Complementary expression patterns of six nonessential Caenorhabditis elegans core 2/I N-acetylglucosaminyltransferase homologues.

The Caenorhabditis elegans genome contains 18 sequences related to mammalian core 2/I N-acetylglucosaminyltransferases. The six most closely related genes (gly-1 and gly-15 to gly-19) likely encode active enzymes, because are all transcribed and do not appear to be pseudogenes. Polypeptide divergence and the gene structures are both concordant with a common ancestor at the time of radiation from mammals that underwent three rounds of duplication and, most recently, a tandem duplication. Polypeptide alignments with mammalian homologues do not indicate whether the enzyme specificities are core 2, 4, or I-like or novel, but do clearly demonstrate the secondary structure characteristics of glycosyltransferases. The six homologues have essentially nonoverlapping expression patterns, unrelated by tissue type or cell lineage. The extent varies widely; gly-15 is expressed only in two gland cells, whereas gly-18 is broadly expressed in diverse cell types. gly-1, -15, -18 and -19 are expressed during adulthood; gly-16 and gly-17 appear to be restricted to embryonic or early larval stages. The parsimonious interpretation of the expression pattern and sequence data is that the catalytic activities are similar but with diverged promoters. Null alleles of three of the genes were generated without causing gross abnormality in homozygous animals. RNA-mediated interference experiments also failed to induce defects in the four genes tested. Nevertheless, the nematode has evolved six diverged core 2 GlcNAc-T-like genes, and we postulate that these arose in response to selection pressures to which C. elegans is not ordinarily subjected in the laboratory.

Multiple signaling mechanisms of the UNC-6/netrin receptors UNC-5 and UNC-40/DCC in vivo.

Cell and growth cone migrations along the dorsoventral axis of Caenorhabditis elegans are mediated by the UNC-5 and UNC-40 receptor subtypes for the secreted UNC-6 guidance cue. To characterize UNC-6 receptor function in vivo, we have examined genetic interactions between unc-5 and unc-40 in the migrations of the hermaphrodite distal tip cells. We report that cell migration defects as severe as those associated with a null mutation in unc-6 are produced only by null mutations in both unc-5 and unc-40, indicating that either receptor retains some partial function in the absence of the other. We show that hypomorphic unc-5 alleles exhibit two distinct types of interallelic genetic interactions. In an unc-40 wild-type genetic background, some pairs of hypomorphic unc-5 alleles exhibit a partial allelic complementation. In an unc-40 null background, however, we observed that unc-5 hypomorphs exhibit dominant negative effects. We propose that the UNC-5 and UNC-40 netrin receptors can function to mediate chemorepulsion in DTC migrations either independently or together, and the observed genetic interactions suggest that this flexibility in modes of signaling results from the formation of a variety of oligomeric receptor complexes.

Spinobulbar muscular atrophy can mimic ALS: the importance of genetic testing in male patients with atypical ALS.

The clinical presentation of amyotrophic lateral sclerosis (ALS) is variable and overlaps with that of other motor neuron diseases such as spinobulbar muscular atrophy (SBMA; Kennedy disease). With the identification of disease-specific mutations such as the CAG repeat expansion in the androgen receptor in SBMA, an accurate molecular diagnosis can be made in some patients with motor neuron disease. To determine the extent of misdiagnosis of ALS we screened 147 male ALS patients and 100 unrelated male patients from 100 familial ALS (FALS) kindreds for the presence of the SBMA mutation using polymerase chain reaction methods. We show that ALS was clinically misdiagnosed in 2% of sporadic cases and in two of the 100 FALS kindreds. This study underscores the difficulty in distinguishing SBMA from ALS clinically, particularly in patients who lack the classic signs of each disease.

SOD1 mutation is associated with accumulation of neurofilaments in amyotrophic lateral sclerosis.

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene are found in 15 to 20% of patients with familial amyotrophic lateral sclerosis (FALS). Increased levels of neurofilament subunits in transgenic mouse models of ALS also suggests a key role for these proteins in the pathogenesis of the disease. We report the coexistence of an Ile113-->Thr substitution in exon 4 of the SOD1 gene and marked neurofilamentous pathology in the same FALS patient. These observations suggest that two mechanisms, SOD1-induced toxicity and neurofilament disruption, are acting together.

Identification of new mutations in the Cu/Zn superoxide dismutase gene of patients with familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons. Although most cases of ALS are sporadic, approximately 10% are inherited as an autosomal dominant trait. Mutations in the Cu/Zn superoxide dismutase gene (SOD 1) are responsible for a fraction of familial ALS (FALS). Screening our FALS kindreds by SSCP, we have identified mutations in 15 families, of which 9 have not been previously reported. Two of the new mutations alter amino acids that have never been implicated in FALS. One of them affects a highly conserved amino acid involved in dimer contact, and the other one affects the active-site loop of the enzyme. These two mutations reduce significantly SOD 1 enzyme activity in lymphoblasts. Our results suggest that SOD 1 mutations are responsible for > or = 13% of FALS cases.

Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder primarily affecting motor neurons. The etiology of the majority of cases remains unknown. Recent findings from several laboratories suggest a role for neurofilaments in the development of motor neuron disorders. The C-terminal region of the human neurofilament heavy subunit (NEFH) contains a unique functional domain consisting of 43 repeat motifs of the amino acids Lys-Ser-Pro (KSP). This C-terminal region of NEFH forms the sidearm projections which cross-link the neurofilaments. Previously, we have demonstrated polymorphism in the C-terminal region of the human NEFH: an allelic variant of a slightly larger molecular size, containing an additional KSP phosphorylation motif. Novel mutations in this region were found in five ALS patients. We propose that changes in the KSP-repeat domain may affect the cross-linking properties of the heavy neurofilament subunit and perhaps contribute to the development of neurofilamentous swellings in motor neurons, a hallmark of ALS.

Identification of flanking markers for the familial amyotrophic lateral sclerosis gene ALS1 on chromosome 21.

Amyotrophic lateral sclerosis (ALS) is a progressive, adult-onset, neurodegenerative disorder characterized by the death of large motor neurons from the cerebral cortex, brainstem, and spinal cord. The etiology of ALS remains unknown; however, approximately 10% of the cases are familial in nature. In the majority of these families, the mode of transmission is autosomal dominant. Recently, linkage of an autosomal dominant familial ALS (FALS) gene to the locus ALS1 on chromosome 21q was established. In addition, evidence was provided for genetic heterogeneity, with approximately 55% of families most likely linked to chromosome 21. The development of a number of highly informative simple sequence repeat polymorphisms in the region of linkage-21q21 through 21q22.1-has permitted us to confirm both the assignment of ALS1 to 21q and the genetic heterogeneity of FALS. In addition, we have been able to refine the mapping of ALS1, based on recombination events in two of the linked families. Flanking markers for the FALS gene are D21S213 on the centromeric side and D21S219 on the telomeric side. The candidate region is approximately 4 Mb and contains the genes copper/zinc superoxide dismutase (CuZnSOD); the fourth member of the class II cytokine receptor family (CRF2-4); and the interferon-alpha receptor (IFNAR).

Molecular analysis of the malic enzyme gene (mae2) of Schizosaccharomyces pombe.

Sequence analysis of a 4.6-kb HindIII fragment containing the malic enzyme gene (mae2) of Schizosaccharomyces pombe, revealed the presence of an open reading frame of 1695 nucleotides, coding for a 565 amino acid polypeptide. The mae2 gene is expressed constitutively and encodes a single mRNA transcript of 2.0 kb. The mae2 gene was mapped on chromosome III by chromoblotting. The coding region and inferred amino acid sequence showed significant homology with 12 malic enzyme genes and proteins from widely different origins. Eight highly homologous regions were found in these malic enzymes, suggesting that they contain functionally conserved amino acid sequences that are indispensable for activity of malic enzymes. Two of these regions have previously been reported to be NAD- and NADP-binding sites.

The Cu/Zn superoxide dismutase gene in ALS and parkinsonism-dementia of Guam.

Guam is one of three endemic foci whose indigenous (Chamorro) people have an unusually high incidence of fatal neurodegenerative disorders, amyotrophic lateral sclerosis (ALS) and Parkinsonism-dementia (PD). Recently, mutations in the Cu/Zn superoxide dismutase (SOD-1) gene have been identified in some familial cases of ALS. To investigate if mutations in the SOD-1 gene are also involved in the pathogenesis of ALS and PD of Guam, we analyzed the SOD-1 gene in Chamorros. No mutations were found in Chamorros with ALS or PD, indicating that mutations in the SOD-1 gene do not underlie the high-incidence neurodegenerative disorders of Guam.

Polymorphism in the multi-phosphorylation domain of the human neurofilament heavy-subunit-encoding gene.

The C-terminal region of the human neurofilament heavy subunit (NEFH) contains a unique functional domain consisting of 43 repeat motifs of the amino acids (aa) Lys-Ser-Pro (KSP) with either 3- or 5-aa spacers in between. Past studies have demonstrated that the serine in these KSP motifs can be phosphorylated, resulting in heavy phosphorylation of this domain. Recent studies provide strong evidence for a role of neurofilament phosphorylation in the establishment of neurofilament density and axonal caliber. Since it may be hypothesized that mutations in the phosphorylated region are a basis for neuropathological conditions, and since regions of the human genome containing repeat motifs have been demonstrated to be significantly polymorphic, we undertook to identify and characterize polymorphism in this region of the human NEFH gene. We were able to identify an allelic variant of a slightly larger molecular size, containing an additional KSP phosphorylation motif. The variant form of NEFH displays Mendelian inheritance and has a widespread population distribution. In addition, we also identified a point mutation in one individual which would result in a Pro-->Leu substitution in one of the repeat motifs.

Mapping of human gamma-glutamyl transpeptidase genes on chromosome 22 and other human autosomes.

gamma-Glutamyl transpeptidase (GGT; EC 2.3.2.2) is a membrane-associated enzyme that plays a role in the metabolism of glutathione and in the transpeptidation of amino acids; changes in GGT activity may reflect preneoplastic or toxic conditions in the liver or kidney. In contrast to the rat, in which GGT is represented by a single gene, at least four GGT genomic sequences have been identified in man and two of these have been localized to two distinct regions of chromosome 22. To characterize this gene/pseudogene family further, we have used somatic cell hybrids to map GGT by hybridization with probes from a human kidney GGT cDNA clone and by amplification of 3' GGT sequence by PCR. We clearly map three GGT loci to chromosome 22: two loci between the centromere and the breakpoint cluster region (BCR) gene and one locus telomeric to the BCR gene. In addition, we have been able to identify GGT-related sequences on chromosomes 18, 19, and 20.

Mutagen content of wines contaminated with common yeasts.

Wines exhibiting a microbial haze were collected and their microbial contents identified. Contaminant yeast species were cultured in grape musts under controlled conditions and extracts of the resultant wines were assayed for mutagen content using four strains of Salmonella in the Ames Salmonella/microsome/faecalase system. The wine extracts exhibited some toxicity to Salmonella but no mutagen content. It would appear that mutagen content is more a function of the grape species and must preparation than of microbial metabolism during fermentation.

Correction factors for the diphenylamine test for deoxyribonucleic acid in yeasts.

The diphenylamine assay for DNA content of yeast is unsatisfactory due to the presence of a yeast component which inhibits the colour reaction. The inhibitor is found in a wide range of yeast species and has not been identified. The relationships between the degree of inhibition and cell concentration, temperature and time of hydrolysis and extraction have been described. A formula for correction of the inhibition has been derived.

Mutagen content of table wines made from various grape species and hybrid cultivars.

The mutagen content of wines produced from the European Vitis vinifera grapes was compared with that of wines produced from hybrids of V. vinifera and species indigenous to North America. Mutagens were extracted on an XAD-2 Amberlite resin column and activated with S-9 and/or faecalase in the Salmonella/microsomal mutagen assay. All white wines had insignificant mutagen levels. The only red wine to produce statistically significant reversion frequencies was that made from the Concord grape. The mutagens were shown to be extracted from the grape skins during fermentation.