Immobilization of the slime mould Dictyostelium discoideum for the continuous production of recombinant human antithrombin III

Recombinant vegetative Dictyostelium discoideum cells were immobilized inside a porous matirx by an inorganic membrane that was permeable to nutrients but not to cells, in order to produce recombinant human antithrombin III. Cells so entrapped could reach up to 15 times higher biomass densities compared with organisms growing freely in suspension. The high cell concentration maintained in the immobilized cell bioreactor caused an increase in specific and volumetric productivity. In continuous operation a maximum volumetric antithrombin productivity of 56 ng h ^–1 ml ^–1 catalyst bulk volume was attained at a dilution rate of 0.016 h ^–1. In addition, the good retention of metabolic activity for several weeks as well as the convenient form of storage and regeneration of the catalytic system were shown. Correspondence to: H. Tiltscher     

Sequence analysis of an actin isoform in the flatworm Diphyllobothrium dendriticum (Cestoda)

We have examined actin cDNA of the flatworm Diphyllobothrium dendriticum (Cestoda). Actin is a contractile protein that has been implicated in a variety of developmental and cellular processes. It is highly conserved and present in all eukaryotic cells. It is of particular interest to analyze evolutionary preserved genes in flatworms, because ancestral flatworms are regarded to play a central role in the evolution of the metazoans (Barnes et al., 1998). Screening a cDNA library of D. dendriticum (UniZap XR, Stratagene) with a human -actin probe resulted in several positive clones. One of the cDNA inserts, Didactl, consisting of 1392 bp was completely sequenced. The established nucleotide sequence revealed a 5 untranslated region of 33 bp, the entire open reading frame of 1128 bp and a 3 untranslated region of 231 bp which ends in a stretch of 21 A residues. The potential polyadenylation signal (AATAAA) is located 14 bp upstream of the poly (A) tail. The deduced amino acid sequence of Didactl is 376 amino acids long. It is a typical invertebrate actin (Fyrberg et al., 1981) resembling more the cytoplasmic than the muscular isoforms of vertebrate actins. Didactl is for example 96% homologous to human cytoplasmic -actin but only 92.6% identical with human smooth muscle -actin. The actin proteins are generally encoded by a multigene family which differs in size from species to species. Most organisms have four to eight genes coding for actin in their genome, but the number of actin genes can also be over 20 (Hamelin et al., 1988). Sequence comparisons of Didactl and the partly sequenced cDNA clones indicate that D. dendriticum has at least four different genes coding for actin in its genome.     

Sequence analysis of the DdPYR5-6gene coding for UMP synthase in Dictyostelium discoideum and comparison with orotate phosphoribosyl transferases and OMP decarboxylases

Summary A Dictyostelium discoideum DNA fragment that complements the ura3 and the ura5 mutants of Saccharomyces cerevisiae has been sequenced. It contains an open reading frame of 478 codons capable of encoding a polypeptide of molecular weight 52475. This gene, named DdPYR5-6, encodes a bifunctional protein composed of the orotate phosphoribosyl transferase (OPRTase) and the orotidine-5-phosphate decarboxylase (OMPdecase) domains described for UMP synthase in mammals. The existence of separate domains for the two activities was suspected because deletion of the N-terminal coding segment of the gene eliminated the ura5 but not the ura3 complementing activity. We have now confirmed that the two parts of the open reading frame share homology with known OPRTase and OMPdecase sequences. Several blocks of sequence are conserved among OPRTase from bacteria, fungi and slime mold and one of them corresponds to the consensus sequence for phosphoribosylbinding sites. The OMPdecase domain shows extensive similarity with the yeast and Neurospora crassa enzymes, suggesting that they have evolved from an ancestral gene which was fused to the OPRTase gene in D. discoideum. It is less related to the bacterial enzyme but all these sequences present conserved blocks of homology which could identify the active site. The codon usage is strongly biased in a manner similar to that found for other D. discoideum genes. The flanking DNA contains homopolymers of A and T and alternating sequences that are characteristic of the gene organization in D. discoideum.     

Cultivation of immobilized <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> for the production of soluble human Fas ligand

Dictyostelium discoideum was immobilized by cultivation on inorganic porous matrices consisting of broken pumice or a ceramic catalyst carrier (CeramTec) to produce human soluble Fas ligand (hFasL). These supports were actively colonized by D. discoideum reaching cell (number) densities 10–20 times higher locally than those observed in suspension culture under similar conditions. In repeated batch or continuous operation, hFasL productivities of up to 15–25 g h^–1 l^–1 pore volume were attained. The immobilized cell densities and hFasL productivities could be kept constant for a long period of time by repeated renewal or continuous feeding of complex or synthetic medium.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Structure of the cDNA coding for conglutin γ, a sulphur-rich protein from Lupinus angustifolius

The sequence of cDNA coding for a sulphur-rich storage protein from Lupinus angustifolius L., conglutin , was determined. The coding region contained an N-terminal leader peptide of 28 amino acids which directly preceded subunits of M _r 28 239 and 16 517. Extensive sequence homology between the protein encoded by conglutin cDNA and basic 7S globulin from soybean was observed. Sequence homology to proteins from other classes of storage proteins, 11S, 7S and 2S, was limited to short and highly fragmented sequences. The amino acid sequence, Asn-Gly-Leu-Glu-Glu-Thr, characteristic of the primary site for post-translational cleavage of the precursors of 11S proteins, was absent from the sequence predicted for prepro-conglutin . It is concluded that conglutin is a representative of a fourth type of storage protein in legumes, distinct from the 11S, 7S and 2S storage protein families.     

Full-length cDNA sequence of X-linked G6PD of an Australian marsupial,the wallaroo

In order to study the mechanism of X Chromosome (Chr) inactivation in marsupials, the cDNA for glucose-6-phosphate dehydrogenase (G6PD) from an Australian marsupial, the wallaroo (Macropus robustus), was cloned. A partial clone containing the 3 half of the cDNA was obtained by screening a liver cDNA library. The majority of the coding region was obtained by polymerase chain reaction of cDNA with primers designed from regions of conservation between human and opossum G6PD. The 5 end was obtained by rapid amplification of cDNA ends. High homology was observed between mammalian species in the coding region. The 5 untraslated region is highly G+C rich, and appears to be part of a CpG island, as is the case in the human and mouse genes. This is the first report of the full sequence of the cDNA for any marsupial X-linked gene.The nucleotide sequence data reported in this paper have been submitted to GenBank and assigned accession number U13899.     

Characterization,expression and phylogenetic study of R2R3-MYB genes in orchid

cDNA fragments representing 21 R2R3-MYB genes were isolated by RT-PCR from the Dendrobiumorchid hybrid Woo Leng. Six full-length cDNA clones were obtained from a flower cDNA library, four of which, DwMYB1, DwMYB2, DwMYB8 and DwMYB10, represent typical plant R2R3-MYB genes. The conceptual DwMYB4 protein is truncated at the C-terminal region and contains the R2 repeat and the N-terminal half of the R3 repeat (R2R3). DwMYB4 expression is restricted to flowers. DwMYB9 contains an 8 amino acid N-terminal deletion in the R2 repeat (R2R3) and is expressed at high levels in mature flower and inflorescence, but at very low levels in young flower buds. DwMYB8 and DwMYB10 show similar expression patterns and share very high sequence similarity in the N-terminal part of the MYB domain. Analysis of amino acid substitution indicated that the pattern and type of substitution between Arabidopsis and maize are quite different. Maize may have more conserved substitution in the MYB^BRH domain than Arabidopsis.     

Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris

Mature human growth hormone (hGH) cDNA was cloned by homologous recombination into the yeast Pichia pastoris genome. The hGH gene expression was placed under the control of the methanol-inducible alcohol oxidase 1 (AOX1) gene promoter and the Saccharomyces cerevisiae -factor signal sequence to direct the secretion of recombinant human growth hormone (rhGH) into the growth medium. O₂-limited induction of recombinant yeast strains in shake tubes with 3 ml of culture medium produced up to 11 mg rhGH l^–1, while high cell density cultures using a 2-l bioreactor produced about 49 mg rhGH l^–1 achieving 40% of total protein of the culture medium supernatant.     

Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 × 10⁶ cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) cross-reacted with re-PNA. The subunit molecular weight (30kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.     

Biosynthesis,cDNA and amino acid sequences of a precursor of conglutin δ, a sulphur-rich protein from Lupinus angustifolius

The biosynthesis of conglutin has been studied in developing cotyledons of Lupinus angustifolius L. Precursors of conglutin formed the major sink for [³⁵S]-cysteine incorporated by developing lupin cotyledons, and these precursors were rapidly sequestered into the endoplasmic reticulum. The sequence of a cDNA clone coding for one such precursor of conglutin was determined. The structure of the precursor polypeptide for conglutin predicted from the cDNA sequence contained an N-terminal leader peptide of 22 amino acids directly preceding a subunit polypeptide of M _r 4520, together with a linking region of 13 amino acids and a subunit polypeptide of M _r 9558 at the C-terminus. The amino acid sequence predicted from the cDNA sequence showed minor variations from that established by sequencing of the protein purified from mature dried seeds (Lilley and Inglis, 1986). These were consistent with the existence of a multi-gene family coding for conglutin . Comparison of the sequences of conglutin with those of other 2S storage proteins showed that the cysteines involved in internal disulphide bridges between the mature subunits of conglutin , were maintained throughout this family of proteins but that little else was conserved either at the protein or DNA level.     

Repeated batch cultivation of rBHK cells on Cytodex 3 microcarriers: antithrombin III,amino acid,and fatty acid metabolic quotients

Anchorage-dependent human antithrombin III-producing recombinant baby hamster kidney (rBHK) cells were cultivated on Cytodex 3 microcarriers in repeated batch mode. During a 3-month experiment four different low-serum (0.025% fetal bovine serum) or serum-free medium formulations were evaluated for (a) the initial growth phase of cells and (b) the subsequent production phase, whereby two free fatty acid (FFA) supplements were examined with respect to their growth-promoting and product-formation-enhancing properties. Selected nutrient and (by)product consumption and production rates (including those for antithrombin III, amino acids, and fatty acids) are reported. The calculated metabolic quotients reflect the prevailing slow growth conditions ( approx. 0.06 day^–1) associated with microcarrier cultures. Specific antithrombin III productivities vary singificantly as a function of the feed medium supplementation with FFA. Correspondence to: G. Schmid     

Improved purification and biochemical characterization of extracellular amylopullulanase from Thermoanaerobacter ethanolicus 39E

A maltose-limited chemostat culture was used to investigate the expression and excretion of amylopullulanase by Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E). In maltose-limited continuous culture, amylopullulanase was produced and secreted at tenfold higher levels than in batch culture. The extracellular amylopullulanase was purified to homonogeneity by using an inhibitor-linked affinity column matrix. The purified amylopullulanase had a specific activity of 480 units (U)/mg protein for pullulanase and 175 U/mg protein for -amylase. -Cyclodextrin inhibited both -amylase and pullulanase activities, with a substrate inhibition constant (K _i) of 0.065 mg/ml.Amylopullulanase had a relative molecular mass (M_r) of 140 000 using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and an M_r of 133 000 using gel-filtration chromatography. The N-terminal sequence of the enzyme was Glu-Thr-Asp-Thr-Ala-Pro-Ala. The purified enzyme displayed Michaelis constant (K _m) values of 0.35 mg/ml for pullulan and 1.00 mg/ml for amylose. The enzyme had an isoelectric point (pI) of 4.0, and displayed an optimum pH for stability and activity of 6.2 and 5.5, respectively. The enzyme was stable up to 85° C in the presence of Ca²⁺, and had a half-life of 40 min at 90° C (pH 6.2). Ca²⁺ was required for thermal stability, but not for activity. Amylose, glycogen, and amylopectin were degrade to maltose, maltotriose, and maltotetraose, whereas only maltotriose was formed from pullulan. Correspondence to: J. G. Zeikus     

1H, 15N, and 13C assignments of the N-terminal activation domain of Dictyostelium discoideum Formin C

Dictyostelium discoideum Formin C (ForC) plays an important role in the fruiting body formation during the multicellular stages of the slime mold. Formins are multidomain proteins that are known to regulate the actin cytoskeleton. Here, we report the assignments of the ¹H, ¹⁵N, and ¹³C nuclei of the N-terminal activation domain (residues 1–100) of ForC. Chemical shifts have been deposited at the BioMagResBank under the BMRB accession number 17,029. The N-terminal region of the 131 kDa ForC protein is supposed to form a GTPase-binding domain required for activation of the formin.     

Cloning of bacteriophage T5 promoters

Summary Bacteriophage T5 was subjected to combined hydrolysis with the restriction endonuclease PstI and HindIII and the resulting fragments were inserted into the plasmid pBR322. Selection of transformants for Ap^s-Tc^r-phenotype made it possible to screen the hybrid plasmids that contained promoter sequences in the cloned fragments.Two PstI/HindIII fragment, 720 bp (51% of the T5 DNA length) and 1,200 bp (70%) were cloned in this study. Tc^r levels for these plasmids were as high as 18 g/ml and 75 g/ml, respectively. The presence of Escherichia coli RNA polymerase binding sites on both fragments was shown using the nitrocellulose filter assay. These binding sites are situated between 35 bp and 95 bp from the HindIII cleavage site on the 1,200 bp fragment; and within 420 bp from the HindIII site on the 720 bp fragment.Abbreviations Ap ampicillin - Tc tetracycline - bp base pairs - NTPs nucleoside triphosphates - PBB polymerase binding buffer     

The Dictyostelium discoideum mitochondrial genome: A primordial system using the universal code and encoding hydrophilic proteins atypical of metazoan mitochondrial DNA

A 3,345-bp fragment of Dictyostelium discoideum mitochondrial DNA (mtDNA) has been sequenced. This fragment contained the 80-kDa subunit of complex I (NADH:ubiquinone oxidoreductase), encoding a predicted amino acid sequence of 688 residues and a molecular mass of 79,805 daltons which is nuclear encoded in other metazoa. The C-terminus of the D. discoideum complex I gene shared a 10-bp overlap with NADH:ubiquinone oxidoreductase chain 5 (ND5), while 21 by 5 were three tRNA genes (two isoleucine and a histidine) and a further 25 by 5 of these genes is the partial sequence (104 residues) of an unidentified open reading frame (ORF104). Both the 80-kDa subunit and the ORF104 were hydrophilic and highly charged, suggesting they are not membrane associated, unlike most mitochondrially encoded proteins in the metazoa. Sequence analysis of the 80-kDa subunit, its adjacent ND5 gene, and ORF104 indicates the universal stop codon TGA, which codes for tryptophan in nearly all nonplant mtDNA, is either unassigned or coding for a stop codon in D. discoideum. The large size of the mitochondrial genome (54 kb), the lack of intergenic sequence, and the apparent use of the universal code suggest D. discoideum mtDNA may encode many primitive genes that are nuclear encoded in higher organisms.Correspondence to: K.L. WilliamsData deposition: GeneBank     

Quantification of citrate lyase by enzyme-linked immunosorbent assay for determining the population of Lactococcus lactis subsp. Lactis biovar diacetylactis in pure and mixed cultures

Citrate lyase production by Lactococcus lactis subsp. lactis biovar diacetylactis DRC2 was quantified by an enzyme-linked immunosorbent assay (ELISA). The citrate lyase reached a concentration equivalent to 41 ± 4 g/ml purified citrate lyase in pure culture. When the strain DRC2, grown in mixed culture with L. lactis subsp. cremoris AM2, represented around 70% (DC culture) or 30% (CD culture) of the total initial population, the level of citrate lyase decreased to 21 ± 7 g/ml and 4.5 ± 1.5 g/ml respectively. The maximum bacterial concentration of strain DRC2 in pure culture reached 2.6 × 10⁹ cfu/ml and decreased to 1.5 (± 0.2) × 10⁹ cfu/ml and 0.5 (± 0.3) × 10⁹ cfu/ml in DC and CD mixed cultures respectively. In mixed cultures, the proportion of the strain DRC2 was 8.5 ± 5.0% lower at the end of the fermentation than immediately after inoculation, thus showing that this strain was clearly inhibited. However, the maximum rate of citrate consumption was the same during pure DRC2 culture and CD mixed culture (2.5 ± 0.3 mmol/h) and slightly highre in DC culture (3.07 mmol/h). The maximum rate of acidification was 0.37 ± 0.04 pH unit/h regardless of the culture. A good correlation was obtained between the population of the strain DRC2 and the citrate lyase concentration determined by ELISA but no relationship was found between citrate consumption and citrate lyase synthesis. Therefore an ELISA test of this kind can be used to monitor the growth of L. lactis subsp. lactis biovar diacetylactis in mixed cultures.     

Secretion of a heat-stable fungalβ-glucanase from transgenic,suspension-cultured barley cells

Transgenic plant cell cultures have a potential for production and secretion of important proteins and peptides. To assess the possibilities of using a stable barley suspension culture for secretion of heterologous proteins in active form, we expressed the cDNA of the thermostable-glucanase (EGI) ofTrichoderma reesei in barley suspension cells. The cDNA coding for EGI and its signal sequence was placed under the control of the CaMV 35S promoter and the construction was transferred to the cells by particle bombardment. Stably transformed lines were obtained by selecting for a cotransformed antibiotic resistance marker. The expression of EGI cDNA led to accumulation of EGI in the culture medium, as shown by analysis with EGI-specific antibodies. Enzymatic assays confirmed that the EGI secreted by the suspension cells retained its activity and thermostable character. Furthermore, it was shown that the enzyme produced by the transgenic suspension culture could be used for degradation of soluble-glucans during mashing.     

The complete amino acid sequence of the murine transplantation antigen H-2Db as deduced by molecular cloning

A mouse cDNA library derived from the EL4 cell line (b-haplotype) was screened with a probe containing a small part of the H-2K^b coding region. One of the clones isolated, pH203, encodes a protein whose deduced amino acid sequence is identical with the known sequence of H-2D^b in 141 of 141 positions available for comparison. The clone, therefore, is believed to code for the H-2D^b transplantation antigen. The cDNA insert of pH203 contains the coding region for residues 82 through the carboxy-terminus of H-2D^b, and includes 476 nucleotides of the 3-untranslated sequence. Comparison between the H-2D^b cDNA clone and a previously isolated H-2K^b cDNA clone shows homologies of 83% and 91% at the amino acid and nucleotide levels, respectively. Analysis of DNA sequences at the 3-coding and untranslated regions suggests that the mRNAs of H-2K^b and H-2D^b are spliced differently at their 3-coding ends.