Suramin-treated HT29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with HT29-D4 cells grown in the absence of glucose

The clonal cell line HT29-D4 is able to differentiate by two different ways: i) by replacing glucose by galactose in the culture medium; ii) by addition of suramin (a drug known to interfere with the growth promoting activity of growth factors) in the medium. In both cases the transition in the organization of the cell monolayer occurred without cell loss. The two ways (i.e., glucose starvation or suramin addition) lead to polarized cells which generate electrically active cell monolayers (Fantini et al., Biol. Cell 65, 163-169 (1989) and this paper). Yet several important differences can be observed at the morphological or at the electrophysiological levels. 1) The suramin-treated cells (HT29-D4-S cells) organized into monolayers of high (40-50 microns) columnar cells while glucose-starved cells (HT29-D4-Gal cells) were rather cuboidal (20-25 microns). 2) HT29-D4-S cells were highly polarized; the nucleus was rejected at the basal side of the cell and lysosomes in the upper part of the cytoplasm. Numerous lipid-like droplets surrounded with glycogen were observed underneath the nucleus. HT29-D4-Gal cells never presented such a degree of organization. 3) The transepithelial resistance and the potential difference of HT29-D4-S monolayers reached values significantly higher than those for HT29-D4-Gal monolayers, reflecting a higher degree of organization. Specific proteins such as sucrase-isomaltase, alkaline phosphatase and carcinoembryonic antigen were localized exclusively on the apical membrane while human lymphocyte antigen (HLA) class I molecules were restricted to the basolateral membrane for both HT29-D4-S and HT29-D4-Gal cells. The present data demonstrate that the same cells can generate a different degree of cellular organization according to the experimental conditions of cell growth, the most elaborate state of differentiation being obtained in the presence of suramin.     

Design of a bubble-swawm bioreactor for animal cell culture

A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22·10^–3–1.45·10^–3 vvm (30–200 ml/h).The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.     

Distribution and conservation of sequences homologous to the 1731 retrotransposon in Drosophila

The distribution of 1731 retrotransposon-hybridizing sequences in the family Drosophilidae has been studied using a 1731 probe from Drosophila melanogaster. Squash blot and Southern blot analyses of 42 species reveal that the 1731 sequences are widespread within both the Sophophora and Drosophila subgenera and are also present in the genera Scaptomyza and Zaprionus. Hence the 1731 retrotransposon family appears to have a long evolutionary history in the Drosophilidae genome. Differences of hybridization signal intensity suggested that the 1731 sequence is well conserved only in the three species most closely related to D. melanogaster (D. simulans, D. mauritiana, and D. sechellia). A survey of insertion sites in numerous different populations of the previous four species by in situ hybridization to polytene chromosomes has shown in all cases both chromocentric hybridizations and a low number of sites (0-5) on the chromosomal arms. This number of sites is among the lowest observed in D. melanogaster and D. simulans when 1731 is compared with other retrotransposon families. In addition, we have observed species-specific patterns of the chromocentric hybridization signal, suggesting rapid modifications of the beta-heterochromatin components since the radiation of the melanogaster subgroup.      

Fission yeast rad17: a homologue of budding yeast RAD24 that shares regions of sequence similarity with DNA polymerase accessory proteins.

Following DNA damage or a block to DNA synthesis, checkpoint pathways act to arrest mitosis and prevent the attempted segregation of damaged or unreplicated DNA. The rad17 locus of Schizosaccharomyces pombe is one of seven known radiation-sensitive (rad) loci which are absolutely required to prevent mitosis following DNA damage in fission yeast. Six of these (rad1, rad3, rad9, rad17, rad26 and hus1) are also required for the checkpoint which prevents mitosis from occurring before DNA replication is complete. We report here that the predicted rad17 gene product is a basic hydrophilic protein of 606 amino acids which contains five domains with sequence homology to replication factor C (RF-C)/activator 1 subunits. Western analysis and fusion with Green Fluorescent Protein indicate that the abundance and electrophoretic mobility of Rad17 is not significantly modified following a block to DNA synthesis or following DNA damage, and that Rad17 is localized in the nucleus. Rad17 function is not essential for growth, but is required for the function of the DNA structure-dependent checkpoints. Site-directed mutagenesis has been used to demonstrate the biological significance of the RF-C/activator 1-related domains. These studies have also defined an element of the radiation sensitivity caused by loss of Rad17 function which is not associated with the radiation-induced G2 arrest defect seen in the rad17.d null mutant cells.     

Ecdysone regulation of the Drosophila Sgs-4 gene is mediated by the synergistic action of ecdysone receptor and SEBP 3.

The steroid hormone 20-hydroxyecdysone controls both induction and repression of the Drosophila 'intermolt gene' Sgs-4. We show here that the ecdysone receptor binds to two sites, element I and element II, in the regulatory region of Sgs-4. A functional analysis revealed that element II appears to be of no importance for Sgs-4 expression, while element I proved to be an ecdysone response element that is necessary, but not sufficient, for induction of Sgs-4 expression. Our results provide no evidence that repression of Sgs-4 expression is mediated by one of the two receptor binding sites. In the close vicinity of elements I and II, we detected two binding sites of secretion enhancer binding protein 3 (SEBP 3). Like receptor element I, one of these sites also proved to be necessary, but not sufficient, for expression of Sgs-4. Therefore, induction of Sgs-4 requires binding of both ecdysone receptor and SEBP 3 to a complex hormone response unit, which also contains binding sites for a third factor, SEBP 2. The SEBP 2 sites coincide with binding sites of products of the Broad-Complex locus, which has been implicated recently with transduction of the hormonal signal. Thus, the available data suggest that induction of Sgs-4, and possibly other 'intermolt genes', is a combination of a primary and a secondary response to the hormone.     

Hot Spots of Recombination in Fission Yeast: Inactivation of the M26 Hot Spot by Deletion of the Ade6 Promoter and the Novel Hotspot Ura4-Aim

The M26 mutation in the ade6 gene of Schizosaccharomyces pombe creates a hot spot of meiotic recombination. A single base substitution, the M26 mutation is situated within the open reading frame, near the 5' end. It has previously been shown that the heptanucleotide sequence 5' ATGACGT 3', which includes the M26 mutation, is required for hot spot activity. The 510-bp ade6-delXB deletion encompasses the promoter and the first 23 bp of the open reading frame, ending 112 bp upstream of M26. Deletion of the promoter in cis to M26 abolishes hot spot activity, while deletion in trans to M26 has no effect. Homozygous deletion of the promoter also eliminates M26 hot spot activity, indicating that the heterology created through deletion of the promoter per se is not responsible for the loss of hot spot activity. Thus, DNA sequences other than the heptanucleotide 5' ATGACGT 3', which must be located at the 5' end of the ade6 gene, appear to be required for hot spot activity. While the M26 hotspot stimulates crossovers associated with M26 conversion, it does not affect the crossover frequency in the intervals adjacent to ade6. The flanking marker ura4-aim, a heterology created by insertion of the ura4(+) gene upstream of ade6, turned out to be a hot spot itself. It shows disparity of conversion with preferential loss of the insertion. The frequency of conversion at ura4-aim is reduced when the M26 hot spot is active 15 kb away, indicating competition for recombination factors by hot spots in close proximity.     

Engineering of zinc finger and MHC motifs to locked-in tertiary folds

The assembly of helical and -sheet peptide blocks containing reactive chain ends results inhighly branched chain architectures (locked-in folds) mimicking native tertiary structures.This molecular kit strategy allows to bypass the protein folding problem in protein de novodesign and gives access to protein mimetics of high thermodynamic stability. The validity ofthis concept is exemplified for the design and synthesis of locked-in folds mimicking the zincfinger and MHC folding motifs.     

Molecular and Cellular Analysis of the DNA Repair Defect in a Patient in Xeroderma Pigmentosum Complementation Group D Who Has the Clinical Features of Xeroderma Pigmentosum and Cockayne Syndrome

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are quite distinct genetic disorders that are associated with defects in excision repair of UV-induced DNA damage. A few patients have been described previously with the clinical features of both disorders. In this paper we describe an individual in this category who has unusual cellular responses to UV light. We show that his cultured fibroblasts and lymphocytes are extremely sensitive to irradiation with UV-C, despite a level of nucleotide excision repair that is 30%–40% that of normal cells. The deficiency is assigned to the XP-D complementation group, and we have identified two causative mutations in the XPD gene: a gly→arg change at amino acid 675 in the allele inherited from the patient's mother and a −1 frameshift at amino acid 669 in the allele inherited from his father. These mutations are in the C-terminal 20% of the 760-amino-acid XPD protein, in a region where we have recently identified several mutations in patients with trichothiodystrophy.