Nuclear matrix involvement in sperm head structural organization

Summary— In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.     

Cloning and functional characterization of LCR-F1: a bZIP transcription factor that activates erythroid-specific,human globin gene expression.

DNase I hypersensitive site 2 (HS 2) of the human beta-globin Locus Control Region (LCR) directs high level expression of the beta-globin gene located 50 kilobases downstream. Experiments in cultured cells and in transgenic mice demonstrate that duplicated AP1-like sites in HS 2 are required for this powerful enhancer activity. A cDNA clone encoding a basic, leucine-zipper protein that binds to these sites was isolated and designated Locus Control Region-Factor 1 (LCR-F1). This protein is a member of a new family of regulatory factors that contain a 63 amino acid ''CNC domain'' overlapping the basic region. This domain is approximately 70% identical in the Drosophila Cap N Collar (CNC) protein, NF-E2 and LCR-F1. LCR-F1 transactivates an HS 2/gamma-globin reporter gene over 170-fold in transient transfection experiments specifically in erythroid cells. These results suggest that LCR-F1 may be a critical factor involved in LCR-mediated, human globin gene expression.     

Pollination Ecophysiology of Mercurialis annua L. (Euphorbiaceae), an Anemophilous Species Flowering all Year Round

Mercurialis annua L. is a dioecious anemophilous species thatflowers all year round in central and southern Italy. The flowersof both sexes are dimorphic: the female flower has a vestigialcalyx; the male flower consists only of a calyx that opens atanthesis. The anthers always dehisce after anthesis. The anthesisof male flowers seems to be temperature dependent, whereas antherdehiscence is related to relative humidity. The pollen grainsvary in volume according to the season: they are smaller whenrelative humidity is low and vice versa. They always decreasein volume after anther dehiscence and have the capacity to varyin volume and reach equilibrium with a changing environment.Viability is high, but may drop suddenly during heavy rain orhail that damage the exposed male flowers. The number of pollengrains per stigma varies from 0 to 300. The data is discussedin relation to the type of pollination and environmental characteristics.Copyright1994, 1999 Academic Press Mercurialis annua, dioecism, anthesis, anther dehiscence, pollen volume, pollen viability, anemophilous pollination, pollination ecology     

Single-cell investigation by laser scanning confocal microscopy of cytochemical alterations resulting from extracellular oxidant challenge

Confocal laser scanning fluorescence microscopy coupled to image analysis was employed in order to develop and evaluate procedures for the appraisal at the single-cell level of: (1) protein-bound 4-hydroxynonenal, the specific product of membrane peroxidation (by means of immunocytochemistry with biotin-avidin revelation); (2) protein oxidation (by reaction of protein carbonyls with 2,4-dinitrophenyl-hydrazine followed by immunocytochemistry of dinitrophenyl moieties); and (3) cellular protein thiols (by direct alkylation of sulfhydryl groups with thiol-specific fluorescent reagents possessing different cell permeabilities). The procedures proved able to reveal the subcellular distribution of cytochemical parameters useful as indices of oxidative stress conditions, and may allow redox phenotyping of isolated cells, which would provide an efficient tool in selected experimental models.     

High molecular weight RNA containing histone messenger in the sea urchin Paracentrotus lividus

Sea urchin RNA extracted from early and mesenchyme blastula embryos and oocytes and fractionated on denaturing sucrose density gradients, was hybridized with histone DNA recombinants of Psammechinus miliaris (clone λh22) and of Paracentrotus lividus (clone pPH70). Histone sequences are found in the 9 S and larger than 9 S regions of the formamide/sucrose density gradients. The melting of the RNA-DNA duplexes obtained by hybridization of polysomal and high molecular weight RNA of embryos of P. lividus at the stage of early blastula, suggests a degree of heterogeneity in the high M_r RNA. The high M_r RNA contains at least four of the five histone gene sequences covalently linked.     

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.     

Three types of ion channels are present on the plasma membrane of Friend erythroleukemia cells

In Friend murine erythroleukemia cells the presence of ion channels was investigated with the patch-clamp technique. During the first 48 hours after cell seeding, three types of ion channels, with the following order of membrane density, were found: i) a Ca2+-dependent K+ channel, fully activated at a cytosolic Ca2+ concentration of 10(-6) M and moderately activated at 10(-7)M; ii) a monovalent cation channel non voltage-activated, with an open-close kinetics dependent on the pressure gradient across the patch; iii) a chloride channel with a slow open-close kinetics. The latter two channels were labile and did not survive during intracellular perfusion. The membrane potential of the leukemia cells was not constant, but underwent large (tens of millivolts) fluctuations due to the opening of a few channels. The average resting membrane potential recorded in this study agrees with that measured in these cells by means of the accumulation ratio of the lipophilic cation Tetraphenylphosphonium.     

An assessment of red list data for the Pezizomycotina (Ascomycota): Umbria (Italy) as a test case

Abstract

A Red List of all 108 Pezizomycotina (Ascomycota) species recorded in Umbria Region (central Italy) is provided. According to the IUCN categories and criteria, 60.18% of the assessed species are classified as threatened, whereas 12.96% are Near Threatened (NT), 1.86% are Least Concerned (LC) and a noteworthy amount of 25% are Data Deficient (DD). As a consequence of the downlisting applied to the majority of the assessed taxa, according to the guidelines for application of IUCN red list criteria at Regional level, only 1.54% of the threatened species is Critically Endangered (CR), while 46.15% are Endangered (EN) and 52.31% are Vulnerable (VU). Given that the present work represents the first complete regional red list of Pezizomycotina in Italy, and that a national, as well as a European red list do not exist to date, it could be considered as a case study for other Italian Regions as well as for other European countries, aiming at the compilation of a national and European red list of this fungal group mostly overlooked in conservation strategies.     

Uncovering the evolutionary origin of blue anthocyanins in cereal grains

Functional divergence after gene duplication plays a central role in plant evolution. Among cereals, only Hordeum vulgare (barley), Triticum aestivum (wheat) and Secale cereale (rye) accumulate delphinidin‐derived (blue) anthocyanins in the aleurone layer of grains, whereas Oryza sativa (rice), Zea mays (maize) and Sorghum bicolor (sorghum) do not. The underlying genetic basis for this natural occurrence remains elusive. Here, we mapped the barley Blx1 locus involved in blue aleurone to an approximately 1.13 Mb genetic interval on chromosome 4HL, thus identifying a trigenic cluster named MbHF35 (containing HvMYB4H, HvMYC4H and HvF35H). Sequence and expression data supported the role of these genes in conferring blue‐coloured (blue aleurone) grains. Synteny analyses across monocot species showed that MbHF35 has only evolved within distinct Triticeae lineages, as a result of dispersed gene duplication. Phylogeny analyses revealed a shared evolution pattern for MbHF35 in Triticeae, suggesting that these genes have co‐evolved together. We also identified a Pooideae‐specific flavonoid 3′,5′‐hydroxylase (F3′5′H) lineage, termed here Mo_F35H2, which has a higher amino acid similarity with eudicot F3′5′Hs, demonstrating a scenario of convergent evolution. Indeed, selection tests identified 13 amino acid residues in Mo_F35H2 that underwent positive selection, possibly driven by protein thermostablility selection. Furthermore, through the interrogation of barley germplasm there is evidence that HvMYB4H and HvMYC4H have undergone human selection. Collectively, our study favours blue aleurone as a recently evolved trait resulting from environmental adaptation. Our findings provide an evolutionary explanation for the absence of blue anthocyanins in other cereals and highlight the importance of gene functional divergence for plant diversity and environmental adaptation.