The nucleotide sequence of the yeast MEL1 gene.

The complete nucleotide sequence of the MEL1 gene of the yeast, Saccharomyces cerevisiae, encoding alpha-galactosidase was determined. The nucleotide sequence contains an open reading frame of 1413 bp encoding a protein of 471 amino acids. Comparison with the known N-terminal amino acid sequence of the mature secreted protein indicated that alpha-galactosidase is synthesized as a precursor with an N-terminal signal sequence of 18 amino acids. The general features of this signal peptide resemble those of other yeast signal peptides. Molecular weight of the mature alpha-galactosidase polypeptide deduced from the nucleotide sequence is 50.049 kd. The 5' regulatory region has sequences in common with other yeast genes regulated by the GAL4-protein.     

Erasure of the memory response in MEL cells

When MEL cells are reexposed to DMSO after an interruption in inducer treatment, they can initiate commitment to differentiation without the lag period observed after the primary exposure to inducer. This property is known as memory. Here we have employed metabolic inhibitors to analyze the basis of the memory response. Treatment of cells with cycloheximide or cordycepin during the inducer withdrawal period causes memory erasure. Cells must recapitulate an entire lag period upon reexposure to DMSO. The memory response is maintained, however, if cells are treated with metabolic inhibitors in the presence of DMSO. Our results suggest that the capacity of MEL cells for memory requires the synthesis of cell components which are normally stable in the absence of DMSO. Experiments involving reciprocal shifts between two different inhibitors have been performed. Evidence is presented that the process leading to the initiation of commitment is composed of at least three components acting in sequence.     

Memory of MEL cells to a previous exposure to inducer.

The mechanism of commitment of murine erythroleukemia (MEL) cells to terminal differentiation has been examined. Before a significant proportion of cells becomes committed, a lag period of at least 9 hr of exposure to inducer is observed. Cells withdrawn from inducer can reinitiate commitment without a lag when reexposed. The proportion of committed cells in a culture discontinuously exposed to inducer is identical to that in a continuously exposed culture even if withdrawal from inducer lasts for 18 hr. The ability to tolerate an interruption in the exposure has been termed "memory." The memory of a previous exposure to inducer is complete up to 18 hr. It is partially erased after 36 hr and completely erased after 72 hr. The length of time the memory persists is not affected by the length of the initial exposure to inducer. These results suggest that a cellular component necessary for the commitment event accumulates in response to inducer and that this component has a decay time on the order of 10 hr.     

Yeast comparative genetics. A new MEL15 alpha-galactosidase gene of Saccharomyces cerevisiae

To supplement the earlier identified European family of the highly homologous alpha-galactosidase MEL1-MEL11 genes and the African family of the divergent MEL12-MEL14 genes, a new MEL gene (MEL15) was found in several Saccharomyces cerevisiae strains isolated from maize dough in Ghana. Southern blotting and restriction enzyme analysis assigned the MEL15 gene to the African family and mapped it to chromosomes IV/XII, which migrate together in electrophoresis. Tetrad analysis ruled out the MEL15 location in the left arm of chromosome IV or the right arm of chromosome XII, which respectively contain the known MEL5 and MEL10 genes.     

Effect of bacitracin on erythroid differentiation of MEL cells

Bacitracin, an antibiotic widely utilized in clinical and veterinary use, was tested on murine erythroleukemia (MEL) cells. Tests were performed to evaluate the capacity of the drug to interfere with erythroid differentiation. Cells were exposed to a single treatment in S phase at sublethal doses of bacitracin. Two responses were found depending on the drug concentration. At higher concentrations (25 g/ml and 250ng/ml) a reduction in number of differentiating cells was observed but the kinetics of the process remained unchanged. At lower concentrations (from 2.5 ng/ml to 2.5 fglml) a dramatic alteration of the dynamic of differentiation was found. These two responses are related to different activities of the DNA repair mechanisms. Higher doses of bacitracin stimulate repair while lower concentrations are not able to activate repair, as demonstrated by tests with hydroxyurea. The bacitracin-induced damage can be considered a stable genetic andlor epigenetic alteration, as demonstrated by the high frequency of mutant clones isolatedfrom low-dose treated cells. The suitability of MEL cells system in evaluating genotoxicity of drugs for veterinary use is underlined.Abbreviations MEL murine erythroleukemia - HU hydroxyurea     

The human gene mutation database.

The Human Gene Mutation Database (HGMD) represents a comprehensive core collection of data on published germline mutations in nuclear genes underlying human inherited disease. By September 1997, the database contained nearly 12 000 different lesions in a total of 636 different genes, with new entries currently accumulating at a rate of over 2000 per annum. Although originally established for the scientific study of mutational mechanisms in human genes, HGMD has acquired a much broader utility to researchers, physicians and genetic counsellors so that it was made publicly available at http://uwcm.ac.uk/uwcm/mg/hgmd0.html in April 1996. Mutation data in HGMD are accessible on the basis of every gene being allocated one web page per mutation type, if data of that type are present. Meaningful integration with phenotypic, structural and mapping information has been accomplished through bi-directional links between HGMD and both the Genome Database (GDB) and Online Mendelian Inheritance in Man (OMIM), Baltimore, USA. Hypertext links have also been established to Medline abstracts through Entrez , and to a collection of 458 reference cDNA sequences also used for data checking. Being both comprehensive and fully integrated into the existing bioinformatics structures relevant to human genetics, HGMD has established itself as the central core database of inherited human gene mutations.     

Selective adhesion of osteoblastic cells to different integrin ligands induces osteopontin gene expression.

Skeletal homeostasis is partly regulated by the mechanical environment and specific signals generated by a cell's adhesion to the matrix. Previous studies demonstrated that osteopontin (OPN) expression is stimulated in response to both cellular adhesion and mechanical stimulation. The present studies examine if specific integrin ligands mediate osteoblast selective adhesion and whether opn mRNA expression is induced in response to these same ligands. Embryonic chicken calvaria osteoblastic cells were plated on bacteriological dishes coated with fibronectin (FN), collagen type I (Col1), denatured collagen/gelatin (G), OPN, vitronectin (VN), laminin (LN) or albumin (BSA). Osteoblastic cells were shown to selectively adhere to FN, Col1, G and LN, yet not to VN, OPN or BSA. Opn mRNA expression was induced by adhesion to Col1, FN, LN and G, but neither OPN nor VN induced this expression. Examination of the activation of the protein kinases A and C second signaling systems showed that only adhesion to FN induced protein kinase A and protein kinase C (PKC) activity while adherence to Col1 induced PKC. Evaluation of the intracellular distribution of focal adhesion kinase (FAK) and p-tyrosine within cells after adherence to FN, VN or BSA demonstrated that adherence to FN stimulated FAK translocation from the nucleus to the cytoplasm and high levels of p-tyrosine localization at the cell surface. However, cell adherence to VN or BSA did not show these morphological changes. These data illustrate that osteoblast selective adhesion is mediated by specific integrin ligands, and induction of intracellular second signal kinase activity is related to the nature of the ligand.     

Cloning, sequence and chromosomal location of a MEL gene from Saccharomyces carlsbergensis NCYC396.

Yeast strains producing alpha-galactosidase (alpha Gal) are able to use melibiose as a carbon source during growth or fermentation. We cloned a MEL gene from Saccharomyces carlsbergensis NCYC396 through hybridization to the MEL1 gene cloned earlier from Saccharomyces cerevisiae var. uvarum. The alpha Gal encoded by the newly cloned gene was galactose-inducible as is the alpha Gal encoded by MEL1. A probable GAL4-protein recognition sequence was found in the upstream region of the NCYC396 MEL gene. The gene was transcribed to a 1.5-kb mRNA which, according to the nucleotide sequence, encodes a protein of 471 amino acids (aa) with an Mr of 52,006. The first 18 aa fulfilled the criteria for the signal sequence, but lacked positively charged aa residues, except the initiating methionine. The enzyme activity was found exclusively in the cellular fraction of the cultures. The deduced aa sequence was compared to the aa sequences of other alpha Gal enzymes. It showed 83% identity with the S. cerevisiae enzyme, but only 35% with the plant enzyme, 30% with the human enzyme and 17% with the Escherichia coli enzyme. With pulsed-field electrophoresis, the MEL gene was located on chromosome X of S. carlsbergensis, whereas the S. cerevisiae var. uvarum MEL1 gene is located on chromosome II.     

Selective enhancement of gene transfer by steroid-mediated gene delivery.

The incorporation of transgenes into the host cells' nuclei is problematic using conventional nonviral gene delivery technologies. Here we describe a strategy called steroid-mediated gene delivery (SMGD), which uses steroid receptors as shuttles to facilitate the uptake of transfected DNA into the nucleus. We use glucocorticoid receptors (GRs) as a model system with which to test the principle of SMGD. To this end, we synthesized and tested several bifunctional steroid derivatives, finally focusing on a compound named DR9NP, consisting of a dexamethasone backbone linked to a psoralen moiety using a nine-atom chemical spacer. DR9NP binds to the GR in either its free or DNA-crosslinked form, inducing the translocation of the GR to the nucleus. The expression of transfected DR9NP-decorated reporter plasmids is enhanced in dividing cells: expression of steroid-decorated reporter plasmids depends on the presence of the GR, is independent of the transactivation potential of the GR, and correlates with enhanced nuclear accumulation of the transgene in GR-positive cells. The SMGD effect is also observed in cells naturally expressing GRs and is significantly increased in nondividing cell cultures. We propose that SMGD could be used as a platform for selective targeting of transgenes in nonviral somatic gene transfer.     

Isolation of the human gene that complements a temperature-sensitive cell cycle mutation in BHK cells.

We have cloned the human genomic DNA and the corresponding cDNA for the gene which complements the mutation of tsBN51, a temperature-sensitive (Ts) cell cycle mutant of BHK cells which is blocked in G1 at the nonpermissive temperature. After transfecting human DNA into TsBN51 cells and selecting for growth at 39.5 degrees C, Ts+ transformants were identified by their content of human AluI repetitive DNA sequences. Following two additional rounds of transfection, a genomic library was constructed from a tertiary Ts+ transformant and a recombinant phage containing the complementing gene isolated by screening for human AluI sequences. A genomic probe from this clone recognized a 2-kilobase mRNA in human and tertiary transformant cell lines, and this probe was used to isolate a biologically active cDNA from the Okayama-Berg cDNA expression library. Sequencing of this cDNA revealed a single open reading frame encoding a polypeptide of 395 amino acids. The deduced BN51 gene product has a high proportion of acidic and basic amino acids which are clustered in four hydrophilic domains spaced at 60- to 80-amino-acid intervals. These domains have strong sequence homology to each other. Thus, the tsBN51 protein consists of periodic repetitive clusters of acidic and basic amino acids.     

LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells.

We have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin promoter and the second intron of the human beta-globin gene, and this expression cassette is then placed downstream of the LCR and transfected into MEL cells. The cDNAs are expressed at levels similar to those of the murine beta-globin in the induced MEL cells. Heterologous genomic sequences can also be expressed at similar levels when linked to to the LCR and beta-globin promoter. In addition we demonstrate that, after induction of differentiation, MEL cells are capable of secreting heterologous proteins over a prolonged time period, making this system suitable for use in continuous production systems such as hollow fibre bioreactors. The utility of the LCR/MEL cell system is demonstrated by the expression of growth hormone at high levels (greater than 100 mg/l) 7 days after induction. Since the expression levels seen do not depend upon gene amplification and are independent of the integration position of the expression cassette, it is possible to obtain clones with stable high-level expression within 3-4 weeks after transfection.     

Cytoplasmic factors involved in erythroid differentiation in mouse erythroleukemia (MEL) cells

In order to identify and characterize intracellular factors involved in in vitro differentiation of mouse erythroleukemia (MEL) cells, the differentiation process was analyzed by cell and cytoplast fusion. The results suggested that the process is not a single cascade of molecular chain reactions, but a synergistic result of two different inducible intracellular reactions. One reaction is induced following damage to DNA (inhibition of DNA replication) and is not specific to MEL cells. The other reaction, which is specific to MEL cells, is fully induced by typical erythroid inducing agents such as dimethylsulfoxide or hexamethylenebisacetamide even at concentrations suboptimal for the erythroid induction. Based upon these data, we searched for the putative trans-acting differentiation-inducing factors and detected two proteinaceous factors (DIF-I and DIF-II) in the cytosol fraction which apparently correspond to these reactions. When, partially purified, either one of these factors was introduced into undifferentiated MEL cells, it triggered erythroid differentiation, provided that the recipient cells had been potentiated by the induction of the other reaction. In this article, we summarize the basic characteristics of these cytoplasmic factors involved in erythroid differentiation in MEL cells.     

Selective electrofusion of conjugated cells in flow.

Using a modified flow cytometer we have induced electrofusion of K562 and L1210 cells in flow. The two cell types are stained with two different fluorescent membrane probes, DiO and DiI, to facilitate optical recognition, and then coupled through an avidin-biotin bridge. In the flow cytometer, the hydrodynamically focused cells and cell pairs are first optically analyzed in a normal flow channel and then forced to flow through a Coulter orifice. If the optical analysis indicates that a cell pair is present, an electric pulse is applied across the orifice to induce fusion. The pulsed cell pairs were subsequently analyzed using normal and confocal microscopy to evaluate fusion induction. It appears that fusion can be induced in about 10% of pulsed cell pairs when one electric pulse with a duration of 10-15 microseconds and an effective electric field strength of 4-8 10(5) V/m is used.     

Recurrent mutation in the human phenylalanine hydroxylase gene.

We report the identification of a missense mutation of Glu280 to Lys280 in the phenylalanine hydroxylase (PAH) gene of a phenylketonuria (PKU) patient in Denmark. The mutation is associated with haplotype 1 of the PAH gene in this population. This mutation has previously been found in North Africa, where it is in linkage disequilibrium with haplotype 38. While it is conceivable that this mutation could have been transferred from one haplotype background to another by a double crossover or gene conversion event, the fact that the mutation is exclusively associated with the two different haplotypes in the two distinct populations supports the hypothesis that these two PKU alleles are the result of recurrent mutations in the human PAH gene. Furthermore, since the site of mutation involves a CpG dinucleotide, they may represent hot spots for mutation in the human PAH locus.     

Synchronization of MEL cell commitment with cordycepin.

The response of differentiating MEL cells to the nucleotide analogue cordycepin reveals a previously unrecognized aspect of the molecular events which cause commitment of these cells to terminal erythroid differentiation. Cordycepin rapidly inhibits commitment of DMSO-treated MEL cells in a dose range which does not cause cytotoxicity. Reversal of cordycepin treatment in the presence of inducer leads to a rapid and synchronous commitment of a significant proportion of cells in the culture. These results suggest that MEL cells can be blocked just prior to the point of commitment by cordycepin treatment.