Polymorphic tandem repeat region in interleukin-1α intron 6

Analysis of polymerase chain reaction amplified products from the sixth intron of the human interleukin-1 gene reveals a high polymorphism (polymorphism information content = 0.51) in a Caucasian population. Altogether, seven alleles have been defined ranging from 620 to 1220bp. This polymorphism is probably attributable to a variable number of 46-bp tandem repeats, each containing potential regulatory sequences.     

Functional cis-element sequence requirements for suppression of gene expression by the TNPA protein of the Zea mays transposon En/Spm

TNPA, one of the two transposition proteins encoded by the En/Spm transposable elements of Zea mays, suppresses the expression of genes that contain an appropriate cis element. Suppression can be monitored in tobacco protoplasts in a transient expression assay as follows. The plant promoter-driven expression of the Escherichia coli-glucuronidase (GUS)-encoding gene, uidA, is repressed in the presence of TNPA if the GUS gene contains a functional cis element in the untranslated RNA leader sequence. Earlier, we found that the minimal cis element is composed of two 12 by sequences in a tail-to-tail inverted orientation. Each 12 by sequence is sufficient to bind TNPA in vitro and can be thought of as a half-site in the cis element. Here, we investigated the sequence requirements of the minimal cis element. Our observations support our expectations that a functional cis element must provide a template to which two TNPA molecules can bind in the correct orientation. Sequences within the half-sites can be altered as long as the eight bases that make up the consensus binding sites are not changed. However, we found the following unexpected sequence specificities. Firstly, some changes to the consensus binding sequence can be tolerated in one half-site, as long as the other site matches the consensus. Secondly, although the region between the half-sites can vary in sequence and in length between two and four bases, a thymidine residue is not tolerated directly 5′ preceding the second half-site. Since many variants of the cis element sequence remain functional, the suppressor response element provides a flexible tool for artificially manipulating the expression of genes.     

Differential expression of two related amino acid transporters with differing substrate specificity in Arabidopsis thaliana

A general amino acid permease cDNA ( AAP2 ) was isolated from Arabidopsis by complementation of a yeast mutant defective in citrulline uptake. Direct transport measurements in yeast show that the protein mediates uptake of l -[¹⁴C]-citrulline and l -[¹⁴C]-proline. Detailed analyses of the substrate specificity by competition studies demonstrate that all proteogenic amino acids are recognized by the carrier, including those that represent the major transport forms of reduced nitrogen in many species, i.e. glutamine, glutamate and asparagine. Thus, AAP2 is less selective as compared with AAP1 and transports basic amino acids such as histidine as shown by expression in a histidine transport-deficient yeast strain. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows significant homologies to the Arabidopsis broad specificity permease AAP1, and a limited homology to bacterial branched chain amino acid transporters, but not to any other known proteins. Alterations in the charged residues as compared with AAP1 in four regions might be involved in the difference in selectivity towards basic amino acids. Both genes are highly expressed in developing pods indicating a role in supplying the developing seeds with reduced nitrogen. AAP2 is selectively expressed in the stem and might therefore play a role in xylem-to-phloem transfer of amino acids during seed filling. Furthermore in situ hybridization shows that both genes are expressed in the vascular system of cotyledons in developing seedlings.     

Comparison between aquatic and terrestrial locomotions of the leatherback sea turle (Dermochelys coriacea)

Kinematic characteristics of the fore- and hindlimb displacements during terrestrial and aquatic locomotions in juvenile marine turtles Dermochelys coriacea are compared. Modulations of the spatial displacements of the limbs and durations of the stance and swing phases are analysed in relationship with the constraints of the aquatic and terrestrial environments. The stance and swing phases used for describing the aquatic locomotion are re-evaluated in the light of the spatial displacements of the forelimbs during complete beating cycles.     

Preparation of Insect Chromosomes for Immunolabeling

We describe a method for isolating chromosomes from testes of the desert locust, Schistocerca gregaria, and their subsequent incubation with antibodies directed against chromosomal proteins. The procedure involves hypotonic pretreatment of the germ cells, centrifugation onto coverslips in a cytocentrifuge and immunolabeling, while still unfixed, using a chromatin-stabilizing buffer. In the present case, an antibody specific for the acetylated isoforms of his tone H4 was tested. After the antibody treatment, the preparations are fixed using formaldehyde, stained with a DNA-specific fluorescent dye and mounted. Analysis of the preparations revealed good preservation of chromosome structure in prophase spermatogonia and late prophase I spermatocytes. Fully condensed chromosomes were not observed and are probably lost during preparation. The bright fluorescence of the autosomes indicates that the reaction between the antibody against acetylated histone H4 and its chromosomal antigen is not impeded. In contrast, the X univalent remained unstained with the exception of a small terminal band. Thus, cytospin preparations of locust germ cells allow high resolution immunolabeling with antibodies against chromosome-associated proteins.     

A Mild Purification Method for Polysaccharide Binding Membrane Proteins: Phase Separation of Digitonin Extracts to Isolate the Hyaluronate Synthase fromStreptococcussp. in Active Form

A new method was developed to purify the streptococcal hyaluronate synthase in active form to electrophoretic homogeneity. The method is based on the extraction of protoplast membranes with digitonin and a phase separation into an aqueous and a detergent phase induced by addition of polyethylene glycol 6000 at 0°C. Proteins bound to hyaluronate were enriched in the aqueous phase, whereas other membrane proteins resided in the detergent phase. Final purification of the hyaluronate synthase was achieved by ion exchange chromatography.     

Repair mechanisms of UV-induced DNA damage in soybean chloroplasts

In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR). The kinetics of damage rapir in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation.     

Characterization of the peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) fromSilene alba cells

The peptide-N ⁴-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992)Glycoconjugate J 9:191–97] was partially purified from culturedSilene alba cells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of unconjugated N-glycans in a suspension medium of culturedSilene alba cells.Abbreviations GlcNAc N-acetylglucosamine - PNGase peptide-N ⁴-(N-acetylglucosaminyl) asparagine amidase - BSA bovine serum albumin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TLC thin layer chromatography - HPAEC-PAD High pH anion exchange chromatography-pulsed amperometric detection