Tomato exo-(1-->4)-beta-D-galactanase. Isolation, changes during ripening in normal and mutant tomato fruit, and characterization of a related cDNA clone.

An exo-(1-->4)-beta-D-galactanase was isolated from ripe tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig and cv Better Boy) using anion-exchange, gel filtration, and cation-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most active fraction revealed a predominant protein band at 75 kD and several minor bands. A 30-amino acid N-terminal sequence from this 75-kD protein showed a high degree of homology with other recently identified beta-galactosidase/ galactanase proteins from persimmon and apple fruits (I.-K. Kang, S.-G. Suh, K.C. Gross, J.-K. Byun [1994] Plant Physiol 105: 975-979; G.S. Ross, T. Wegrzyn, E.A. MacRae, R.J. Redgwell [1994] Plant Physiol 106: 521-528) and with the predicted polypeptide sequence encoded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldsbrough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71). The enzyme focused to a single band of beta-galactosidase activity on an isoelectrofocusing gel at pH 9.8. The enzyme was specific for (1-->4)-beta-D-galactan substrates with a pH optimum of 4.5. The only reaction product detected was monomeric galactose, indicating that the enzyme was an exo (1-->4)-beta-D-galactanase. beta-Galactanase activity increased at the onset of ripening in normal fruit, but no similar increase was detected in the nonripening mutants nor and rin. A tomato homolog (pTombetagal1) was isolated using the SR12 cDNA clone from carnation as a probe. This clone showed 73% identify at the amino acid level with beta-galactosidase-related sequences from apple and asparagus and 66% identity with SR12. pTombetagal1 is a member of a gene family. Northern analysis demonstrated that pTombetagal1 expression was ripening related in normal fruits, with lower levels apparent in the nonsoftening mutants.     

Identification of the most represented repeated motifs in Arabidopsis thaliana microsatellite loci

The major simple sequence repeats present in the Arabidopsis genome were identified by Southern hybridizations with 49 oligonucleotide probes matching all the possible combinations of motifs up to 4 nucleotides long. The method used allowed us to perform all the hybridizations under the same temperature conditions. A good correlation was observed with the data obtained from database analysis, indicating that the method can be useful for identifying the major classes of microsatellite loci in species for which few or no sequence data are available. AG/CT, AAG/CTT, ATG/CAT and GTG/CAC are the major motifs present in the Arabidopsis genome that can be used as convenient probes to isolate microsatellite loci by screening libraries. AAG/CTT is the more frequent of these motifs, and its relative frequency in Arabidopsis is much higher than averagely found in the plant kingdom. About 8% of the cDNA clones from an immature silique library contains AG/CT, AAG/CTT or ATG/CAT microsatellite loci. Several microsatellite loci were isolated by screening genomic and cDNA libraries. Twenty-six tri-nucleotide loci were PCR amplified from four different ecotypes, and polymorphism was observed for 12 of them; 10 loci showing two alleles and 2 loci showing three alleles.     

Proteinase-resistant factors in human erythrocyte membranes mediate CD4-dependent fusion with cells expressing human immunodeficiency virus type 1 envelope glycoproteins.

Murine CD4+ cells are resistant to human immunodeficiency virus type 1 (HIV-1) entry and to fusion with cells expressing HIV-1 envelope glycoproteins (Env). The role of human-specific factors in Env/CD4-mediated fusion is shown by the ability of transient cell hybrids formed between CD4+ murine cells and human HeLa cells to fuse with Env+ cells. Fusion events were observed when other human cells, including erythrocytes, were substituted for HeLa cells in the hybrids. Experiments with erythrocyte ghosts showed that the factors allowing Env/CD4-mediated fusion are located in the plasma membrane. These factors were fully active after extensive digestion of erythrocytes with proteinase K or pronase. Nonprotein components of human plasma membranes, possibly glycolipids, could therefore be required for Env/CD4-mediated fusion and virus entry.     

Athila,a new retroelement from Arabidopsis thaliana

An analysis of Arabidopsis thaliana heterochromatic regions allowed the identification of a new family of retroelements called Athila. These 10.5 kb elements, representing ca. 0.3% of the genome, present several features of retrotransposons and retroviruses. Athila elements are flanked by 1.5 kb long terminal repeats (LTR) that are themselves bounded by 5 bp perfect inverted repeats. These LTRs start and end with the retroviral consensus 5TG...CA3 nucleotides. A putative tRNA-binding site and a polypurine tract are found adjacent to the 5 and 3 LTR respectively. The central domain is composed of two long open reading frames (ORFs) of 935 and 694 amino acids. Despite several indications of recent transposition activity, the translation of these ORFs failed to reveal significant homology with proteins associated to retrotransposition. We suggest that the Athila family could result from the transduction and dispersion of a cellular gene by a retrotransposon.     

The electrophoretic polymorphism of bacterial esterases

Abstract: The specific activities of esterases and certain other molecular properties including immunospecificity indicate that the electrophoretic variations of these enzymes in bacterial populations are the result of allelic variations at specific gene loci. The esterase polymorphism of Enterobacteriaceae and some other species isolated from man or animals demonstrates that esterases can distinguish between bacteria at the species or subspecies level, both by their biochemical properties and by their electrophoretic differences. The esterase data complement DNA hybridization studies and agree with ribosomal DNA polymorphism, especially for delineating a phylogenetically distinct group of highly pathogenic strains in Escherichia coli . A two-dimensional electrophoretic profile obtained by establishing a direct correspondence between homologous esterase bands resolved by independent runs of isoelectric focusing and standard electrophoresis considerably improves the detection of allelic variations, whereas protein titration curves (electrophoresis in pH gradient) can be used to demonstrate the real electrophoretic homogeneity of allozymes or evalue their molecular relationship in terms of apparent amino acid substitutions. This overview establishes that esterases, by their significant electrophoretic polymorphism, are reliable molecular markers for systematics and epidemiology, and are suitable enzyme systems for studying population genetics and phylogeny.     

Multiple extracellular domains of CCR-5 contribute to human immunodeficiency virus type 1 entry and fusion.

Human immunodeficiency virus type 1 (HIV-1) entry is governed by the interaction of the viral envelope glycoprotein (Env) with its receptor. The HIV-1 receptor is composed of two molecules, the CD4 binding receptor and a coreceptor. The seven-membrane-spanning chemokine receptor CCR-5 is one of the coreceptors used by primary isolates of HIV-1. We demonstrate that the mouse homolog of CCR-5 (mCCR-5) does not function as an HIV-1 coreceptor. A set of chimeras of human CCR-5 and mCCR-5 was studied for Env-induced cell fusion and HIV-1 infection. Using the HIV-1ADA envelope glycoprotein in a syncytium formation assay, we show that replacement of any fragment containing extracellular domains of mCCR-5 by its human counterparts is sufficient to allow Env-induced fusion. Conversely, replacement of any fragment containing human extracellular domains by its murine counterpart did not lead to coreceptor function loss. These results show that several domains of CCR-5 participate in coreceptor function. In addition, using a panel of primary nonsyncytium-inducing and syncytium-inducing isolates that use CCR-5 or both CXCR-4 and CCR-5 as coreceptors, we show that the latter dual-tropic isolates are less tolerant to changes in CCR-5 than strains with a more restricted coreceptor use. Thus, different strains are likely to have different ways of interacting with the CCR-5 coreceptor.     

Excess of deletions of maternal origin in the DiGeorge/Velo-cardio-facial syndromes. A study of 22 new patients and review of the literature

We have determined the parental origin of the deleted chromosome 22 in 29 cases of DiGeorge syndrome (DGS) using a CA-repeat mapping within the commonly deleted region, and in one other case by using a chromosome 22 short arm heteromorphism. The CA-repeat was informative in 21 out of 29 families studied and the deleted chromosome was of maternal origin in 16 cases (72%). When these data are pooled with recent results from the literature, 24 de novo DGS, velo-cardio-facial syndrome (VCFS) and isolated conotruncal cardiac disease deletions are found to be of maternal origin and 8 of paternal origin, yielding a ² of 8 with a probability level lower than 0.01. These data, and review of the literature on familial DGS/VCFS and isolated conotruncal cardiopathies suggest that there is a strong tendency for the 22q11.2 deletions to be of maternal origin.     

The role of the germinal vesicle in producing maturation-promoting factor (MPF) as revealed by the removal and transplantation of nuclear material in starfish oocytes

In starfish, oocytes are released from prophase block by a hormone, which has been identified as 1-methyladenine. The action of 1-methyladenine is indirect in inducing oocyte maturation: it acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), the direct trigger of germinal vesicle breakdown (GVBD). Less than 5 min after hormone addition, thus about 10 min before appearance of the cytoplasmic maturation-promoting factor, a factor appears in the germinal vesicle, which triggers the production of cytoplasmic MPF, GVBD, and the subsequent events of meiotic maturation when transferred in the cytoplasm of any fully grown oocyte of the starfishes Marthasterias glacialis and Asterias rubens. Before hormone action, the germinal vesicle also contains a factor capable of inducing meiosis reinitiation in recipient oocytes, but in contrast with nuclear MPF, this factor acts exclusively when transferred in the cytoplasm of a special category of oocytes (the “competent” oocytes). In contrast to other oocytes (the “incompetent” oocytes) the competent oocytes are capable of producing MPF to some extent after enucleation, upon hormonal stimulation. Transfer of either nuclear or cytoplasmic MPF initially produced in hormone-treated maturing oocytes triggers the production of both cytoplasmic and nuclear MPF in non-hormone-treated recipient oocytes of both categories.