Effects of plasmid presence on growth and enzyme activity of Escherichia coli DH5α

Summary The effects of recombinant DNA propagation and gene expression on the physiology of the host cell was investigated using a series of copy number mutant plasmids. The plasmids at copy numbers of 30, 57, 115 and 501 per chromosome equivalent encoded constitutive production of the enzyme -lactamase. Ribose phosphate isomerase activity was relatively unaffected by plasmid presence, and glucose-6-phosphate dehydrogenase, fructose 1,6-diphosphate aldolase and fructose 1,6-diphosphatase activities were lower in plasmid-containing cells than in the plasmid-free host strain. Increasing copy number resulted in increased depression of enzyme activity levels. The results indicate that plasmid presence mediates subtle changes in the net expression of host enzymes involved in carbon metabolism. Responses of Escherichia coli DH5 in Evans medium to these plasmids differed substantially from responses of E. coli HB101 in rich medium.Offprint requests to: J. E. Bailey     

Nucleotide sequence of a cDNA encoding porcine testis 17 alpha-hydroxylase cytochrome P-450.

We describe the isolation and characterization of a cDNA encoding the complete porcine neonatal testis 17 alpha-hydroxylase/C-17,20-lyase cytochrome P-450. The deduced amino acid sequence is 509 amino acids in length.     

Ligneous membranitis in Scottish Terriers is associated with a single nucleotide polymorphism in the plasminogen (PLG) gene

Ligneous membranitis (LM) is a rare chronic inflammatory condition of the mucous membranes associated with plasminogen (encoded by PLG) deficiency in affected humans and dogs. In human, the condition is genetic in nature with numerous mutations and polymorphisms in PLG identified in affected individuals and related family members. The condition is uncommonly reported in dogs and, to date, no genetic studies have been performed. We identified related Scottish Terriers (littermates) with severe LM and unaffected relatives (sire, dam and a sibling from a previous litter). Plasma plasminogen activity was below normal in one affected dog but within normal reference intervals for the other. Sequencing of PLG from the affected dogs revealed a homozygous A>T single nucleotide polymorphism in an intron donor site (c.1256+2T>A). The related, unaffected dogs displayed heterozygous alleles at this position (c.1256+2T/A), whereas no mutation was detected in unaffected, non‐related control dogs. This is the first report to identify gene polymorphisms associated with LM in dogs.     

Role of the nucleophosmin (NPM) portion of the non-Hodgkin's lymphoma-associated NPM-anaplastic lymphoma kinase fusion protein in oncogenesis.

The NPM-ALK fusion gene, formed by the t(2;5)(p23;q35) translocation in non-Hodgkin's lymphoma, encodes a 75-kDa hybrid protein that contains the amino-terminal 117 amino acid residues of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase ALK (anaplastic lymphoma kinase). Here, we demonstrate the transforming ability of NPM-ALK and show that oncogenesis by the chimeric protein requires the activation of its kinase function as a result of oligomerization mediated by the NPM segment. Sedimentation gradient experiments revealed that NPM-ALK forms in vivo multimeric complexes of approximately 200 kDa or greater that also contain normal NPM. Cell fractionation studies of the t(2;5) translocation-containing lymphoma cell line SUP-M2 showed NPM-ALK to be localized within both the cytoplasmic and nuclear compartments. Immunostaining performed with both polyclonal and monoclonal anti-ALK antibodies confirmed the dual location of the oncoprotein and also indicated that NPM-ALK is abundant within both the nucleoplasm and the nucleolus. An intact NPM segment is absolutely required for NPM-ALK-mediated oncogenesis, as indicated by our observation that three different NPM-ALK mutant proteins lacking nonoverlapping portions of the NPM segment were each unable to form complexes, lacked kinase activity in vivo, and failed to transform cells. However, NPM could be functionally replaced in the fusion protein with the portion of the unrelated translocated promoter region (TPR) protein that activates the TPR-MET fusion kinase by mediating dimerization through its leucine zipper motif. This engineered TPR-ALK hybrid protein, which transformed cells almost as efficiently as NPM-ALK, was localized solely within the cytoplasm of cells. These data indicate that the nuclear and nucleolar localization of NPM-ALK, which probably occur because of transport via the shuttling activity of NPM, is not required for oncogenesis. Further, the activation of the truncated ALK protein by a completely heterologous oligomerization domain suggests that the functionally important role of the NPM segment of NPM-ALK in transformation is restricted to the formation of kinase-active oligomers and does not involve the alteration of normal NPM functions.     

Analysis of helicase gene mutations in Japanese Werner’s syndrome patients

The profile of helicase gene mutations was studied in 89 Japanese Werner’s syndrome (WRN) patients by examining the previously described mutations 1– 4 as well as a new mutation found during this study, designated mutation 5. Of 178 chromosomes (89 patients), 89 chromosomes (50%) had mutation 4, 11 (6.2%) chromosomes had mutation 1, and two chromosomes (1.1%) contained mutation 5. Mutations 2 and 3 were not observed in this patient population. The remaining 76 (42.7%) chromosomes had none of these mutations. A significant fraction of all patients (22 total patients, 24.7%) appear to be compound heterozygotes, including those carrying mutations of both types 1 and 4. The genotype analysis of the markers surrounding the WRN helicase gene strongly suggests that most of the chromosomes carrying either mutation 1 or 4 were derived from two single founders. Received: 25 July 1996 / Revised: 20 September 1996     

Large P body-like RNPs form in C. elegans oocytes in response to arrested ovulation, heat shock, osmotic stress, and anoxia and are regulated by the major sperm protein pathway

As Caenorhabditis elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) foci form in these arrested oocytes that contain RNA-binding proteins and translationally masked maternal mRNAs. Within 65 min of mating, the RNP foci dissociate and fertilization proceeds. The majority of arrested oocytes with foci result in viable embryos upon fertilization, suggesting that foci are not deleterious to oocyte function. We have determined that foci formation is not strictly a function of aging, and the somatic, ceh-18, branch of the major sperm protein pathway regulates the formation and dissociation of oocyte foci. Our hypothesis for the function of oocyte RNP foci is similar to the RNA-related functions of processing bodies (P bodies) and stress granules; here, we show three orthologs of P body proteins, DCP-2, CAR-1 and CGH-1, and two markers of stress granules, poly (A) binding protein (PABP) and TIA-1, appear to be present in the oocyte RNP foci. Our results are the first in vivo demonstration linking components of P bodies and stress granules in the germ line of a metazoan. Furthermore, our data demonstrate that formation of oocyte RNP foci is inducible in non-arrested oocytes by heat shock, osmotic stress, or anoxia, similar to the induction of stress granules in mammalian cells and P bodies in yeast. These data suggest commonalities between oocytes undergoing delayed fertilization and cells that are stressed environmentally, as to how they modulate mRNAs and regulate translation.     

Purification of Canine Plasma Renin by Affinity Chromatography

An affinity column for the purification of canine plasma renin was prepared using goat anti-renin (dog kidney) γG globulins. The antiserum was prepared against a purified kidney renin preparation. The anti-renin globulins were coupled to cyanogen bromide-activated Sepharose. Using the anti-renin globulin-coupled Sepha-rose as an immuno-adsorbant, a method was devised allowing purification of plasma renin to a 1, 000-fold purity.     

Dolichol and N-linked oligosaccharide synthesis in the rat testis: interaction between Sertoli and spermatogenic cells, evidence for paracrine effects.

The relative contribution of the Sertoli cell and the pachytene spermatocyte to dolichol and N-linked oligosaccharide biosynthesis within the seminiferous tubule was investigated. Evidence is presented to show that the interaction between these two cell types affects dolichol and N-linked oligosaccharide biosynthesis. Analysis of the dolichol content of Sertoli cultures confirms earlier data suggesting that the Sertoli cell constitutes the major pool of dolichols within the seminiferous tubule. [14C]Acetate incorporation studies suggest that the Sertoli cell in culture synthesizes dolichol much more rapidly than does the isolated pachytene spermatocyte. This information, in addition to previous data in the literature, infers an interactive effect whereby the presence of the spermatogenic cell in the tubule stimulates dolichol synthesis in the Sertoli cell. The absence of normal Sertoli-spermatocyte interactions in in vitro incubations may also limit dolichol synthesis in the pachytene spermatocyte. The distribution of dolichol kinase between the Sertoli and the pachytene spermatocyte was also examined. The concentration of this enzyme in the Sertoli cell suggests the presence of an active salvage pathway within that cell. The correlation between the appearance of the pachytene spermatocyte and the previously described peak of dolichol kinase activity in the seminiferous tubules of the prepubertal animal implies cell-cell interactions. Radiolabelling studies of N-linked oligosaccharides were conducted using [3H]mannose and concanavalin A affinity chromatography to identify multiantennary, biantennary, and high-mannose oligosaccharide pools. An in vitro bicameral coculture system was used to demonstrate that pachytene spermatocytes stimulate incorporation of [3H]mannose into Sertoli cell oligosaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)