Absence of expression of the FMR-1 gene in fragile X syndrome

We previously reported the isolation of a gene (FMR-1) expressed in brain at the fragile X locus. One exon of this gene lies within an EcoRI fragment that exhibits length variation in fragile X patients. This exon also contains the CGG repeat within the CpG island hypermethylated in fragile X patients. To study the involvement of the FMR-1 gene in the fragile X syndrome, its expression was studied in lymphoblastoid cell lines and leukocytes derived from patients and normal controls. FMR-1 mRNA was absent in the majority of male fragile X patients, suggesting a close involvement of this gene in development of the syndrome. Normal individuals and carriers all show expression. The methylation status of the BssHII site at the CpG island was also studied by Southern blot analysis of DNA from patients, carriers, and controls. The minority of fragile X affected males that show expression of FMR-1 demonstrated an associated incomplete methylation of the BssHII site.     

Production of nonclassical inclusion bodies from which correctly folded protein can be extracted

Human granulocyte-colony stimulating factor (hG-CSF), an important biopharmaceutical drug used in oncology, is currently produced mainly in Escherichia coli. Expression of human hG-CSF gene in E. coli is very low, and therefore a semisynthetic, codon-optimized hG-CSF gene was designed and subcloned into pET expression plasmids. This led to a yield of over 50% of the total cellular proteins. We designed a new approach to biosynthesis at low temperature, enabling the formation of "nonclassical" inclusion bodies from which correctly folded protein can be readily extracted by nondenaturing solvents, such as mild detergents or low concentrations of polar solvents such as DMSO and nondetergent sulfobetaines. FT-IR analysis confirmed different nature of inclusion bodies with respect to the growth temperature and indicated presence of high amounts of very likely correctly folded reduced hG-CSF in nonclassical inclusion bodies. The yield of correctly folded, functional hG-CSF obtained in this way exceeded 40% of the total hG-CSF produced in the cells and is almost completely extractable under nondenaturing conditions. The absence of the need to include a denaturation/renaturation step in the purification process allows the development of more efficient processes characterized by higher yields and lower costs and involving environment-friendly technologies. The technology presented works successfully at the 50-L scale, producing nonclassical inclusion bodies of the same quality. The approach developed for the production of hG-CSF could be extended to other proteins; thus, a broader potential for industrial exploitation is envisaged.     

RNA interference can be used to disrupt gene function in tardigrades

How morphological diversity arises is a key question in evolutionary developmental biology. As a long-term approach to address this question, we are developing the water bear Hypsibius dujardini (Phylum Tardigrada) as a model system. We expect that using a close relative of two well-studied models, Drosophila (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda), will facilitate identifying genetic pathways relevant to understanding the evolution of development. Tardigrades are also valuable research subjects for investigating how organisms and biological materials can survive extreme conditions. Methods to disrupt gene activity are essential to each of these efforts, but no such method yet exists for the Phylum Tardigrada. We developed a protocol to disrupt tardigrade gene functions by double-stranded RNA-mediated RNA interference (RNAi). We showed that targeting tardigrade homologs of essential developmental genes by RNAi produced embryonic lethality, whereas targeting green fluorescent protein did not. Disruption of gene functions appears to be relatively specific by two criteria: targeting distinct genes resulted in distinct phenotypes that were consistent with predicted gene functions and by RT-PCR, RNAi reduced the level of a target mRNA and not a control mRNA. These studies represent the first evidence that gene functions can be disrupted by RNAi in the phylum Tardigrada. Our results form a platform for dissecting tardigrade gene functions for understanding the evolution of developmental mechanisms and survival in extreme environments.     

Absence of expression from RNA internalised into electroporated mammalian mitochondria

Transfection of mammalian mitochondria has proved to be notoriously difficult. Whilst there have been sporadic reports of import of foreign nucleic acids into isolated organelles, these imported nucleic acids have never been demonstrated to be functional. Inability to manipulate mitochondrial gene expression has hampered our understanding of RNA processing, maturation and translation in mitochondria. In an attempt to establish a model system for mt-RNA expression, we have electroporated rat liver mitochondria and mitoplasts in the presence of various RNA constructs built around the mitochondrial reporter gene mt-luciferase. Following electroporation, a fraction of the RNA was shown to be stably maintained, mitochondria remained coupled for oxidative phosphorylation and intramitochondrial protein synthesis was unaffected. In no case, however, was this RNA translated.     

Escherichia coli 4.5S RNA gene function can be complemented by heterologous bacterial RNA genes.

The essential 4.5S RNA gene of Escherichia coli can be complemented by 4.5S RNA-like genes from three other eubacteria, including both gram-positive and gram-negative organisms. Two of the genes encode RNAs similar in size to the E. coli species; the third, from Bacillus subtilis, specifies an RNA more than twice as large. The heterologous genes are expressed efficiently in E. coli, and the product RNAs resemble those produced by cognate cells. We conclude that the heterologous RNAs can replace E. coli 4.5S RNA and that the essential function of 4.5S RNA is evolutionarily conserved. A consensus structure is presented for the functionally related 4.5S RNA homologs.     

Protein expression can be monitored in yeast by peptide-mediated induction of TetR-controlled gene expression

The rapidly increasing number of completed genome sequences urgently calls for convenient and efficient methods for analysis of gene function and expression. TetR-inducing peptides (TIP) can induce reporter gene expression controlled by Tet repressor (TetR) when fused to a protein of choice which makes them a highly valuable tool for monitoring expression in vivo. However, TIP functionality has only been demonstrated in bacteria so far. Here, we report that TIP is also functional in yeast. An mCherry-TIP fusion that locates to the nucleus induces TetR-controlled gfp+ expression in a dose-dependent manner. This opens up potential applications in proteome research in which the expression of proteins can be analyzed in vivo by fusing TIP to proteins of choice in conjunction with a Tet-controlled reporter system.     

The early expression of glycoprotein B from herpes simplex virus can be detected by antigen-specific CD8+ T cells

The immune response to cutaneous herpes simplex virus type 1 (HSV-1) infection begins with remarkable rapidity. Activation of specific cytotoxic T lymphocytes (CTL) begins within hours of infection, even though the response within the draining lymph nodes peaks nearly 5 days later. HSV gene products are classified into three main groups, alpha, beta, and gamma, based on their kinetics and requirements for expression. In C57BL/6 mice, the immunodominant epitope from HSV is derived from glycoprotein B (gB(498-505)). While gB is considered a gamma or "late" gene product, previous reports have indicated that some level of gene expression may occur soon after infection. Using brefeldin A as a specific inhibitor of viral antigen presentation to major histocompatibility complex class I-restricted CTL, we have formally addressed the timing of gB peptide expression in an immunologically relevant manner following infection. Presentation of gB peptide detected by T-cell activation was first observed within 2 h of infection. Comparison with another viral epitope expressed early during infection, HSV-1 ribonucleotide reductase, demonstrated that gB is presented with the same kinetics as this classical early-gene product. Moreover, this rapidity of gB expression was further illustrated via rapid priming of na?ve transgenic CD8(+) T cells in vivo after HSV-1 infection of mice. These results establish that gB is expressed rapidly following HSV-1 infection, at levels capable of effectively stimulating CD8(+) T cells.     

Modifications of gene expression detected in peripheral blood after brain ischemia treated with remote postconditioning

Molecular Biology Reports - A stroke is an acute damage to a certain area of a nerve tissue of the brain. In developed countries, it ranks second among the most often causes of death and is also...     

Mannitol-specific carrier protein from the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system can be extracted as a dimer from the membrane

The association state of the mannitol-specific enzyme II (EIIMtl) has been studied both in the purified form and embedded in the cytoplasmic membrane. Membrane fragments obtained from mannitol-grown Escherichia coli catalyze the phosphoenolpyruvate- (PEP) dependent phosphorylation of both glucose and mannitol; thus they contain both the glucose- and mannitol-specific enzymes II. The autoradiogram of an electrophoresed mixture of [32P]PEP, EI, HPr, and membrane fragments shows bands at 58 and 116 kilodaltons, in addition to the bands of P-EI and P-HPr. In an analogous experiment with purified EIIMtl, suspended in detergent micelles, only a 58 000-dalton band and the P-HPr and P-EI bands were found. Treatment of the phosphorylated membranes with mannitol results in an immediate substantial decrease in the radioactivity in the 58- and 116-kilodalton bands. A similar treatment of the phosphorylated membranes with glucose had no direct effect on the autoradiogram. We conclude therefore that the 58- and 116-kilodalton bands originate from enzyme IIMtl monomers and dimers, respectively. The interaction between the subunits of the dimer is not abolished by the addition of up to 5% sodium dodecyl sulfate. However, the nonionic detergent Lubrol PX, which is present during the purification of EIIMtl, is capable of transforming the enzyme IIMtl dimers into monomers.