The spore coat of a fucosylation mutant in Dictyostelium discoideum

Strain HL250 of Dictyostelium discoideum cannot convert GDP-mannose to GDP-fucose, resulting in an inability to fucosylate protein. This affects a group of proteins which are normally fucosylated intracellularly and then secreted via prespore vesicles to become part of the outer lamina of the spore coat. We have found that strain HL250 nevertheless accumulates typical amounts of these proteins, stores them normally in prespore vesicles, and secretes them normally to become a part of the spore coat. However, affected proteins are proteolyzed after germination, the spore coat is more accessible to penetration by a macromolecular probe, and germination is inefficient in older spores. These findings can be explained by a dependence of the integrity of the outer layer of the spore coat on protein-linked fucose.     

Instability of orthophthalaldehyde reagent for amino acid analysis

Determination of amino acids by reversed-phase chromatography of the adduct with orthophthalaldehyde and a thiol is rapid and sensitive. The major recognized adverse feature of this method is the instability of the reaction product, which requires precise control of reaction timing and chromatographic parameters for reliable quantitative application. We report another source of major variability: reagent instability. Deterioration of reagent was noted as low peak heights and peak broadening and was predictable if the premixed reagent was left at room temperature. Restoration of sharp chromatograms was accomplished by addition of mercaptoethanol or sodium metabisulfite. Reagent which was chromatographically inert contained minimal free thiol by direct assay. Free thiol disappearance was markedly slowed by addition of a chelating agent. Excess mercaptoethanol was deleterious. We conclude that reagent deterioration represents oxidation of the thiol, may be reversed by rereduction with minimal thiol or bisulfite, and may be minimized by inclusion of a metal chelator in the reagent.     

Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase delta

Twenty-three pyrophosphate analogues were screened as inhibitors of proliferating cell nuclear antigen independent DNA polymerase delta (pol delta) derived from calf thymus. Carbonyldiphosphonate (COMDP), also known as alpha-oxomethylenediphosphonate, inhibited pol delta with a potency (Ki = 1.8 microM) 20 times greater than that displayed for DNA polymerase alpha (pol alpha) derived from the same tissue. Characterization of the mechanism of inhibition of pol delta indicated that COMDP competed with the dNTP specified by the template and was not competitive with the template-primer. In the case of pol alpha, COMDP did not compete with either the dNTP or the polynucleotide substrate. COMDP inhibited the 3'----5' exonuclease activity of pol delta weakly, displaying an IC50 greater than 1 mM.     

Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae

Recombinant hirudin was produced by the yeast Saccharomyces cerevisiae using the alpha-pheromone prepro sequence to direct its secretion into the culture medium. The secreted hirudin was isolated to greater than or equal to 95% purity as measured by 205-nm absorbance integration from a reverse-phase chromatogram. One major activity peak corresponding to the complete, correctly processed molecule and two minor activity peaks corresponding to C-terminally truncated forms were identified. The primary structure of the major peak, determined by N-terminal sequencing of tryptic peptides, was that predicted from the cDNA sequence, and the molecular mass analyzed by fast atom bombardment mass spectrometry (FAB-MS) was 6892.6 (calculated 6892.5). UV spectral analysis suggested that, in contrast to the natural molecule, recombinant hirudin produced by S. cerevisiae is not sulfated.     

Risk of nonocular cancer in first-degree relatives of retinoblastoma patients

Summary The increased risk of nonocular cancer seen consistently in studies of survivors of retinoblastoma may be caused in part by the presence of a retinoblastoma gene that also predisposes to other cancers. It has been claimed that this gene also increases the risk for cancer among unaffected relatives of genetic retinoblastoma probands. We report here a population-based study of the risk of nonocular cancer in parents and siblings of persons notified to the Danish Cancer Registry with retinoblastoma during 1943–84. No excess was observed among first degree relatives of 61 genetic retinoblastoma probands, whereas a slight (10%) excess was seen among the parents of 115 nongenetic probands. The latter was the result of significant excesses of malignant melanoma (4 observed, 0.4 expected), multiple myeloma (2 observed, 0.2 expected) and osteogenic sarcoma (1 observed, 0.03 expected). The observed risk pattern cannot be explained by the presence of the retinoblastoma gene.     

Amylase gene expression in intraspecific and interspecific somatic transformants of Drosophila.

The Amylase locus in Drosophila melanogaster normally contains two copies of the structural gene for alpha-amylase, a centromere-proximal copy, Amy-p, and a distal copy, Amy-d. Products of the two genes may display discrete electrophoretic mobilities, but many strains known to carry the Amy duplication are characterized by a single amylase electromorph, e.g., Oregon-R, which produces the mobility variant AMY-1. A transient expression assay was used in somatic transformation experiments to test the functional status of the Amy genes from an Oregon-R strain. Plasmid constructs containing either the proximal or distal copy were tested in amylase-null hosts. Both genes produced a functional AMY-1 isozyme. Constructs were tested against an AMY-3 reference activity produced by a coinjected plasmid that contains the Amy-d3 allele from a Canton-S strain. With reference to the internal control, the Amy-p and Amy-d genes from Oregon-R expressed different relative activity levels for AMY-1 in transient assays. The transient expression assay was successfully used to test the functional status of Amy-homologous sequences from strains of other species of Drosophila characterized by a single amylase elctromorph, namely, Drosophila pseudoobscura ST and Drosophila miranda S 204. The amylase-null strain of D. melanogaster provided the hosts for these interspecific somatic transformation experiments.     

Differential methylation of the hypervariable locus DXS255 on active and inactive X chromosomes correlates with the expression of a human X-linked gene

Consistent differences in methylation of particular cytosine residues in the DNA of active and inactive X chromosomes can be used for rapid, direct analysis of X-inactivation patterns in different female tissues. We have studied methylation of the highly polymorphic DXS255 locus in tissues from patients with deficiency of the E1 alpha subunit of the pyruvate dehydrogenase complex in whom the results can be correlated directly with total enzyme activity, levels of immunoreactive protein, and patterns of cell mosaicism. The results confirm that methylation of the DXS255 locus correlates with X-chromosome expression. In patients and normal controls, the pattern of X inactivation varied widely from tissue to tissue and often deviated markedly from a 50:50 proportion. These deviations are likely to reflect small numbers of tissue-specific stem cells at the time of random X inactivation and cannot be taken alone as evidence for selection or "nonrandom" inactivation.