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.

Shuttling of Preribosomal Subunits during Sea Urchin Development

Mung Bean nuclease mapping of RNAs 3' end

Abstract A method is described that allows an accurate mapping of 3' ends of RNAs. In this method a labeled DNA probe, containing the presumed 3' end of the RNA under analysis is allowed to anneals to the RNA itself. Mung-bean nuclease is then used to digest single strands of both RNA and DNA. Electrophoretic fractionation of "protected" undigested, labeled DNA is than performed using a sequence reaction of a known DNA as length marker. This procedure was applied to the analysis of both a polyA RNA (Interleukin 10 mRNA) and non polyA RNAs (sea urchin 18S and 26S rRNAs). This method might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pat…

Orthogonal electrophoretic fractionation of DNA in agarose gels.

We developed an electrophoretic procedure, using Voltage Gradient Gel Electrophoresis (VGGE), which allows to obtain both an improvement of the resolution power of the system in orthogonal fractionation of DNA and, mainly, an about fourfold enhancement of hybridization signals in Southern blotting applications.

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…

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

Abstract 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′- and 3′-untranslated regions (UTR's) of mRNAs, and as the 3′-UTR regions are mainly involved in the stability and decay rate of mRNAs, the 5′-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…

A homemade cytospin apparatus

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.