Expression of engrailed proteins in arthropods, annelids, and chordates

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.     

Drosophila neuroglian: a member of the immunoglobulin superfamily with extensive homology to the vertebrate neural adhesion molecule L1

Drosophila neuroglian is an integral membrane glycoprotein that is expressed on a variety of cell types in the Drosophila embryo, including expression on a large subset of glial and neuronal cell bodies in the central and peripheral nervous systems and on the fasciculating axons that extend along them. Neuroglian cDNA clones were isolated by expression cloning. cDNA sequence analysis reveals that neuroglian is a member of the immunoglobulin superfamily. The extracellular portion of the protein consists of six immunoglobulin C2-type domains followed by five fibronectin type III domains. Neuroglian is closely related to the immunoglobulin-like vertebrate neural adhesion molecules and, among them, shows most extensive homology to mouse L1. Its homology to L1 and its embryonic localization suggest that neuroglian may play a role in neural and glial cell adhesion in the developing Drosophila embryo. We report here on the identification of a lethal mutation in the neuroglian gene.     

Newly recognized ectrodactyly/deafness syndrome

A 7-year-old non-Ashkenazi Jewish girl is described having asymmetrical ectrodactyly (split hand and foot deformity), short stature, mental retardation, sensorineural deafness, and abnormal facies. Because this constellation of findings has not been reported previously, the authors believe that this represents a new congenital malformation syndrome, most probably of genetic etiology.     

Purification and partial characterization of 1-aminocyclopropane-1-carboxylate synthase from tomato pericarp

1-Aminocyclopropane-1-carboxylate synthase was purified 5000-fold from LiCl-induced tomato fruit slices by conventional and high-performance liquid chromatography. The final preparation was estimated to be between 25% and 50% pure. Two-dimensional gel electrophoresis indicates that 1-aminocyclopropane-1-carboxylate synthase activity is associated with a 45-kDa polypeptide, with a pI of 5.8 +/- 0.2. The enzyme is inactivated both by its substrate, S-adenosyl-L-methionine (AdoMet) and by one of its products, 1-aminocyclopropane-1-carboxylate. Due to the extremely low abundance of the protein it was necessary to scale up the extraction in order to obtain reasonable amounts for sequence analysis. Therefore, 200 kg tomatoes were extracted on semi-industrial scale and 1-aminocyclopropane-1-carboxylate synthase purified. This yielded approximately 150 micrograms enzyme.     

Genetic and Molecular Characterization of Suppressors of Sir4 Mutations in Saccharomyces Cerevisiae

In order to learn more about other proteins that may be involved in repression of HML and HMR in Saccharomyces cerevisiae, extragenic suppressor mutations were identified that could restore repression in cells defective in SIR4, a gene required for function of the silencer elements flanking HML and HMR. These suppressor mutations, which define at least three new genes, SAN1, SAN2 and SAN3, arose at the frequency expected for loss-of-function mutations following mutagenesis. All san mutations were recessive. Suppression by san1 was allele-nonspecific, since san1 could suppress two very different alleles of SIR4, and was locus-specific since san1 was unable to suppress a SIR3 mutation or a variety of mutations conferring auxotrophies. The SAN1 gene was cloned, sequenced, and used to construct a null allele. The null allele had the same phenotype as the EMS-induced mutations and exhibited no pleiotropies of its own. Thus, the SAN1 gene was not essential. SAN1-mediated suppression was neither due to compensatory mutations in interacting proteins, nor to translational missense suppression. SAN1 may act posttranslationally to control the stability or activity of the SIR4 protein.     

Comparison of polymerase insertion and extension kinetics of a series of O2-alkyldeoxythymidine triphosphates and O4-methyldeoxythymidine triphosphate

The effect of alkyl group size on ability to act as deoxythymidine triphosphate (dTTP) has been studied for the carcinogen products O2-methyl-, O2-ethyl-, and O2-isopropyl-dTTP by using three types of nucleic acids as template and DNA polymerase I (Pol I) or Klenow fragment as the polymerizing enzymes. Apparent Km and relative Vmax values were determined in primer extension on M13 DNA at a single defined site, in poly[d(A-T)], and in nicked DNA. These data are the basis for calculation of the relative rate of insertion opposite A, relative to dTTP. The insertion rate for any O2-alkyl-dTTP is much higher than for a mismatch between unmodified dNTPs. Unexpectedly, O2-isopropyl-dTTP is more efficiently utilized than O2-methyl-dTTP or O2-ethyl-dTTP on each of the templates. O2-isopropyl-dTTP also substitutes for dTTP over extended times of DNA synthesis at a rate only slightly lower than that of dTTP. Parallel experiments using O4-methyl-dTTP under the same conditions show that it is incorporated opposite A more frequently than is O2-methyl-dTTP. Therefore, both the ring position and the size of the alkyl group influence polymerase recognition. Once formed, all O2-alkyl-T.A termini permit elongation, as does O4-methyl-T.A. In contrast to the relative difficulty of incorporating the O-alkyl-dTTPs, formation of the following normal base pair (C.G) occurs rapidly when dGTP is present. This indicates that a single O-alkyl-T.A pair does not confer significant structural distortion recognized by Pol I.     

The immunocytochemical localization of superoxide dismutase in the enterocytes of the avian intestine: The effect of vitamin D3

Summary Both light microscopical and electron microscopical immunocytochemical techniques were utilized to localize CuZnsuperoxide dismutase (SOD) in the duodenum of normal, rachitic and vitamin-D₃-replete chicks. This enzyme catalyses the dismutation of the superoxide anion, a toxic free radical generated during the normal aerobic metabolism of most respiring cells. Light microscopy showed no SOD activity associated with the duodenal enterocytes of normal and rachitic chicks. However, in rachitic animals subsequently treated with vitamin D, i.e. vitamin-D-replete chicks, intense immunoreactivity for the enzyme was seen in association with the apical border of the duodenal absorptive cells. Immunostaining for SOD was not seen in goblet cells. With electron microscopy, immunostaining for SOD activity was identified in association with the apical microvilli and, to a lesser degree, with the terminal web, a well as in association with both lysosomes and peroxisomes. From this report it appears that there is a physiological relationship between vitamin D, SOD and the intestinal absorptive cell. However, the precise relationship must await further clarification.     

Kinetic analysis of an E.coli phenylalanine-tRNA synthetase mutant.

A mutation in the pheS gene, encoding phenylalanyl-tRNA synthetase, in E. coli NP37 confers temperature-sensitivity on the organism. A five-fold increase in tRNA(phe) levels complements the mutation. Analysis of the kinetic properties of the mutant enzyme indicates that the KM is 20-fold higher than the wild-type and the dissociation constant of the tRNA(phe)-synthetase complex for the mutant is at least 10-fold higher. These results indicate that the mutation in E. coli NP37 directly affects the tRNA(phe) binding site on the cognate synthetase.