Molecular characterization of the lysosomal acid phosphatase fromDrosophila melanogaster

InDrosophila, unlike humans, the lysosomal acid phosphatase (Acph-1) is a non-essential enzyme. It is also one of the most rapidly evolving gene-enzyme systems in the genus. In order to determine which parts of the enzyme are conserved and which parts are apparently under little functional constraint, we cloned the gene fromDrosophila melanogaster via a chromosomal walk. Fragments from the gene were used to recover an apparently full-length cDNA. The cDNA was subcloned into aDrosophila transformation vector where it was under the control of the 5′ promoter sequence of thehsp-70 gene. Three independent transformants were obtained; in each, Acph-1 expression from the cDNA was constitutive and not dependent on heat shock, as determined by densitometric analyses of the allozymic forms of the enzyme. The pattern of expression indicates thehsp-70 and endogenousAcph-1 promoters act together in some, but not all, tissues. The sequence of the cDNA was determined using deletions made with exonuclease III, and primers deduced from the cDNA sequence were used to sequence the genomic clone. Five introns were found, and putative 5′ up-stream regulatory sequences were identified. Amino acid sequence comparisons have revealed several highly conserved motifs betweenDrosophila Acph-1 and vertebrate lysosomal and prostatic acid phosphatases.     

A radiation hybrid map spanning the entire human X Chromosome integrating YACs,genes, and STS markers

We present a radiation hybrid (RH) map of human Chromosome (Chr) X, using 50 markers on 72 radiation hybrids. The markers, obtained from the consensus map, form a grid spanning the entire chromosome. To check the RH map, the marker order was determined by analysis of presence or absence of retained human DNA fragments in the RHs; the comparison with the consensus showed a similar order. Any STSs, microsatellites, genes, and clones can be positioned and ordered relative to the marker grid. This approach integrates genetic, physical, and large-scale clone mapping and is used to link YAC contigs containing data from various experimental sources. Received: 14 February 1996 / Accepted: 20 May 1996     

Sequential assignment of 1H, 13C and 15N resonances of human carbonic anhydrase I by triple-resonance NMR techniques and extensive amino acid-specific 15N-labeling

Summary The backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific ¹⁵N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.The chemical shift data have been deposited in the BioMagResBank in Madison, WI, U.S.A.     

Characterization of anther-expressed genes encoding a major class of extracellular oleosin-like proteins in the pollen coat of Brassicaceae†

A large, heterogeneous, highly expressed gene family encoding oleosin-like proteins is described in the Brassicaceae. íeven related cDNA sequences were isolated from Brassica napus anther mRNA using RACE-PCR and compared with other recently described anther-specific oleosin-like genes from B. napus. The expression patterns of four representative members of this diverse gene family were analyzed by Northern blotting and in situ hybridization. In all cases, the genes were expressed specifically in the tapetum of 3–5 mm B. napus buds, which contained microspores at the late-vacuolate and bicellular stages of development. The predicted protein products are ordered into subclasses, each of which has a characteristic C-terminal domain, containing different amino acid motifs or repeated residues. Tryphine (pollen coat) fractions from mature B. napus pollen were found to be particularly enriched in polypeptides of apparent molecular weights 32–38 kDa, plus numerous less abundant polypeptides of less than 15 kDa. The N-terminal 15–20 residues of three of these polypeptides (12, 32 and 38 kDa) were found by microsequencing to be identical to parts of the predicted amino acid sequences of three of the tapetal-expressed oleosin-like genes. This indicates the possibility of post-translational modification of these proteins resulting in a cleavage of the primary translation products in order to generate the mature tryphine polypeptides. These data imply that a large and diverse group of oleosin-like proteins is synthesized in the tapeturn of B. napus anthers and that following tapetal degradation, these proteins, possibly in modified form, then relocate to the developing microspores where they eventually constitute some of the major components of the extracellular tryphine of mature pollen grains. These proteins share a conserved 70 amino acid residue hydrophobic domain and are related structurally to the seed-specific intracellular oleosins, although their biological function may be different.     

Gibberellins and pea seed development

The gibberellin (GA)-biosynthesis mutations, lh ⁱ , ls and Ie ⁵⁸³⁹ have been used to investigate the role(s) of the GAs in seed development of the garden pea (Pisum sativum L.). Seeds homozygous for lh ⁱ possess reduced GA levels, are more likely to abort during development, and weigh less at harvest, compared with wild-type seeds due to expression of the lh ⁱ mutation in the embryo and/ or endosperm. Compared with wild-type seeds, the lh ⁱ mutation reduces endogenous GA₁ and gibberellic acid (GA₃) levels in the embryo/endosperm a few days after anthesis and fertilizing lh ⁱ plants with wild-type pollen dramatically increases GA₁ and GA₃ levels in the embryo/ endosperm and restores normal seed development. By contrast, the ls and le ⁵⁸³⁹ mutations do not appear to reduce GA levels in the embryo/endosperm of seeds a few days after anthesis, and do not affect embryo or endosperm development. However, both the ls and lh ⁱ mutations substantially reduce endogenous GA levels in embryos at contact point (the first day the liquid endosperm disappears). Levels of GAs in seeds from crosses involving the ls and lh ⁱ mutations suggest that GAs are synthesised in both the embryo/endosperm and testa and that the expression of ls depends on the tissue and developmental stage examined. These results suggest that GAs (possibly GA₁ and/or GA₃) play an important role early in pea seed development by regulating the development of the embryo and/or endosperm. By contrast, the high GA levels found in wild-type seeds at contact point (and beyond) do not appear to have a physiological role in seed development.Abbreviations GA_n gibberellin A_n - DAA days after anthesis - WT wild-type We thank Noel Davies, Katherine McPherson and Peter Bobbi for technical assistance, Professor L. Mander (ANU, Canberra) for dideuterated GA standards, and the Australian Research Council and Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN, Japan), for financial support.     

Barley anther culture using membrane rafts

When compared to agarose solidified media in small petri dishes, membrane rafts used in conjunction with liquid induction media significantly improved anther culture response in the Australian, malting-quality, spring barley cultivar Clipper. In contrast, the German cultivar Gimpel did not show an increased response on rafts.Abbreviations BA 6-benzylaminopurine - IAA indoleacetic acid - DH doubled haploid     

The transfer of pyrethroid resistance resulting from crosses between resistant German cockroaches and susceptible Asian cockroaches

Crosses were made between the Asian cockroach,Blattella asahinai Mizukubo, and resistant strains of the German cockroach,B. germanica (L.), to assess the transfer of pyrethroid resistance to the progeny and to study the inheritance mechanism(s) involved. It was shown that the strain of Asian cockroaches studied was susceptible to four pyrethroids. F₁ progeny were essentially susceptible to the same compounds. Tests with F₂ progeny and those from backcrosses to the resistant parent indicated that the data for each pyrethroid fit an hypothesis of simple, autosomal, nearly completely recessive inheritance. The results are discussed from the standpoint of the impact of the Asian genome on the inheritance mechanism(s).     

Gene inactivation in the plant pathogen Glomerella cingulata: three strategies for the disruption of the pectin lyase gene pnIA

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnIA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA ^– );both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried a 5and 3truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA-transformants. pnlA-transformants failed to produce a PNLA protein with a pI identical to one normally detected in wild-type isolates by silver and activity staining of isoelectric focussing gels. Pathogenesis on Capsicum and apple was unaffected by disruption of the pnlA gene, indicating that the corresponding gene product, PNLA, is not essential for pathogenicity. Gene disruption is a feasible method for selectively mutating defined loci in G. cingulata for functional analysis of the corresponding gene products.     

Hierarchical Analysis of Genetic Structure in Native Fire Ant Populations: Results from Three Classes of Molecular Markers

We describe genetic structure at various scales in native populations of the fire ant Solenopsis invicta using two classes of nuclear markers, allozymes and microsatellites, and markers of the mitochondrial genome. Strong structure was found at the nest level in both the monogyne (single queen) and polygyne (multiple queen) social forms using allozymes. Weak but significant microgeographic structure was detected above the nest level in polygyne populations but not in monogyne populations using both classes of nuclear markers. Pronounced mitochondrial DNA (mtDNA) differentiation was evident also at this level in the polygyne form only. These microgeographic patterns are expected because polygyny in ants is associated with restricted local gene flow due mainly to limited vagility of queens. Weak but significant nuclear differentiation was detected between sympatric social forms, and strong mtDNA differentiation also was found at this level. Thus, queens of each form seem unable to establish themselves in nests of the alternate type, and some degree of assortative mating by form may exist as well. Strong differentiation was found between the two study regions using all three sets of markers. Phylogeographic analyses of the mtDNA suggest that recent limitations on gene flow rather than longstanding barriers to dispersal are responsible for this large-scale structure.     

Glucose Transporter (GLUT-4) Is Targeted to Secretory Granules in Rat Atrial Cardiomyocytes

The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the transGolgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulindependent movement to the cell surface. To examine the relationship between these GLUT-4–containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed.