Applications of quantitative PCR in the biosafety and genetic stability assessment of biotechnology products

High throughput screening, increased accuracy and the coupling of real-time quantitative PCR (Q-PCR) to robotic set-up systems are beginning to revolutionise biotechnology. Applications of Q-PCR within biotechnology are discussed with particular emphasis on the following areas of biosafety and genetic stability testing: (a) determination of the biodistribution of gene therapy vectors in animals; (b) quantification of the residual DNA in final product therapeutics; (c) detection of viral and bacterial nucleic acid in contaminated cell banks and final products; (d) quantification of the level of virus removal in process validation viral clearance studies; (e) specific detection of retroviral RT activity in vaccines with high sensitivity; and (f) transgene copy number determination for monitoring genetic stability during production. Methods employed for Q-PCR assay validation as required in ICH Topic Q2A Validation of Analytical Methods: Definitions and Terminology (1st June 1995) are also reviewed.     

Functional defects of SKAP-55-deficient T cells identify a regulatory role for the adaptor in LFA-1 adhesion

The ADAP-SKAP-55 module regulates T-cell receptor (TCR)-induced integrin clustering and adhesion in T cells. However, it has been unclear whether ADAP and/or SKAP-55 is an effector of the response. ADAP controls SKAP-55 expression such that ADAP(-/-) T cells are also deficient in SKAP-55 expression. In this study, we report the phenotype of the SKAP-55-deficient mouse. SKAP-55(-/-) T cells retain ADAP expression yet show defects in beta1 and beta2 integrin adhesion, leukocyte function-associated antigen 1 (LFA-1) clustering, production of the cytokines interleukin-2 and gamma interferon, and proliferation. This dependency was also reflected in more-transient conjugation times in response to the superantigen staphylococcal enterotoxin A on dendritic cells and a reduced number of cells with TCR/CD3 microcluster localization at the immunological synapse. SKAP-55(-/-) T cells showed the same general impairment of function as ADAP(-/-) T cells, indicating that SKAP-55 is an effector of the ADAP-SKAP-55 module. At the same time, the requirement for ADAP and SKAP-55 was not absolute, since a subset of peripheral T cells adhered with loss of expression of either adaptor. Further, dependency on SKAP-55 or ADAP differed with the strength of the TCR signal. As with the ADAP(-/-) mouse, SKAP-55-deficient mice showed no major effects on lymphoid development or the appearance of peripheral T cells, B cells, and NK cells. Our findings identify a clear effector role for SKAP-55 in LFA-1 adhesion in peripheral T cells and demonstrate that dependency on SKAP-55 and ADAP differs among T cells and differs with the strength of the TCR signal.     

Exposure of Petunia Seedlings to Ethylene Decreased Apical Dominance by Reducing the Ratio of Auxin to Cytokinin

Seedlings of Petunia x hybrida ‘Orchid’ treated with the ethylene-releasing compound ethephon at 0.9, 1.7, and 3.5 mM evolved ethylene at a higher rate as the concentration of ethephon increased. Regardless of the concentration of ethephon applied, ethylene evolution peaked 6 to 8 h following application. Evidence that ethephon application decreased apical dominance included an increase in the number of new nodes on the main stem and a sustained increase in the length of new and existing lateral shoots compared to the control (no ethephon). Plants treated with 3.5 mM ethephon developed mild chlorosis, whereas a concentration of 1.7 mM ethephon decreased apical dominance without phytotoxic effects. The auxin/cytokinin ratio decreased in the apical shoot section as early as 1 h after ethephon treatment. In contrast, a decrease in the ratio in the subapical shoot section was not detected until 24 h after ethephon application. Reduction in auxin/cytokinin ratio was a result of a decrease in indole-3-acetic acid (IAA) and an increase of zeatin riboside (ZR), but not isopentenyladenosine (iPA). These results suggest that exposing ‘Orchid’ petunia seedlings to ethylene via ethephon lowers the auxin/cytokinin ratio, thereby promoting the outgrowth of lateral shoots.     

Detection of infectious Bovine polyomavirus

Bovine polyomavirus (BPyV) is a member of the Polyomaviridae, a virus that was originally thought to be of simian origin but was later shown to be of bovine origin, the primate cultures having been contaminated through the use of foetal bovine serum. The significance of this agent to the biotechnology industry cannot be underestimated. The presence of BPyV in serum batches poses a serious risk for the contamination of human therapeutic products. The current PCR based assays provide a means of detecting virus sequences but give no indication as to the infectious nature of the virus. The communication reports the successful development of an assay to detect infectious BPyV using an in vitro amplification system followed by PCR. A lengthy culture period on bovine cells was required before replicating BPyV could be detected and distinguished from non-replicating virus in the cell culture supernatant. A mock-test assay using foetal bovine serum positive for BPyV showed that there was no evidence of replicating BPyV in the serum sample. The BPyV spiked serum control showed that replicating virus was present thus confirming that the serum itself did not inhibit replication of the virus. Cells harvested during the culture period were subjected to fixation, embedding and sectioning and examined by electron microscopy. Intact virus-like particles of approximately 40-50nm were observed in the nucleus of the bovine kidney cells, the site of polyomavirus replication.     

The ‘greenhouse effect’ and UK agriculture

Book Review

The greenhouse effect and UK agricultureR.M. Bennett (Ed.), Reading: Centre for Agricultural Strategy, 1989. 144 pages, paperback. £15.00. ISBN 0-7049-0988-X     

Regulation of Purine Metabolism in Intact Leaves of Coffea arabica

The capacity of Coffea arabica leaves (5- x 5-mm pieces) to synthesize de novo and catabolize purine nucleotides to provide precursors for caffeine (1,3,7-trimethylxanthine) was investigated. Consistent with de novo synthesis, glycine, bicarbonate, and formate were incorporated into the purine ring of inosine 5[prime]-monophosphate (IMP) and adenine nucleotides ([sigma]Ade); azaserine, a known inhibitor of purine de novo synthesis, inhibited incorporation. Activity of the de novo pathway in C. arabica per g fresh weight of leaf tissue during a 3-h incubation period was 8 [plus or minus] 4 nmol of formate incorporated into IMP, 61 [plus or minus] 7 nmol into [sigma]Ade, and 150 nmol into caffeine (the latter during a 7-h incubation). Coffee leaves exhibited classical purine catabolism. Radiolabeled formate, inosine, adenosine, and adenine were incorporated into hypoxanthine and xanthine, which were catabolized to allantoin and urea. Urease activity was demonstrated. Per g fresh weight, coffee leaf squares incorporated 90 [plus or minus] 22 nmol of xanthine into caffeine in 7 h but degraded 102 [plus or minus] 1 nmol of xanthine to allantoin in 3 h. Feedback control of de novo purine biosynthesis was contrasted in C. arabica and Cucurbita pepo, a species that does not synthesize purine alkaloids. End-product inhibition was demonstrated to occur in both species but at different enzyme reactions.     

Separate de Novo and Salvage Purine Pools Are Involved in the Biosynthesis of Theobromine but Not Caffeine in Leaves of Coffea arabica L

We used a video digitizer system to (a) measure changes in the pattern of longitudinal surface extension in primary roots of maize (Zea mays L.) upon application and withdrawal of auxin and (b) compare these patterns during gravitropism in control roots and roots pretreated with auxin. Special attention was paid to the distal elongation zone (DEZ), arbitrarily defined as the region between the meristem and the point within the elongation zone at which the rate of elongation reaches 0.3 of the peak rate. For roots in aqueous solution, the basal limit of the DEZ is about 2.5 mm behind the tip of the root cap. Auxin suppressed elongation throughout the elongation zone, but, after 1 to 3 h, elongation resumed, primarily as a result of induction of rapid elongation in the DEZ. Withdrawal of auxin during the period of strong inhibition resulted in exceptionally rapid elongation attributable to the initiation of rapid elongation in the DEZ plus recovery in the main elongation zone. Gravistimulation of auxin-inhibited roots induced rapid elongation in the DEZ along the top of the root. This resulted in rapid gravitropism even though the elongation rate of the root was zero before gravistimulation. The results indicate that cells of the DEZ differ from cells in the bulk of the elongation zone with respect to auxin sensitivity and that DEZ cells play an important role in gravitropism.     

Alfalfa (Medicago sativa) carbamoylphosphate synthetase gene structure records the deep lineage of plants

Given the central role of carbamoylphosphate synthetases in pyrimidine and arginine metabolism in all living organisms, the absence of fundamental information regarding plant CPSase genes is a striking omission [Lawson et al., Mol. Biol. Evol. 13 (1996) 970-977; van den Hoff et al., J. Mol. Evol. 41 (1995) 813-832]. Whereas CPSase gene architecture and aa sequence have proven to be useful characters in establishing ancient and modern genetic affinities, phylogenetic analysis cannot be completed without the inclusion of plant CPSases. We describe the first isolation by molecular cloning of a plant CPSase gene (CPAII) derived from alfalfa (Medicago sativa). DNA sequence analysis reveals a proteobacterial architecture, namely closely linked carA and carB coding domains separated by a short intergenic region, and transcribed as a polycistronic mRNA. CPAII encodes the amino acid residues that typify a CPSase type II enzyme. In addition, an ancient internal duplication has been retained in the plant carB sequence. Partial nucleotide sequencing of additional clones reveals that the alfalfa genome contains multiple CPSase II gene copies which may be tissue-specific in their expression. It appears that with respect to CPSase genes, CPAII resembles the carAB gene of bacteria, and may have preserved much of this ancient gene structure in the alfalfa genome.