Induction of S.cerevisiae MAG 3-methyladenine DNA glycosylase transcript levels in response to DNA damage.

We previously showed that the expression of the Saccharomyces cerevisiae MAG 3-methyladenine (3MeA) DNA glycosylase gene, like that of the E. coli alkA 3MeA DNA glycosylase gene, is induced by alkylating agents. Here we show that the MAG induction mechanism differs from that of alkA, at least in part, because MAG mRNA levels are not only induced by alkylating agents but also by UV light and the UV-mimetic agent 4-nitroquinoline-1-oxide. Unlike some other yeast DNA-damage-inducible genes, MAG expression is not induced by heat shock. The S. cerevisiae MGT1 O6-methylguanine DNA methyltransferase is not involved in regulating MAG gene expression since MAG is efficiently induced in a methyltransferase deficient strain; similarly, MAG glycosylase deficient strains and four other methylmethane sulfonate sensitive strains were normal for alkylation-induced MAG gene expression. However, de novo protein synthesis is required to elevate MAG mRNA levels because MAG induction was abolished in the presence of cycloheximide. MAG mRNA levels were equally well induced in cycling and G1-arrested cells, suggesting that MAG induction is not simply due to a redistribution of cells into a part of the cell cycle which happens to express MAG at high levels, and that the inhibition of DNA synthesis does not act as the inducing signal.     

Multidimensional flow cytometric blood cell differentiation without erythrocyte lysis.

Forward light scattering, orthogonal light scattering, and the fluorescence intensities of unlysed peripheral blood cells, labeled with CD45-phycoerythrin and the nucleic acid dyes LDS-751 and thiazole orange, were measured simultaneously, utilizing a flow cytometer. Erythrocytes, reticulocytes, platelets, neutrophils, eosinophils, basophils, monocytes, lymphocytes, nucleated erythrocytes, and immature nucleated cells occupied unique positions in the five-dimensional space created by the listmode storage of the five independent parameters. A software program was developed which identified and enumerated each of these cell populations. Platelets in this study were identified by LDS-751 staining, in addition to their forward and orthogonal light-scattering characteristics. Validation of this approach was obtained by demonstrating that all CD41- or CD42-expressing platelets also stained with LDS-751. Furthermore, the staining by LDS-751 did not change following platelet activation with ADP. The quantification of erythrocytes, platelets, neutrophils, eosinophils, monocytes, and lymphocytes correlated well with data obtained with a commercial hematology whole blood analyzer (H-1). Reproducibility of the identification of these populations was shown by repeated measurement of the same sample and by staining and analysis of multiple aliquots of identical blood samples. Stability studies demonstrated that 8 hours after blood collection, the number of damaged cells increased. This could be measured by a greater thiazole orange uptake by the damaged cells. This investigation demonstrates the feasibility of multidimensional flow cytometric blood cell differentiation for an automated whole blood cell analysis without the necessity of erythrocyte lysis. The ability to simultaneously identify reticulocytes, nucleated erythrocytes, and immature nucleated cells in one measurement is unique and promises to be a powerful tool for the assessment of abnormal blood samples.     

High-level secretion of correctly processed recombinant human interleukin-1 beta in Kluyveromyces lactis.

The lactose-assimilating yeast, Kluyveromyces lactis, has been developed as a microbial host for the synthesis and secretion of human proteins. Here, we report the use of multi-copy vectors based on the 2 mu-like plasmid pKD1 from Kluyveromyces drosophilarum [Chen et al., Nucleic Acids Res. 14 (1986) 4471-4481] for the secretion of recombinant human interleukin-1 beta (reIL-1 beta). High levels of reIL-1 beta were secreted into the growth medium when the structural gene was fused in-frame to a synthetic secretion signal derived from the 'pre'-region of the K. lactis killer toxin. N-terminal sequencing of the excreted protein showed highly efficient (greater than 95%) maturation of the signal sequence. Synthesis as prepro-IL-1 beta, the 'pro'-sequence being derived from the human serum albumin-encoding gene, resulted in equally efficient secretion of mature IL-1 beta. Cytoplasmic production of Met-IL-1 beta, without a secretion signal, was found to be toxic to K. lactis. As in Saccharomyces cerevisiae [Baldari et al., EMBO J. 6 (1987) 229-234], but unlike native human IL-1 beta, K. lactis reIL-1 beta is glycosylated. This glycosylation led to a 95% loss of its biological activity. Removal of the carbohydrate chains by endo-beta-N-acetyl-glucosamidase H treatment fully restored the biological activity. A modified form of IL-1 beta (Asn7----Gln7), in which the unique site for Asn-linked glycosylation was deleted, exhibited the same biological activity as native IL-1 beta. The level of secretion of mature recombinant IL-1 beta ws glycosylation-independent.     

The use of field-inversion gel electrophoresis for deletion detection in Duchenne muscular dystrophy.

Deletion is a common cause of Duchenne muscular dystrophy (DMD). Field-inversion gel electrophoresis, in conjunction with Southern blot hybridization, was used to detect large SfiI DNA fragments in the DMD locus. Two unrelated boys with DMD were found to have abnormal sized DNA fragments resulting from deletions. Some of the female relatives of these patients were also shown by this method to have deletions in the DMD locus.     

An insertion within the factor IX gene: hemophilia BEl Salvador.

A patient with moderate to severe hemophilia B has been found to have a large insertion within his factor IX gene. The site of insertion is located in a DNA segment of approximately 0.8 kb between exon IV and an EcoRI site within intron D. The size of the DNA insertion is approximately 6 kb, and it contains at least two TaqI sites, two EcoRI sites, and one HindIII site. The insert probably originates from outside the FIX gene and does not represent an internal duplication. We propose that this abnormal FIX gene be called FIX El Salvador in recognition of the birthplace of the patient.     

Design and synthesis of metabolically stable atrial natriuretic factor analogs. Amino- and carboxy-terminal stabilization

Two analogs of rat atrial natriuretic factor, rANF7-28-NH2 and [Mpr7,Ala20,D-Arg27]rANF7-27-NH2, were prepared by the solid-phase method. These peptides had 2-fold and 7-fold less affinity, respectively, than rANF1-28 in binding to membranes prepared from cultured aortic smooth muscle cells, and both peptides were 5-fold less potent than rANF1-28 in relaxing serotonin-contracted rabbit aortic rings. rANF7-28-NH2 was rapidly degraded by rat kidney homogenates but [Mpr7,Ala20,D-Arg27]rANF7-27-NH2 had enhanced stability against rat kidney homogenate degradation. However, this in vitro stability did not translate into an extended duration of action in vivo.     

Recombinant interferon-beta 2 (interleukin-6) induces myeloid differentiation

Human IFN-beta 2 cytokine produced in E. coli was purified to homogeneity by immunoaffinity and ion-exchange chromatography. The cytokine inhibits the growth of myeloleukemic M1 cells and induces their morphological and functional differentiation into macrophages. Differentiation was also observed in the histiocytic lymphoma U937 cells. The effect on U937 was synergized by IFN-gamma and under these conditions IFN-beta 2 produced the induction of (2'-5') oligo(A) synthetase typical to IFN action and to differentiation.