Mapping of the structural gene of pseudorabies virus glycoprotein A and identification of two non-glycosylated precursor polypeptides.

Cell-free translation of pseudorabies virus RNA isolated during the late phase of the infectious cycle yielded a variety of polypeptides. A monoclonal antibody directed against one of the major viral glycoproteins, gA, immunoprecipitated two polypeptides ranging in molecular weight from 78K to 83K. To localize the structural gene for gA, we used cloned BamHI fragments of the viral DNA to select specific mRNA species and immunoprecipitated their in vitro translation products with the anti-gA monoclonal antibody. This allowed us to map the genomic region encoding the mRNA for the gA within the short unique region of the viral genome on BamHI fragments 7 and 12. Additional polypeptides encoded by this region were characterized by their electrophoretic mobility. In three virus strains tested a similar, but strain-specific, pattern of the two gA precursors was found which was not dependent on the host cell or the state of infection after reaching the late phase.     

Efficiency of the cyclic batch antibiotic fermentation

For a simple mathematical model of microbial product formation, the productivities of stationary and nonstationary cultivation methods (chemostat and cyclic batch) are compared. Conditions of superiority of the cyclic batch cultivation are characterized. The model includes substrate inhibition of the product formation and the age-dependent loss of productivity of the culture.     

Kluyveromyces lactis cells entrapped in Ca-alginate beads for the continuous production of a heterologous glucoamylase

Viable cells of Kluyveromyces lactis, transformed with the glucoamylase gene from Arxula adeninivorans, were entrapped in beads of Ca-alginate and employed on a lab scale in a continuous stirred and a fluidised bed reactor (FBR), both fed with a rich medium (YEP) containing lactose as carbon source. Experiments with freely suspended cells in batch and chemostat had demonstrated that glucoamylase production was favoured in the presence of lactose and YEP medium. Employing controlled-sized beads having a 2.13 mm diameter, specific glucoamylase productivity was higher in the stirred reactor (CSTR) than in the FBR; in the latter a higher volumetric productivity was achieved, due to the lower void degree. The performance of the immobilised cell systems, in terms of specific glucoamylase productivity, was strongly affected by mass transfer limitations occurring throughout the gel due to the high molecular weight of the product. In the perspective to improve and scale-up the immobilised cell system proposed, a mathematical model, which takes into account substrate transfer limitations throughout the gel, has been developed. The effective lactose diffusivity was related to the bead reactive efficiency by means of the Thiele modulus. The regression of the model parameters on the experimental data of substrate consumption obtained both in the CSTR and in the FBR allowed to estimate lactose diffusivity and the kinetic parameters of the immobilised yeast.     

Effects of high cell density on growth-associated monoclonal antibody production by hybridoma T0405 cells immobilized in macroporous cellulose carriers

Relationship between monoclonal antibody (MAb) productivity and growth rate, and effects of high cell density on MAb production of hybridoma T0405 cells immobilized in macroporous cellulose carriers were investigated in continuous and batch cultures. The results showing, that the specific MAb production rate increased with increasing specific growth rate in both suspended and immobilized continuous cultures indicate a positively growth-associated relationship between MAb productivity and growth rate. Moreover, the specific production rate was higher in the immobilized cell culture than that in suspended one at all dilution rates. In order to clarify these phenomena, MAb mRNA expression and cell cycle distribution were investigated in batch cultures with immobilized cells and suspended cells. RT-PCR was used for observation of MAb mRNA expression and a two-color bromode-oxyuridine (BrdU)/propidium iodide (PI) flow cytometry method for determination of cell cycle distribution. The results revealed that MAb mRNA expression reached the peak during the exponential growth phase, suggest a positively growth-associated MAb production. And the immobilized cells continued the MAb mRNA expression until dead phase, which was longer than that in suspended cells. The cell cycle distribution patterns were observed almost the same for both immobilized and suspended cells. Such results may imply that a high cell density state has positive influence on the mRNA expression and on growth-associated MAb productivity of T0405 cells.     

The role of the cell cycle in determining gene expression and productivity in CHO cells

Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Specific monoclonal antibody productivity and the cell cycle-comparisons of batch,continuous and perfusion cultures

A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G₁ phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of G₁ phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.     

By-product formation in cell-recycled continuous culture of Lactobacillus casei

While the volumetric productivity of lactic acid increased in continuous culture of Lactobacillus casei with cell recycle, enhanced formation of by-products such as acetate, formate, ethanol, and D-lactate was observed in the cell-recycled fermenter compared with a simple chemostat at a similar range of dilution rate. The increased formation of by-product which was significantly dependent on substrate limitation resulted from a lower dilution rate rather than a high cell concentration in the cell-recycled fermenter.     

Performance evaluation of ethanol fermentor systems using a vector-valued objective function

It is shown that the performance evaluation using a vector-valued objection function whose components are the product productivity, the product concentration, and the substrate conversion is quite useful in getting deeper insight into the development of new processes and in determining the operating point. Particular attention is focused on the ethanol fermentation using variety of systems such as the conventional chemostat system, multiple fermentor system, cell recycle system, extractive fermentor system, cell recycle system, extractive fermentor system, and immobilized cell system. The contour map and the projection of the noninferior set are used in investigating the performance improvement and the trade-offs among performance indexes.     

Construction of cDNA coding for human von Willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene.

Von Willebrand Factor (vWF) mRNA was identified in fractionated polyA+ RNA preparations isolated from cultured human endothelial cells. Micro-injection of specific polyA+ RNA fractions in Xenopus laevis oocytes provoked the synthesis of a vWF-like product which could be detected with an immunoradiometric assay relying on Sepharose-linked monoclonal anti-vWF IgG and different radiolabeled monoclonal anti-vWF IgGs. A vWF-mRNA-containing polyA+ RNA preparation served as substrate for a size-selected cDNA-expression library of 60 000 colonies which was screened for the synthesis of antigens related to vWF, using polyclonal anti-vWF IgG and a second antibody conjugated with peroxidase. Eight positive colonies were detected of which two reacted strongly in the enzyme-linked assay. Immunoblotting of bacterial extracts of "expression clones" with a monoclonal anti-vWF IgG revealed polypeptides which size fits within the length of the cDNA insertions. Northern blotting of human endothelial RNA, employing fragments of vWF cDNA as probes, showed specific hybridization with a mRNA of about 9000 nucleotides. DNA-sequence analysis of a vWF-cDNA insertion revealed an open reading frame followed by a translation stopcodon. It is argued that the cDNA insertions encode the carboxy-terminal part of the vWF protein. vWF-cDNA probes were employed to map the von Willebrand factor gene on chromosome 12 using a panel of 35 human-rodent somatic cell hybrids.     

A U-rich element in the 5' untranslated region is necessary for the translation of p27 mRNA

Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5' untranslated region (5'UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5'UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth- and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5'UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals.