Chromosome localization and characterization of a family of long interspersed repetitive DNA elements from the genus Zea

This paper describes the characterization and chromosomal distribution of new long repetitive sequences present in all species of the genus Zea. These sequences constitute a family of moderately repetitive elements ranging approximately from 1350 to 1700 copies per haploid genome in modern maize (Zea mays ssp. mays) and teosinte (Zea diploperennis), respectively. The elements are long, probably larger than 9 kb, and they show a highly conserved internal organization among Zea subspecies and species. The elements are present in all maize chromosomes in an interspersed pattern of distribution, are absent from centromeric and pericentric heterochromatin, and with some clustering in the distal regions of chromosome arms.     

Genomic structure and chromosomal location of the mouse pre-T-cell receptor alpha gene

The mouse pre-T-cell receptor alpha (pT) chain is a 33 000 M _r glycoprotein expressed on the surface of immature thymocytes as a disulfide-linked heterodimer with the T-cell receptor beta (TCR) chain, and in association with proteins of the CD3 complex. The cDNA for pT, isolated previously, encodes a type I transmembrane protein that is a member of the immunoglobulin (Ig) superfamily. Here we report the complete nucleotide sequence, the exon/intron structure, and the chromosomal location of the pTa gene. The gene spans about 8.4 kilobases (kb) and consists of four exons. Exon 1 encodes the 5 untranslated region, the leader peptide, and the first three amino acids of the mature protein. This exon is followed by a relatively long intron of 4.9 kb that contains many short interspersed repeats (SINEs) of the B1 and B2 family. The second exon encodes the extracellular Ig-like domain and exon 3 with just 45 base pairs the connecting peptide (CP), including the cysteine required for heterodimer formation. A similar exon/intron structure encoding corresponding parts of the mature polypeptide is found both in the Tcra and Tcrd constant region genes. The last exon encodes the transmembrane portion, the cytoplasmic tail, and about 540 nucleotides of 3 untranslated sequence, including a B2 repetitive element. In situ hybridization maps the pTa gene to the D/E1 region of mouse chromosome 17.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U27268     

Analysis of human V H gene repertoire expression in peripheral CD19+ B cells

Using CD19 B-cell selection and polymerase chain reaction-amplified cDNA libraries, we analyzed the peripheral immunoglobulin heavy chain variable repertoire of three healthy adult donors. Here we report that most of the CD19+ circulating B cells expressed unmutated V _H-D-J_H rearrangements. By specific V _H family hybridization, we show that V _H gene family utilization in the periphery roughly corresponds to the complexity of these families in the germline and appears to be relatively constant among the analyzed subjects. However, sequence data of clones picked at random from one IgM cDNA library reveals that in spite of this random utilization, the V _H gene expression in naive circulating B cells is highly biased towards the expression of a limited set of V _H genes. As previously reported by others, this restricted mechanism is also found for the D and J _H segments.The nucleotide sequence data reported in this paper have been submitted to the GenBank/EMBL nucleotide sequence database and have been assigned the accession numbers Z47213-Z47243 and Z47349     

In vitro influence of zinc and magnesium on the deformability of red blood cells artificially hardened by heating

Trace elements have been shown to improve red blood cell (RBC) deformability: zinc in sickle cell disease and magnesium in an in vitro model of chemically rigidified erythrocytes. In this study, we investigated the effect and the influence of incubation time of zinc or magnesium on an in vitro model of rigidified RBCs by heating. Erythrocyte rigidity was determined by viscosimetry at high shear rate by a falling ball viscosimeter MT 90. In the first part of the study, six normal volunteers participated. Viscosimetry was performed on native blood before and after heating the sample for 10 min at 50°C. Therefore, increasing concentrations of zinc gluconate (final concentration: 0.5–4 g/L) or isotonic NaCl as control medium were added to the sample. Heating induced a twofold increase in all indices of RBC rigidity (p<0.05). At all these concentrations of zinc, a highly significant, dose-related fluidifying effect was observed (40–70%): this effect was immediately obtained and did not change over 60 min. Even at the highest concentration, recovery was not complete. In the second part of the study, we studied magnesium’s effects on blood. In a first protocol, whole blood was rigidified by heating at 56°C for 10 min, and the correcting effect of 5 min of incubation at 37°C of RBCs in 150 mmol/L NaCl, MgSO₄, magnesium acetate, and magnesium gluconate was investigated. In a second protocol, the same incubation with NaCl and magnesium salts was made on blood that had not been previously heated. In a third protocol, the correcting effect of magnesium gluconate on heated red blood cells was tested at four concentrations (75, 150, 225, and 300 mmol/L) over 1 h, for evaluating the effects of both concentration and time. Erythrocyte rigidity by heating is corrected by the three salts employed in protocol 1 (compared to sodium). In protocol 2, the deformability of normal (nonheated) red cells is not modified by magnesium. In protocol 3, no marked modification over 1 h is observed. The correcting effect is not complete for 75 mmol/L Mg, but remains the same at the three other concentrations. This study shows that zinc and magnesium at supraphysiological concentration are able to reverse RBC’s rigidification induced by heating, but that magnesium does not modify the flexibility of normal RBCs. This article suggests that zinc and magnesium may be studied in vivo as potential pharmacologic tools for improving hemorheologic disturbances.     

Chemical synthesis,expression and mutagenesis of a gene encoding β-cryptogein,an elicitin produced by Phytophthora cryptogea

Elicitins are 10 kDa holoproteins secreted by Phytophthora fungi, that elicit an incompatible hypersensitive reaction, leading to resistance against fungal and bacterial plant pathogens. Comparison of primary sequences of -elicitins and -elicitins indicated several potential necrotic activity-determining residues. All of the highly necrotic -elicitins have a hydrophilic residue (usually lysine) at position 13, whereas in the less necrotic -elicitins this residue is replaced by a valine. Here, we report the synthesis and expression of a gene encoding a highly necrotic elicitin, -cryptogein, and we show that the substitution of Lys-13 of this recombinant protein by a valine leads to a drastic alteration to the necrotic activity of the recombinant protein.     

Characterisation of protoplasts from hypocotyls of Helianthus annuus in relation to their tissue origin

Cells and protoplasts isolated from three different tissues of sunflower hypocotyls and cultured either in liquid or agarose medium were compared in terms of their volume, DNA content, division potential and embryoid formation. Epidermal and external cortical cells differ from other tissue cells by their small size, their weak response to plasmolysis and their low DNA content (around 1C). They contribute only very weakly to the dividing protoplast population. In contrast, protoplasts from cortical and medullar cells both have similar division potential, reaching 50%. The nuclear DNA content of these two cell types, as well as their corresponding protoplasts, has a 2C value, taking root tip cells in G0 phase as standard. The culture conditions induce the same specific response in protoplasts isolated from both tissues: exclusively loose colony formation in liquid medium, and mainly production of embryoids in agarose medium.     

Speculations on the origin of life and thermophily: Review of available information on reverse gyrase suggests that hyperthermophilic procaryotes are not so primitive

All present-day hyperthermophiles studied so far (eitherBacteria orArchaea) contain a unique DNA topoisomerase, reverse gyrase, which probably helps to stabilize genomic DNA at high temperature. Herein the data relating this enzyme is reviewed and discussed from the perspective of the nature of the last detectable common ancestor and the origin of life. The sequence of the gene encoding reverse gyrase from an archaeon,Sulfolobus acidocaldarius, suggests that this enzyme contains both a helicase and a topoisomerase domains (Confalonieriet al.,Proc. Natl. Acad. Sci., 1993, 90, 4735). Accordingly, it has been proposed that reverse gyrase originated by the fusion of DNA helicase and DNA topoisomerase genes. If reverse gyrase is essential for life at high temperature, its composite structure suggests that DNA helicases and topoisomerases appeared independently and first evolved in a mesophilic world. Such scenario contradicts the hypothesis that a direct link connects present day hyperthermophiles to a hot origin of life. We discuss different patterns for the early cellular evolution in which reverse gyrase appeared either before the emergence of the last common ancestor ofArchaea, Bacteria andEucarya, or in a lineage common to the two procaryotic domains. The latter scenario could explain why all today hyperthermophiles are procaryotes.     

The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold.

The 3-D structure of Bacillus cereus (569/H/9) beta-lactamase (EC 3.5.2.6), which catalyses the hydrolysis of nearly all beta-lactams, has been solved at 2.5 A resolution by the multiple isomorphous replacement method, with density modification and phase combination, from crystals of the native protein and of a specially designed mutant (T97C). The current model includes 212 of the 227 amino acid residues, the zinc ion and 10 water molecules. The protein is folded into a beta beta sandwich with helices on each external face. To our knowledge, this fold has never been observed. An approximate internal molecular symmetry is found, with a 2-fold axis passing roughly through the zinc ion and suggesting a possible gene duplication. The active site is located at one edge of the beta beta sandwich and near the N-terminal end of a helix. The zinc ion is coordinated by three histidine residues (86, 88 and 149) and a water molecule. A sequence comparison of the relevant metallo-beta-lactamases, based on this protein structure, highlights a few well-conserved amino acid residues. The structure shows that most of these residues are in the active site. Among these, aspartic acid 90 and histidine 210 participate in a proposed catalytic mechanism for beta-lactam hydrolysis.