The promoter of the beta-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae

Summary The relationship between the promoter length of the Kluyveromyces fragilis -glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless -galactosidase gene of Escherichia coli. The removal of a region from position-425 to-232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed -glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the -glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.     

A T cell surface molecule different from CD2 is involved in spontaneous rosette formation with erythrocytes

Increasing evidence indicates that rosettes which spontaneously from between human T cells and E might be of physiologic relevance. We show here that another T cell-surface molecule than CD2 is involved in rosette formation. Four mAb have been obtained reacting with human T cells that block rosettes with E from many species, including autologous cells. They react with a molecule, we termed E2, which is actively synthetized by T and monocytic cells. Immunoprecipitation revealed a major 32-kDa band. Immunoblots revealed a major 32-kDa band and a minor 20-kDa band. This molecule was detected on all T cells tested--and present at high densities on corticothymocytes, but at low densities on medullary thymocytes. It was also found on monocytes but not on B cells. However B-CLL cells did carry this molecule. E2 molecules were also detected on nonhematologic cells. Together with the recent evidence that 3 molecules from the erythrocyte surface are also involved for rosettes, intricate molecular interactions would account for adhesion of T cells to autologous E and possibly autologous nucleated cells.     

Subculture of rabbit articular chondrocytes within a collagen gel: growth and analysis of differentiation

Primary cultures of rabbit articular chondrocytes have been subcultured within three-dimensional (3D) collagen gels. Under these conditions, the cells remained viable and divided, but with a lower proliferation rate than that observed in control monolayer cultures. Flow cytometric analysis of progression of the cells into the cell cycle has confirmed and extended these findings. Also the cellular volume was decreased in 3D-culture, being in the same range as thein vivo size of cartilage cells. Specific staining for proteoglycans and type II collagen immunolocalization on sections of gels showed the expression of differentiated phenotypes and revealed the accumulation of these matrix components in the immediate surroundings of the cells. The use of Ultroser G (a serum substitute) improved the conditions for 3D- culture of rabbit articular chondrocytes.     

The root-knot nematode resistance gene (Mi) in tomato: construction of a molecular linkage map and identification of dominant cDNA markers in resistant genotypes

A dominant allele at the Mi locus on chromosome 6 of tomato (Lycopersicon esculentum Mill) confers resistance to three species of root-knot nematodes (Meloidogyne). The resistance, which is associated with a localized necrotic response, was originally introduced into tomato from the wild species Lycopersicon peruvianum. As a step towards the molecular cloning of Mi, we have identified closely linked DNA markers from both cDNA and genomic DNA libraries as restriction fragment length polymorphisms (RFLPs). DNA from tomato populations segregating for nematode resistance was analyzed to generate a high-resolution genetic map of this region. Additional information on gene order was obtained by comparing the size of the introgressed L. peruvianum chromosomal segment within a collection of nematode-resistant tomato lines. Among the four cDNA markers that are tightly linked to Mi, three are dominant, i.e. L. peruvianum-specific. One cDNA marker corresponds to a gene family comprising 20-30 members, one of which is diagnostic for all nematode-resistant genotypes tested. The presence of non-homologous sequences around the Mi gene may contribute to the suppression of recombination in this region of the genome in crosses heterozygous for Mi. The potential of 'walking' from closely linked markers to Mi is discussed.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Clinical and molecular heterogeneity of phenylalanine hydroxylase deficiencies in France

RFLPs of 68 normal and 74 mutant alleles at the phenylalanine hydroxylase (PAH) locus were determined in 37 French kindreds. A total of 23 haplotypes, including 18 normal and 16 mutant alleles, were observed. Two-thirds of all mutant alleles were confined within only four haplotypes, while the last third was accounted for by 12 haplotypes, including eight haplotypes absent from Caucasian pedigrees reported thus far. Several mutant haplotypes were present in typical phenylketonuria only, others were present in variants only, and some were present in both. In addition, a particular mutant haplotype (haplotype 2) was found to harbor different mutations in our series, resulting in either typical phenylketonuria or in mild hyperphenylalaninemias. The diploid combination of so many mutant haplotypes in PAH-deficient patients and of compound heterozygosity at the PAH locus in southern Europe might account for the broad spectrum of individual phenotypes observed in France.     

Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta

Cultured smooth muscle cells from pig aorta arrested in G0 phase by serum deprivation were stimulated to proliferate by replacing the medium with one containing 10% serum. Studies in DNA replication and proliferation of cells showed a relatively good synchrony: 90% of the cells were in G1 phase for 16 h after addition of serum; they entered S phase between 18 and 24 h, completed S phase and traversed G2 phase between 24 and 30–32 h; 75% of these cells multiplied after 30–32 h and the remainder were blocked at the end of G2 phase. The synthesis and secretion of sulfated proteoglycans were examined throughout a full cell cycle using metabolic labelling with [³⁵S]sulfate. Smooth muscle cells in G1 or G2 phase synthesized and secreted sulfated proteoglycans with a possible pause at the end of the G2 phase but at the beginning of the S phase and during mitosis the incorporation of [³⁵S]sulfate into these macromolecules stopped entirely. Structural characteristics of sulfated proteoglycans secreted into the medium during G1 phase and an entire cell cycle were investigated. The proportion of proteoglycan complexes and the relative hydrodynamic size of monomers and of constituent subunits of complexes were determined after chromatography on Sepharose CL-2B and CL-6B columns run under both associative and dissociative conditions. No significant differences were observed for the periods of the cell cycle that were studied:

1. 1. [³⁵S]Proteoglycan complexes represented at the end of G1 phase and of the cell cycle respectively 19 and 16% of the total [³⁵S]proteoglycans secreted into the medium.

2. 2. More than 90% of the subunits, obtained after dissociation of complexes, were characterized by a similar k_av after chromatography on Sepharose CL-2B columns eluted under dissociative conditions (k_av 0.68 at the end of G1 phase and 0.65 at the end of full cell cycle).

3. 3. About 95% of monomers synthesized at the two stages of the cell cycle were eluted at k_av 0.25 after chromatography on Sepharose CL-6B column run under associative conditions and were characterized by a similar glycosaminoglycan distribution. These results suggest that smooth muscle cells in culture liberate similar populations of proteoglycans into the medium during the G1 and G2 phases.

     

Improved Agar Bottle Plate for Isolation of Methanogens or Other Anaerobes in a Defined Gas Atmosphere

A bottle plate for the cultivation of methanogens or other organisms in a defined pressurized-gas atmosphere was developed. The bottle provides the convenience of an agar streak plate, solves the problem of the water exudate from agar medium, and provides a convenient way of adding or sampling a defined gas atmosphere.