Modulation of chaperone gene expression in mutagenized Saccharomyces cerevisiae strains developed for recombinant human albumin production results in increased production of multiple heterologous proteins.

The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regulating the ATPase cycle of Kar2p, is increased in a proprietary yeast strain, developed by several rounds of random mutagenesis and screening for increased production of recombinant human albumin (rHA). To establish whether this expression contributes to the enhanced-production phenotype, these genes were overexpressed both individually and in combination. The resultant strains showed significantly increased shake-flask production levels of rHA, granulocyte-macrophage colony-stimulating factor, and recombinant human transferrin.

A chlamydial type III translocated protein is tyrosine-phosphorylated at the site of entry and associated with recruitment of actin.

The obligate intracellular bacterium Chlamydia trachomatis rapidly induces its own entry into host cells. Initial attachment is mediated by electrostatic interactions to heparan sulfate moieties on the host cell, followed by irreversible binding to an unknown secondary receptor. This secondary binding leads to the recruitment of actin to the site of attachment, formation of an actin-rich, pedestal-like structure, and finally internalization of the bacteria. How chlamydiae induce this process is unknown. We have identified a high-molecular-mass tyrosine-phosphorylated protein that is rapidly phosphorylated on attachment to the host cell. Immunoelectron microscopy studies revealed that this tyrosine-phosphorylated protein is localized to the cytoplasmic face of the plasma membrane at the site of attachment of surface-associated chlamydiae. The phosphoprotein was isolated by immunoprecipitation with the antiphosphotyrosine antibody 4G10 and identified as the chlamydial protein CT456, a hypothetical protein with unknown function. The chlamydial protein (Tarp) appears to be translocated into the host cell by type III secretion because it is exported in a Yersinia heterologous expression assay. Phosphotyrosine signaling across the plasma membrane preceded the recruitment of actin to the site of chlamydial attachment and may represent the initial signal transduced from pathogen to the host cell. These results suggest that C. trachomatis internalization is mediated by a chlamydial type III-secreted effector protein.

Chlamydia trachomatis type III secretion: evidence for a functional apparatus during early-cycle development.

The obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole termed an inclusion. During its intracellular developmental cycle, C. trachomatis maintains this intracellular niche, presumably by expressing a type III secretion system, which deploys a set of host cell-interactive proteins including inclusion membrane-localized proteins termed Incs. Some Incs are expressed and secreted by 2 h (early cycle) after infection, whereas the expression of type III-specific genes is not detectable until 6-12 h (mid-cycle). To resolve this paradox, we investigated the presence of a type III apparatus on elementary bodies (EBs) that might function early in infection. We demonstrate the existence of the type III secretory apparatus by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and immunoblot analyses of purified EB extracts. Immunoblots using polyclonal antibodies specific for the core apparatus component CdsJ identified this protein in both EB and reticulate body (RB) extracts. Furthermore, CdsJ-specific signals were detected by immunoblot of whole infected-culture extracts and by indirect immunofluorescence of infected monolayers at times before the detection of cdsJ-specific message. Finally, expression of IncC, expressed by 2 h after infection during C. trachomatis infections, in Yersinia pseudotuberculosis resulted in its secretion via the Yersinia type III apparatus. Based on these data, we propose a model in which type III secretion pores are present on EBs and mediate secretion of early Incs and possible additional effectors. Mid-cycle expression of type III genes would then replenish secretion apparatus on vegetative RBs and serve as a source of secretion pores for subsequently formed EBs.

Effect of pruning on nitrogen dynamics within crowns of Pinus radiata.

Stem injection of (15)N-labeled ammonium sulfate was used to determine effects of pruning on canopy nitrogen dynamics in open-grown Pinus radiata D. Don in New Zealand. Trees were planted in July 1990 and the isotope introduced in December 1994. Tree crowns were divided into three zones: base section, from which branches of pruned trees were removed; mid section, between the pruned zone and the height of the trees at the start of the year in which they were pruned; and top section, which grew predominantly after the isotope was applied. Pruning removed 32% of the green crown length, representing 75% of foliage biomass. Needles were sampled from each region of the crown until July 1996. Branch growth was used to predict foliage biomass for each sampling occasion. Approximately 45% of the applied isotope was recovered from needles sampled in December 1994 (1 week after application and immediately before pruning), two-thirds of which occurred in needles in the base section. Thereafter, changes in isotope content of needles in the base section of unpruned trees largely reflected foliage biomass fluctuations and dilution of the isotope by continued uptake from the unlabeled soil nitrogen pool. Recovery of isotope in needles from the mid-crown section increased by 58 and 86% from December 1994 to July 1995 in control and pruned trees, respectively. Within this crown section, there was evidence of isotope translocation from old to new needles, with both isotope dilution and efflux observed in the needle cohorts that had been present at the time the isotope was applied. Therefore, isotope dynamics did not reflect the dynamics of the total nitrogen pool in the mid-crown section. By July 1996, a small proportion of the applied isotope was recovered from the new foliage formed in the top section of the crown. Within the top section, isotope dynamics closely matched total nitrogen fluxes. Pruning the lower crown did not affect nitrogen dynamics elsewhere in the crown for the following 18 months.

Cloning, sequencing, and expression of a gene from Campylobacter jejuni encoding a protein (Omp18) with similarity to peptidoglycan-associated lipoproteins.

A Campylobacter jejuni genomic plasmid library was screened with antiserum generated against whole C. jejuni, revealing two immunoreactive clones. Sequence analysis of the recombinant plasmids revealed a common open reading frame of 498 nucleotides encoding a protein of 165 amino acids with a calculated molecular mass of 18,018 Da. The recombinant product partitioned to the outer membrane fractions of Escherichia coli transformants and has been designated Omp18. The deduced amino acid sequence of the cloned C. jejuni gene exhibits considerable similarity to peptidoglycan-associated lipoproteins from other gram-negative bacteria.

Site-directed mutagenic alteration of potential active-site residues of the A subunit of Escherichia coli heat-labile enterotoxin. Evidence for a catalytic role for glutamic acid 112.

Escherichia coli heat-labile enterotoxin (LT) and the related cholera toxin exert their effects on eukaryotic cells through the ADP-ribosylation of guanine nucleotide-binding proteins of the adenylate cyclase complex. The availability of the crystal structure for LT has permitted the tentative identification of residues that lie within or are vicinal to a presumptive NAD(+)-binding site and thus may play a role in substrate binding or catalysis. Using a plasmid clone encoding the A subunit of LT, we have introduced substitutions at such potential active-site residues and analyzed the enzymatic properties of the resultant mutant analogs. Enzymatic analyses, employing both transducin and agmatine as acceptor substrates, revealed that substitutions at serine 61, glutamic acid 110, and glutamic acid 112 resulted in reduction of enzyme activity to < 10% of wild-type levels. Kinetic analyses indicated that alteration of these sites affected the catalytic rate of the enzyme and had little or no effect on the binding of either NAD+ or agmatine. Of the mutant analogs analyzed, only glutamic acid 112 appeared to represent an essential catalytic residue as judged by the relative effects on kcat and kcat/Km. The results provide formal evidence that glutamic acid 112 of the A subunit of LT represents a functional homolog or analog of catalytic glutamic acid residues that have been identified in several other bacterial ADP-ribosylating toxins and that it may play an essential role in rendering NAD+ susceptible to nucleophilic attack by an incoming acceptor substrate.

Increase in copy number of an integrated vector during continuous culture of Hansenula polymorpha expressing functional human haemoglobin.

Recombinant human haemoglobin A (rHbA) was produced by a leucine-requiring strain of Hansenula polymorpha which had been transformed with an integration vector containing the Saccharomyces cerevisiae LEU2 gene and cDNAs for the expression of alpha and beta globin each driven by the H. polymorpha MOX promoter. After 40 generations in a chemostat it was found that the integrated vector had become amplified in the host strain. In some cases this led to an increase in LEU2 gene dosage, but a loss of globin expression cassettes. In other cases the globin gene dosage also increased. These changes coincided with an increase in rHbA production in the culture, which was reversed when the dilution rate was increased. Isolates from a chemostat culture producing elevated levels of rHbA were grown in fed-batch fermentations, resulting in higher productivities than when inoculated with the parent strain. The rHbA produced was purified and characterized. Oxygen binding studies and electrospray mass spectrometry showed that the rHbA had been processed and assembled correctly, and behaved as a fully functional co-operative tetramer.

Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Campylobacter jejuni.

Campylobacter jejuni is a significant cause of bacterial enteritis in humans. Three of seven C. jejuni isolates examined were found to contain fragmented 23S rRNA. The occurrence of fragmented 23S rRNA correlated with the presence of an intervening sequence (IVS) within the 23S rRNA genes. The IVS is 157 nucleotides in length and replaces an eight nucleotide sequence in the 23S rRNA genes of C. jejuni isolates that contain intact 23S rRNA. The two ends of the IVS share 31 bases of complementarity that could form a stem-loop structure. Fragmentation of the 23S ribosomal RNA results from the excision of the IVS from the transcribed RNA; the 3' cleavage site maps within the putative stem-loop formed by the IVS. Southern hybridization analysis revealed that the IVS is not present in the genomes of isolates that contain intact 23S rRNA, suggesting that the IVS is not derived from Campylobacter chromosomal sequences. The C. jejuni IVS is located at a position analogous to that of the IVSs found in both Salmonella and Yersinia spp.

Cloning and expression of the hup encoding a histone-like protein of Campylobacter jejuni.

A Campylobacter jejuni gene, designated hup, that appears to encode a homolog of the histone-like DNA-binding protein, HU, has been cloned, sequenced and expressed in Escherichia coli. Immunoblotting and in vitro transcription/translation analyses revealed a 11-kDa protein that was produced by recombinant plasmids containing hup. The gene contains an open reading frame (ORF) sufficient to encode a protein of 98 amino acids (aa) with a calculated molecular mass of 10,267 Da and a predicted isoelectric point of 10.1. The deduced aa sequence of the protein, designated HCj, exhibits considerable sequence identity with members of the HU family of proteins from other eubacterial species. The transcription start point was identified by primer extension analysis and appropriately spaced promoter sequences were found which exhibit considerable similarity to E. coli and Bacillus promoters. Southern hybridization analyses indicate that C. jejuni has a single copy of hup.

Distribution and retranslocation of (15)N lodgepole pine over eight growing seasons.

We studied the distribution and retranslocation of N in 11-year-old Pinus contorta Dougl. trees following a winter application of N at 100 kg ha(-1) as (15)N-urea, (15)NH(4)NO(3) or NH(4) (15)NO(3). In all treatments, there was little uptake of (15)N after the first growing season although labeled N was still present in the soil. In subsequent years, (15)N in the trees was partly retranslocated, and, at the same time, it was diluted by uptake of unlabeled N from the soil. Between Years 1 and 8 after N fertilization, net retranslocation of (15)N from the lower crown (branches formed before fertilization) was 14%, and 18-25% of the (15)N in the trees was translocated to the upper and mid-crown. Overall, uptake of (15)N from nitrate was less than from urea or ammonium. However, when compared with the urea- and ammonium-N sources, (15)N from the nitrate source initially moved as rapidly into the foliage, but a greater proportion of it was retranslocated from the foliage during the second growing season. Nitrogen in foliage and wood formed in the growing season following fertilization was more highly labeled (measured as % N derived from the fertilizer) than in recently formed tissues. Labeling was substantially higher in foliage formed before fertilization than in wood of a similar age. In contrast, N in foliage formed after fertilization had only slightly higher labeling than wood of a similar age, indicating a relatively stable labeling throughout the trees once (15)N uptake had ceased. The concentrations of total and labeled N were substantially higher in foliage than in either wood or bark. There was evidence of N movement into wood tissues formed before fertilization, presumably along rays, and also of N retranslocation out of xylem cells as they matured. This study of internal N cycles was facilitated by the use of (15)N labeling because there was little uptake of labeled N after the first growing season, whereas interpretation based on total N was obscured by substantial uptake of N from the soil. We conclude that retranslocation studies based on measurements of total N content should be avoided.

Kinetic and antigenic characterization of altered protein synthesis by Campylobacter jejuni during cultivation with human epithelial cells.

Cultivation of Campylobacter jejuni with INT 407 cell monolayer cultures results in new or enhanced synthesis of a number of proteins compared with bacteria cultured in the absence of the epithelial cells. These proteins were detected within 60 min after the addition of the bacteria to the epithelial cell cultures, and their synthesis was temporally associated with an increase in C. jejuni internalization. A rabbit antiserum raised against bacteria that were cultivated with INT 407 cells recognized nine proteins that were not recognized by an antiserum against C. jejuni cultivated in medium alone. The former antiserum inhibited the internalization, but not the binding, of Campylobacter jejuni in a dose-dependent fashion. The results suggest that one or more of the proteins synthesized by C. jejuni in response to cocultivation with epithelial cells plays a role in facilitating internalization.

Phospholipid methylation in brain membrane preparations: kinetic mechanism.

The methylation reactions which convert phosphatidylethanolamine (PE) to phosphatidylcholine (PC) have been studied kinetically using exogenously added intermediates and crude membrane preparations from brain. The addition of exogenous PE resulted in no change in the methylation rates compared to that of endogenous PE. The addition of the two intermediates, monomethylphosphatidylethanolamine (PMME) and dimethylphosphatidylethanolamine (PDME), resulted in significantly increased rates of methylation and allowed the kinetic analysis of these latter two methylation reactions. The mechanism for this enzyme appears to be similar to human RBC (Reitz et al. (1989) J. Biol. Chem. 264, 8097-8106) which was a rapid-equilibrium random Bi-Bi sequential mechanism. There were some slight differences between the brain enzyme and that from the RBC, but there is little reason to suggest a fundamentally different mechanism. It is more likely that the differences may relate to an additional dead-end complex for the enzyme from brain such that saturation with AdoMet cannot eliminate AdoHcy inhibition. The KM values for the two phospholipid substrates were 41-44 microM and 39 microM for the methylation of PMME and PDME, respectively. The KM for S-adenosylmethionine (AdoMet) was 7-9 microM with PMME and 4 microM with PDME as the other substrates. The Ki(lipid) varied from 54 microM with PMME to 225 microM with PDME, and the Ki(AdoMet) was 11 microM with PMME and 21 microM with PDME. The product from the use of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a noncompetitive inhibitor of both lipid substrates as well as AdoMet. The methylation of PMME was somewhat higher in cerebellum and brain stem compared to cortex and striatum, but the methylation of PDME was similar in cerebellum, brain stem and cortex.

Translocation of Campylobacter jejuni across human polarized epithelial cell monolayer cultures.

The ability of Campylobacter jejuni isolates to translocate across an epithelial cell barrier was investigated by using polarized Caco-2 cell monolayers grown on microporous membrane filters. The 4 C. jejuni isolates tested all traversed the Caco-2 cell monolayers and displayed similar translocation kinetics. The number of bacteria crossing the polarized cell monolayers continued to increase with time until 4 h after inoculation, at which time a maximum rate of translocation was observed. Transmission electron microscopy revealed that C. jejuni translocated across polarized Caco-2 cell monolayers by passing both through and between cells. Chloramphenicol, an inhibitor of bacterial protein synthesis, reduced the translocation of C. jejuni. Bacterial attachment, internalization, and translocation were inhibited at low temperature. These data indicate that adherence, penetration, and translocation of C. jejuni require active bacterial and target cell processes and further suggest a role for cellular translocation in the pathogenesis of C. jejuni-mediated enteritis.

Phosphatidylethanolamine N-methyltransferase in human red blood cell membrane preparations. Kinetic mechanism.

The successive methylations of phosphatidylethanolamine to form phosphatidylcholine were measured using exogenously added intermediates and membrane preparations from human red blood cells. The addition of phosphatidylethanolamine resulted in no increase in methylation rate over that with endogenous substrate; however, the addition of monomethylphosphatidylethanolamine (PME) and dimethylphosphatidylethanolamine (PDE) markedly increased the reaction rate and allowed studies into the kinetic mechanism for the second and third methylation reactions. The data are consistent with catalysis of the last two methylations being by a single enzyme with a random Bi-Bi sequential mechanism. Analysis of PDE:phosphatidylcholine product ratios indicates that the enzyme can conduct multiple methylations of enzyme-bound phospholipid. The nature of the acyl chain (16:0 versus 18:1) of the phospholipid had only a small effect on the value of the kinetic constants. The maximal velocities obtained with the 18:1 substrate were less than 5% lower than those obtained with the 16:0 substrate. The Km values for the two phospholipids were 20-45 and 10-14 microM for the methylation of PME and PDE, respectively. The Km for S-adenosylmethionine (AdoMet) was 5-9 microM with PME and 4 microM with PDE as substrates. Depending on the acyl chain and the phospholipid, the Ki(AdoMet) varied from 8 to 19 microM, the Ki(PME) from 41 to 82 microM, and the Ki(PDE) from 35 to 61 microM. The Ki for S-adenosylhomocysteine (AdoHcy) was between 1.0 and 1.4 microM depending upon the variable substrate. The endogenous concentrations of PME and PDE in red blood cell membranes were estimated to be 0.49 and 0.24 mumol/liter packed cells, respectively. The product from the utilization of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a competitive inhibitor of its precursor, AdoMet, and a noncompetitive inhibitor of the two phospholipid substrates.

The yeast 2 micron plasmid: strategies for the survival of a selfish DNA.

The designation of the yeast 2 mu circle as a "selfish" DNA molecule has been confirmed by demonstrating that the plasmid is lost with exponential kinetics from haploid yeast populations grown in continuous culture. We show that plasmid-free yeast cells have a growth rate advantage of some 1.5%-3% over their plasmid-containing counterparts. This finding makes the ubiquity of this selfish DNA in yeast strains puzzling. Two other factors probably account for its survival. First, the rate of plasmid loss was reduced by allowing haploid populations to enter stationary phase periodically. Second, it was not possible to isolate a plasmid-free segregant from a diploid yeast strain. Competition experiments demonstrated that stability in a diploid is conferred at the level of segregation and that plasmid-free diploid cells are at a selective advantage compared with their plasmid-containing counterparts. Yeast cells in nature are usually homothallic and must frequently pass through both diploid and stationary phases. The 2 mu plasmid appears to have evolved a survival strategy which exploits these two features of its host's life cycle.

Purification and properties of a double-stranded ribonuclease from the yeast Saccharomyces cerevisiae.

A double-stranded ribonuclease has been purified more than 90-fold to near homogeneity from the yeast, Saccharomyces cerevisiae. The enzyme shows a high specificity for double-stranded RNA as its substrate. It has a molecular weight of 27000 as determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The enzyme degrades dsRNA optimally at 30 degrees C; it is stimulated by KCl and by the -SH reagent, dithiothreitol. In contrast to RNase III from Escherichia coli, the yeast enzyme is inhibited by divalent cations. Physiological studies have demonstrated that in vivo levels of the enzyme activity fell during the latter part of the exponential growth phase but rose during stationary phase. The specific activity of the enzyme in nitrogen-starved yeast cells was 2-3-fold higher than in non-starved cells. The enzyme could be detected in yeast strains containing both, one or none of the species of cytoplasmic dsRNA (L and MdsRNAs) and may, therefore, have some wider role.