Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes.

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.     

A dimorphic Alu Sb-like insertion in COL3A1 is ethnic-specific

Alu elements are a class of repetitive DNA sequences found throughout the human genome that are thought to be duplicated via an RNA intermediate in a process termed retroposition. Recently inserted Alu elements are closely related, suggesting that they are derived from a single source gene or closely related source genes. Analysis of the type III collagen gene (COL3A1) revealed a polymorphic Alu insertion in intron 8 of the gene. The Alu insertion in the COL3A1 gene had a high degree of nucleotide identity to the Sb family of Alu elements, a family of older Alu elements. The Alu sequence was less similar to the consensus sequence for the PV or Sb2 subfamilies, subfamilies of recently inserted Alu elements. These data support the observations that at least three source genes are active in the human genome, one of which is distinct from the PV and Sb2 subfamilies and predates either of these two subfamilies. Appearance of the Alu insertion in different ethnic populations suggests that the insertion may have occurred in the last 100,000 years. This Alu insert should be a useful marker for population studies and for marking COL3A1 alleles.     

Demonstration of pH control in a commercial immobilized glucose isomerase

The synthesis of a variety of important biochemicals involves multistep enzyme-catalyzed reactions. In many cases, the optimal operating pH is much different for the individual enzymatic steps of such synthesis reactions. Yet, it may be beneficial if such reaction steps are combined or paired, allowing them to occur simultaneously, in proximity to one another, and at their respective optimal pH. This can be achieved by separating the micro-environments of the two steps of a reaction pathway using a thin urease layer that catalyzes an ammonia-forming reaction. In this article, the pH control system in a commercial immobilized glucose (xylose) isomerase pellet, which has an optimal pH of 7.5, is demonstrated. This system allows the glucose isomerase to have near its optimal pH activity when immersed in a bulk solution of pH 4.6. A theoretical analysis is also given for the effective fraction of the immobilized glucose isomerase, which remains active when the bulk pH is at 4.6 in the presence of 20 mM urea versus when the bulk pH is at its optimal pH of 7.5. Both theoretical and experimental results show that this pH control system works well in this case. (c) 1996 John Wiley & Sons, Inc.     

The Ubc3 (Cdc34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo.

The transition from G1 to S phase of the cell cycle in Saccharomyces cerevisiae requires the activity of the Ubc3 (Cdc34) ubiquitin-conjugating enzyme. S. cerevisiae cells lacking a functional UBC3 (CDC34) gene are able to execute the Start function that initiates the cell cycle but fail to form a mitotic spindle or enter S phase. The Ubc3 (Cdc34) enzyme has previously been shown to catalyze the attachment of multiple ubiquitin molecules to model substrates, suggesting that the role of this enzyme in cell cycle progression depends on its targeting an endogenous protein(s) for degradation. In this report, we demonstrate that the Ubc3 (Cdc34) protein is itself a substrate for both ubiquitination and phosphorylation. Immunochemical localization of the gene product to the nucleus renders it likely that the relevant substrates similarly reside within the nucleus.     

Osteogenesis imperfecta type I: molecular heterogeneity for COL1A1 null alleles of type I collagen.

Osteogenesis imperfecta (OI) type I is the mildest form of inherited brittle-bone disease. Dermal fibroblasts from most affected individuals produce about half the usual amount of type I procollagen, as a result of a COL1A1 "null" allele. Using PCR amplification of genomic DNA from affected individuals, followed by denaturing gradient gel electrophoresis (DGGE) and SSCP, we identified seven different COL1A1 gene mutations in eight unrelated families with OI type I. Three families have single nucleotide substitutions that alter 5' donor splice sites; two of these unrelated families have the same mutation. One family has a point mutation, in an exon, that creates a premature termination codon, and four have small deletions or insertions, within exons, that create translational frameshifts and new termination codons downstream of the mutation sites. Each mutation leads to both marked reduction in steady-state levels of mRNA from the mutant allele and a quantitative decrease in type I procollagen production. Our data demonstrate that different molecular mechanisms that have the same effect on type I collagen production result in the same clinical phenotype.     

Insertional Mutations in the Yeast Hop1 Gene: Evidence for Multimeric Assembly in Meiosis

The HOP1 gene of Saccharomyces cerevisiae has been shown to play an important role in meiotic synapsis. In this study we analyzed the mechanism of this function by phenotypic characterization of novel in-frame linker-insertion mutations located at various sites throughout the HOP1 coding sequence. Among 12 mutations found to cause defects in meiotic recombination and spore viability, three were temperature-sensitive for the spore viability defect. Although substantial meiotic recombination was found for these conditional alleles at the restrictive temperature, the level of exchange measured in spo13 meiosis was reduced in some of the monitored intervals, indicating that nondisjunction resulting from a deficit in crossing over could account for SPO13 spore inviability. Intragenic complementation between linker-insertion alleles was assessed by testing the viability of spores generated from heteroallelic diploids after SPO13 meiosis. Complex patterns of complementation and enhancement of the spore-inviability phenotype indicate that HOP1 functions in a multimeric complex. In addition, the ability of alleles which map near the carboxyl terminus to complement several other alleles provides evidence for a functional domain in this region of the protein. Two previously identified multicopy suppressors of the conditional hop1-628(ts) allele were tested for their effects in cells bearing the linker-insertion hop1 alleles. Overexpression of REC104 from a 2μ plasmid was shown to enhance the spore viability of every allele tested, including a hop1 disruption allele. On the other hand, suppression by overexpression of RED1 from a 2μ plasmid was found only for allele hop1-628(ts). Surprisingly, similar overexpression of RED1 in strains bearing several other conditional hop1 linker-insertion alleles caused enhanced spore lethality. This finding, in conjunction with the evidence for a carboxy-terminal domain, provides new insight into the nature of interactions between the HOP1 and RED1 products in meiosis.     

Diversity of siderophore genes encoding biosynthesis of 2,3-dihydroxybenzoic acid in Aeromonas spp.

Most species of the genus Aeromonas produce the siderophore amonabactin, although two species produce enterobactin, the siderophore of many enteric bacteria. Both siderophores contain 2,3-dihydroxybenzoic acid (2,3-DHB). Siderophore genes (designated aebC, -E, -B and -A, for aeromonad enterobactin biosynthesis) that complemented mutations in the enterobactin genes of the Escherichia coli 2,3-DHB operon, entCEBA(P15), were cloned from an enterobactin-producing isolate of the Aeromonas spp. Mapping of the aeromonad genes suggested a gene order of aebCEBA, identical to that of the E. coli 2,3-DHB operon. Gene probes for the aeromonad aebCE genes and for amoA (the entC-equivalent gene previously cloned from an amonabactin-producing Aeromonas spp.) did not cross-hybridize. Gene probes for the E. coli 2,3-DHB genes entCEBA did not hybridize with Aeromonas spp. DNA. Therefore, in the genus Aeromonas, 2,3-DHB synthesis is encoded by two distinct gene groups; one (amo) is present in the amonabactin-producers, while the other (aeb) occurs in the enterobactin-producers. Each of these systems differs from (but is functionally related to) the E. coli 2,3-DHB operon. These genes may have diverged from an ancestral group of 2,3-DHB genes.     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

A new variety of spondyloepiphyseal dysplasia characterized by punctate corneal dystrophy and abnormal dermal collagen fibrils

Summary Several individuals from one family are described with a unique form of spondyloepiphyseal dysplasia. Characteristic features include shorttrunked short stature, punctate corneal dystrophy and marked disorganization of dermal collagen fibrils when examined by transmission electron microscopy. Inheritance is compatible with either dominance and a variable expression or X-linkage. Although the basic defect has not been determined, the tissue distribution is consistent with a defect in a non-collagenous component that affects collagen fibril formation or stability.