Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

Effects of medium composition on extracellular proteinase stability and yield in batch cultures of a Thermus sp.

Summary Thermus sp. Rt41A produces an extracellular proteinase that is produced concomitant with growth and l-glutamate catabolism. Calcium-chelating medium components were shown to decrease the half-life of the proteinase in growing cultures. Medium modifications avoiding these components resulted in an increase in the half-life and in the peak level of proteinase. By adding the inorganic phosphate requirement for growth in anabolic amounts to pH-controlled batch cultures, stability of the proteinase in the medium was greatly enhanced and there was consequent improvement in the total proteinase yield. This approach also allowed a balanced increase in substrate and phosphate concentrations to increase the cell and proteinase yield in batch culture in an almost stoichiometric manner.Offprint requests to: H. W. Morgan     

Expression of a neuromodulin-β-galactosidase fusion protein in primary cultured neurons and its accumulation in growth cones

Cultured embryonic neurons share a number of characteristic morphological and physiological properties with their counterparts in vivo. For example, differentiating hippocampal neurons in culture develop two distinct classes of processes that serve as dendrites and axons. It has also been shown that the microtubule organization and composition in axons differs from those in dendrites, which may contribute to differential transport of macromolecules into axons or dendrites. We have expressed a neuromodulin--galactosidase fusion gene in cultured mesencephalic neurons in order to study the transport of the neurospecific protein neuromodulin into neurite growth cones. When -galactosidase alone was expressed in neurons, it was found in the cell bodies with diffuse neurite staining. In marked contrast, the neuromodulin--galactosidase fusion protein was rapidly transported into neurites and was concentrated in the growth cones. This system may provide a useful model for studying the structural domain(s) of neuromodulin that are required for transport and accumulation of neuromodulin in the growth cones of neurons.     

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.     

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Osteogenesis imperfecta due to recurrent point mutations at CpG dinucleotides in the COL1A1 gene of type I collagen

Summary Most individuals with osteogenesis imperfecta (OI) are heterozygous for dominant mutations in one of the genes that encode the chains of type I collagen. Each of the more than 30 mutations characterized to date has been unique to the affected member (s) of the family. We have determined that two individuals with a progressive deforming variety of OI, OI type III, have the same new dominant mutation [1(I)gly154 to arg] and that two unrelated infants with perinatal lethal OI, OI type II, share a second new dominant muation [1(I)gly1003 to ser]. These mutations occurred at CpG dinucleotides, in a manner consistent with deamination of a methylated cytosine residue, and raise the possibility that CpG dinucleotides are common sites of recurrent mutations in collagen genes. Further, these findings confirm that the OI type-III phenotype, previously thought to be inherited in an autosomal recessive manner, can result from new dominant mutations in the COL1A1 gene of type-I collagen.     

Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA

Electrotransformation was used to introduce both plasmid and linear DNA into Streptococcus pyogenes. The method was optimized using strain NZ131, for which transformation frequencies up to 10(7) per micrograms of plasmid DNA were obtained. A linear fragment of DNA, containing the streptokinase gene (ska) in which an internal fragment had been replaced with an erythromycin resistance gene (erm), was transformed into strain NZ131 with a frequency of 10(3) per micrograms DNA. The introduction of linear DNA into S. pyogenes by electrotransformation should be useful for future genetic analyses as well as targeted gene replacement.     

Encapsulation of eggs of the encyrtid wasp, Metaphycus swirskii, by the hemispherical scale, Saissetia coffeae: Effects of host age and rearing temperature

The combined effects of host age and rearing temperature upon the encapsulation of eggs of Metaphycus swirskii Annecke & Mynhardt (Hymenoptera: Encyrtidae) by the hemispherical scale, Saissetia coffeae (Walker) (Homoptera: Coccidae), were determined. Encapsulation incidence at 16, 20, 24 and 28°C increased with the age of the host, being 5.5–19.6% in 3rd instar nymphs, 45.4–74.2% in young female scales and 88.8–100% in preovipositing female scales. Within each host physiological age range, encapsulation frequency was not materially influenced by most of the temperatures. At 32°C, regardless of host age, S. coffeae encapsulated 99.5 to 100% of parasitoid eggs. Complete immunity to successful parasitism by M. swirskii occurred at 20, 24, 28 and 32°C in preovipositing female scales and at 32°C in 3rd-instar nymphs and young female scales of S. coffeae.

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Résumé Cette étude a déterminé les effets combinés de l'âge de l'hôte et de la température sur l'encapsulation des oeufs de M. swirskii Annecke & Mynhardt (Hym. Encyrtidae) par S. coffeae Walker (Homopt. Coccidae). La fréquence d'encapsulation à 16, 20, 24 et 28°C a augmenté avec l'âge de l'hôte, 5,5–19,6% dans les larves de 3ème stade, 45,4–74,2% chez les jeunes femelles et 88,8–100% chez les femelles avant la ponte. Dans chaque gamme d'âge physiologique de l'hôte, la fréquence d'encapsulation n'est pas influencée par la plupart des températures. A 32°C, quel que soit l'âge de l'hôte, S. coffeae a encapsulé 99,5–100% des oeufs du parasitoïde. Une immunité complète au parasitisme par M. swirskii a été obtenue à 20, 24, 28 et 32°C pour les femelles de cochenilles dans la période précédant la ponte, et à 32°C pour les larves de 3ème stade et les jeunes femelles de S. coffeae.

     

Oligosaccharides from placenta: early diagnosis of feline mannosidosis

High-pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of alpha-mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N-acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.     

The species composition,occurrence and temporal stability of submerged aquatic macrophyte patches along the main channel border of pool 5A,Upper Mississippi River

Species composition, relative abundance, distribution and physical habitat associations of submerged aquatic macrophytes in the main channel border (MCB) habitat of Pool 5A, Upper Mississippi River (UMR) were investigated during the summers of 1980 and 1983. The submerged aquatic macrophytes in Pool .5A MCB were a small and stable component of the river ecosystem. Submerged plants occurred primarily in small, monospecific clumps. Clumps in close proximity to each other formed plant patches. Plant patches were stable in location and number between 1980 and 1983; 82.5% of the patches first observed in 1980 were present in 1983. Submerged macrophytes covered about 10–12 ha of the 201 ha MCB in Pool 5A. Submerged plants were most common in the lower two-thirds of the pool. Ten species of aquatic macrophytes occurred on rock channel-training structures and eleven occurred on non-rock substrates in the MCB. The most common submerged plants, in order of abundance, were Vallisneria americana Michx., Heteranthra dubia Jacq., Potamogeton pectinatus L., Ceratophyllum demersum L. and Potamogeton americanus C. & S.