Rainer Barbieri

Ribosomal DNA Location in the Scarab Beetle Thorectes Intermedius (Costa) (Coleoptera: Geotrupidae) Using Banding and Fluorescent in-situ Hybridization

Mitotic metaphase chromosomes of the scarab beetle Thorectes intermedius (Costa) (Coleoptera Scarabaeoidea: Geotrupidae) were analyzed using various banding methods and fluorescent in-situ hybridization (FISH) with a ribosomal probe. The results obtained indicate that silver and CMA3 staining are unable to localize the chromosome sites of nucleolar organizer regions (NORs). Such an inadequacy is a consequence of the extensive silver and CMA3 stainability of both constitutive heterochromatin and heterochromatin associated to the NORs.

High heterochromatin content in somatic chromosomes of two unrelated species of Diplopoda (Myriapoda)

For the first time, a conventional analysis of C-banded karyotypes was carried out in two distantly related diplopod species; this revealed an impressive percentage of heterochromatin in both genomes. In Acanthopetalum sicanum (Order Callipodida) (2n = 12), heterochromatin constitutes about 60% of the total DNA in females and 56% in males, whereas in Enologus oxypygum (Order Julida) (2n = 22) it is about 67% in both sexes. Heterochromatin of the two species was found to be similar in base composition (AT rich) and heterochromatin distribution, indicating that it has accumulated in a species-specific manner. Sex-determining mechanisms of the XY type were detected in both A. sicanum and E. oxypygum. In A. sicanum, the Y presented the lowest heterochromatic content of all chromosomes in the karyotype, whereas the X presented the highest.

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.

Organometallic Complexes with Biological Molecules: VIII. Synthesis, Solid State and in vivo Investigation of Triorganotin(IV) Derivatives of l-Homocysteic Acid

Several new triorganotin(IV) derivatives of L-homocysteic acid (L-HCAH) with formula R3Sn(L-HCA) (R=Me, nBu, Ph) have been synthesized. Their solid-state configurations were determined by IR and Mossbauer spectroscopy. The tin(IV) atom is five-coordinated in all the complexes, with the L-homocysteic acid behaving as a monoanionic bidentate ligand coordinating the tin(IV) atom through a chelating or bridging carboxylate group. The sulfonate (SO3−) and NH3+ groups of L-homocysteic acid maintain their free acid configuration and hence do not participate to the coordination of the tin(IV) atom. Coordination hypotheses have been checked through the correlation between the Mossbauer parameter isomer shift, δ, and partial atomic charge on the tin atoms, QSn, performed, for all the new organotin(IV) compounds, on the basis of an equalization procedure applied to idealized trigonal-bipyramidal structures for R3Sn(L-HCA). 1H and 13C NMR spectra of the complexes show that pentacoordination of the tin atom, with R groups in the equatorial plane of a trigonal bipyramid, is retained in DMSO solution. The NMR data confirm also that the uncoordinated NH3+ group of the ligand is still present in solution. Results gathered after exposure of two- to four-cell embryos of the sea urchin Paracentrotus lividus (Echinodermata) to the triorganotin(IV) L-homocysteate derivatives as well as to the parent triorganotin(IV) chlorides document cytotoxicity of the complexes, while free L-homocysteic acid exerts no significant toxic activity. The trimethyltin(IV) L-homocysteate derivative seems to exert a lower cytotoxicity than the tributyl- and triphenyl-tin(IV) ones. Different structural lesions have been identified by comparative analysis of mitotic chromosomes from untreated embryos (negative controls) and embryos treated with triorganotin(IV) L-homocysteate derivatives, such as (1) suppression of the stretch among sister chromatids at the beginning of anaphase stage; (2) deeply stained zones mainly located at the telomeric regions of chromosomes; (3) arm breakages; and (4) chromosome bridges among daughter chromosomes at anaphase stage. A colchicine-like effect of triorganotin(IV) L-homocysteate derivatives was observed.

Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

An inexpensive Plexiglas apparatus which allows a simple and rapid preparation of horizontal polyacrylamide gels of different dimensions for different purposes, is described. Preparation of such gels is as easy and rapid as agarose gel preparation, and polymerized polyacrylamide gels are used to fractionate proteins or small DNA fragments using a common horizontal electrophoretic tank. This apparatus was used to electrophoretically fractionate proteins or DNA for immuno-blot analyses, particularirly in the study of the allergenic response to Parietaria judaica pollen in senescence, for Southern-blot hybridizations and in the study of DNA polymorphisms.

Sequence analysis of the rDNA spacer of Paracentrotus lividus and observations about pre-rRNA processing: NTS sequence of Paracentrotus lividus rDNA

We have isolated and sequenced one intergenic region and a small part of the flanking regions (18S and 26S rRNA coding regions) of the rRNA-encoding genes (rDNA) from the sea urchinParacentrotus lividus. This region is about 3.8 Kb long. Northern blot hybridizations and S1 mapping experiments demonstrated the presence of a partially processed 21S rRNA precursor which has the same 5′ terminus as the 32S primary precursor, also in developmental stages characterized by a low rate of rRNA synthesis.

A method for eluting DNA in a wide range of molecular weights from agarose gels

We have developed a simple and rapid method for recovering DNAs of a wide range of molecular weights from agarose gels. A DNA-containing gel slice is placed on a Parafilm sheet in the center of a circular (positive) electrode and covered with a drop of buffer, while a linear (negative) electrode is placed on the top of the gel and driven about 1 mm into the gel itself. When a continuous current is applied, the DNA migrates into the buffer toward the circular electrode. We have obtained almost total recovery of DNAs up to 10 kb in size. Our method may also be used, under appropriate conditions, for higher molecular weight DNAs. The yield and all the biological assays performed on the DNAs obtained by our method recommend it for routine laboratory use.

Modified apparatus for voltage gradient gel electrophoresis

We built a modified version of voltage gradient gel electrophoresis system to correct distortions in nucleic acids electrophoretic migration patterns occurring at the edges of the gel when the original voltage gradient apparatus is used. The new device allows correct fractionation of nucleic acids also when electrophoresis is performed at high voltages.

Methods for recovering nucleic acid fragments from agarose gels

Agarose gel electrophoresis is a powerful technique for the separation of nucleic acids on the basis of their size and conformation. The development of methods to recover size-fractionated nucleic acids molecules from agarose gels has greatly facilitated recombinant DNA technologies. Although several methods for recovering DNA and RNA molecules have been developed during the past fifteen years, none of them has been universally accepted. In this review we describe, discuss and evaluate the most common procedures with which we have had experience. Our evaluation is based on the criteria of yield, purity, speed, simplicity and low cost. We have considered three different approaches to the problem of recovering nucleic acids: chemical gel dissolution, physical gel disruption and physical extrusion from intact gels.

Polymorphisms in the intergenic region of the sea urchin Paracentrotus lividus ribosomal DNA

Abstract Blot-hybridizations of the sea urchin Paracentrotus lividus genomic DNA with ribosomal DNA (rDNA) probes revealed individual variations in the length and in the sequence of the non-transcribed spacer (NTS) region. The number of rDNA repeat subclasses distinguishable within any individual sea urchin is usually limited (1 to 3) with respect to the widest polymorphism of the population as a whole. The heterogeneity in sequence is revealed by the presence or the absence of specific restriction sites in the spacer region. The data obtained by the intensity of the polymorphic bands indicate that different mechanisms bring about these two types of polymorphism. Preliminary data also indicate different methylation patterns in single individuals.