A new method for the mapping of 5' ends of RNAs

In this article, we describe a new procedure to map 5′ ends of RNAs. The procedure consists in the use of specific RNase H digestion of a hybrid formed by the RNA and a complementary DNA oligonucleotide. Northern blot hybridization of the resulting RNA fragment allows an accurate measurement of its length. Although we generally use this procedure as a control of previously performed primer extension analyses, the absence of nonspecific bands, which often occur in primer extensions on RNA templates with extended secondary structures, suggests that our method may be preferable when these difficult templates are analyzed

A homemade device for linear sucrose gradients

We have developed a simple and inexpensive device to obtain linear sucrose gradients with commonly used laboratory materials--a syringe, a flask, a plastic tube, and a piece of Pongo (Play-Doh). Refractive index values measured on sucrose fractions collected using our system demonstrate both the linearity and reliability of the gradients obtained

Chromosomal localization and molecular characterization of three different 5S ribosomal DNA clusters in the sea urchin Paracentrotus lividus.

In this paper the chromosomal localization and molecular cloning and characterization of three 5S rDNA clusters of 700 bp (base pairs), 900 bp, and 950 bp in the sea urchin Paracentrotus lividus are reported. Southern blot hybridization demonstrated the existence of three 5S rDNA repeats of differing length in the P. lividus genome. Fluorescence in situ hybridization analysis, performed in parallel on both haploid and diploid metaphases and interphase nuclei using different 5S rDNA units as probes, localized these 5S rDNA clusters in 3 different pairs of P. lividus chromosomes. This is the first complete gene mapping not only in a sea urchin but also in the phylum of echinoderms as a whole

Improved resolution power of electrophoretic fractionation of DNA using a voltage gradient up and down application

The improved resolution power of electrophoretic fractionation of DNA in a wide range of molecular masses is demonstrated using an "up and down" application of voltage gradient gel electrophoresis (VGGE). This application also allows separation of different DNA fragments which are poorly fractionated in conventional electrophoresis

Increasing voltage gradient electrophoresis of DNA

We developed a method which allows electrophoretic fractionation of DNA in an agarose matrix according to an increasing current gradient, using a previously designed [R. Barbieri, V. Izzo, M.A. Costa, G. Giudice, G. Duro, Anal. Biochem. 212 (1993) 168; M.R. Asaro, V. Izzo, R. Barbieri, J. Chromatogr. A 855 (1999) 723] voltage gradient apparatus. This method allows the separation of different DNA fragments by increasing the distances of the components fractionated in the gel, revealing small differences in the length of different DNA components

Increased susceptibility to streptozotocin and impeded regeneration capacity of beta-cells in adult offspring of malnourished rats.

Background: Epidemiological studies related poor maternal nutrition and subsequent growth retardation in the progeny to the development of diabetes later in life. Low-protein diet during gestation altered the beta-cell development of the rat progeny by decreasing beta-cell proliferation and increasing their sensitivity to nitric oxide and cytokines in the foetus. This disturbed maternal environment had long-lasting consequences because the higher beta-cell vulnerability was maintained at adulthood. Aim: The aim of this study was to determine whether early malnutrition influences the vulnerability and the regeneration capacity of beta-cells after streptozotocin (STZ) damage at adulthood. Methods: Gestating rats were fed either a control or a low-protein diet until weaning. Adult female offspring received injections of Freund's adjuvant weekly for 5 weeks followed 24 h later by STZ. Half of the cohort was killed at d34, whereas the other half was maintained until d48 to analyse the regeneration capacity of the beta-cells. Results: Although control and low-protein rats had equivalent pancreatic insulin content and beta-cell volume density at d34, hyperglycaemia appeared earlier and was more dramatic in low-protein rats than in control rats. STZ treatment increased beta-cell proliferation similarly in both groups. At d48, apoptotic rate was higher in the low-protein group. Regeneration appeared in control, but not in the low-protein rats, where beta-cell aggregates/surface area and Reg1-positive area were decreased compared to control. Conclusion: Maternal malnutrition programmes a more vulnerable endocrine pancreas in the progeny which is unable to regenerate after injury, therefore predisposing it to develop glucose intolerance and diabetes later in life

Characterization of two alternative Interleukin(IL)-10 5_UTR mRNA sequences, induced by lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells

IL-10 production shows a broad-spectrum of individual response, suggesting a genetic component of approximately 75%. Different polymorphisms located close to, or within the IL-10 gene has been demonstrated to influence its transcription rate whereas the post-transcriptional regulation of IL-10 production has not well elucidated. The main responsible elements at this control level are both the 5\u2032- and 3\u2032-untranslated regions (UTR's) of mRNAs, and as the 3\u2032-UTR regions are mainly involved in the stability and decay rate of mRNAs, the 5\u2032-UTR regions mediate the binding rate of the molecule with ribosomal 40S subunit as a cis-acting element. Herein are report data on the identification of two IL10 mRNA that differ by the length of respective 5\u2032UTR regions (160 and 288 nucleotides, respectively; EMBL accession nrs: EU751618 and EU751619) produced after stimulation of human blood samples with bacterial lipopolysaccharide (LPS). The longer 5\u2032UTR is constitutively expressed in unstimulated PBMC cells cultured at 37 \ub0C for 24 h, while in LPS stimulated cells an additional IL-10 mRNA molecule, containing a shorter 5\u2032UTR, is synthesized. RNADRAW software (http://www.rnadraw.com/) analysis have indicated that the secondary structures of the shorter 5\u2032UTR IL-10 mRNA region is more available for the binding to the 40S ribosomal subunit. In conclusion, our data seem to suggest that LPS could influence the post-transcriptional control of IL-10 production inducing an alternative mRNA immediately available in response to the inflammatory stimulation