 0.05), but lower (P < 0.05) than those in the control group (29%). Exposing GV oocytes to anisotonic conditions increased polyspermic fertilization (P < 0.05), although MII oocytes were not similarly affected (P > 0.05). These data support the hypothesis that osmotic stress is detrimental to bovine oocytes and must be considered when developing optimized cryopreservation procedures for these cells. Mol. Reprod. Dev. 55:212-219, 2000.