Multiallelic synthetic quality control material: lessons learned from the cystic fibrosis external quality assessment scheme.

AIM: With the arrival of increasingly complex molecular tests, we are obliged to create new ways to monitor and troubleshoot the underperformance of these multiplex assays. A synthetic multiallelic quality control material has been designed to augment genomic DNA controls. We aimed to evaluate the control on a large scale, testing it on a wide variety of oligonucleotide ligation assays, test protocols, and analysis software. In addition, we investigated how laboratories treat untried and complex materials. METHODS: The synthetic control monitored 32 cystic fibrosis transmembrane conductance regulator mutations and polymorphisms simultaneously. Participants of a cystic fibrosis external quality assessment scheme were invited to analyze the quality control. RESULTS: In total, 58 laboratories participated in this study. Twenty-seven (47%) laboratories detected 32 variants; another 27 laboratories (47%) detected from 31 to 4 variants and 4 participants reported no variants (6%). The main observations included administrative errors when indicating variants on a checklist, errors caused by misreading the instructions for use of the control or assay, and technical problems related to the assay used. CONCLUSION: Synthetic quality control materials proved to be valuable in troubleshooting underperforming assays and complement existing genomic controls. The study also revealed a strong need for increased quality control in the postanalytical phase of testing.

Functional characterization of human cytochrome P450 2S1 using a synthetic gene-expressed protein in Escherichia coli.

Human cytochrome P450 2S1 was recently identified and shown to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and hypoxia. It is highly expressed in epithelial cells of tissues that are exposed to the environment and in many tumors of epithelial origin. The biological function of CYP2S1 has not yet been determined, although its possible role in carcinogen metabolism has been suggested. In this report, we investigated its ability to metabolize carcinogens. To obtain a large quantity of active enzyme for substrate screening, we overexpressed CYP2S1 in Escherichia coli (200 nM culture), using a synthetic gene approach. High-level expression allowed us to achieve purification of CYP2S1 with high specific content and purity (16 nmol/mg). Despite high-level expression, we found that CYP2S1 was not readily reduced by cytochrome P450 reductase, and thus no activity was found using NADPH. However, the oxidative activity of CYP2S1 was supported by cumene hydroperoxide or H(2)O(2), such that CYP2S1 oxidized many important environmental carcinogens, including benzo[a]pyrene, 9,10-dihydro-benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene-7,8-dihydrodiol, aflatoxin B1, naphthalene, and styrene, with high turnover. Most substrates tested were converted to detoxification products, except in the case of benzo[a]pyrene-7,8-dihydrodiol, which was converted into the very potent carcinogenic metabolite 7,8-dihydrodiol-trans-9,10-epoxide at a relatively efficient rate (K(m) = 12.4 +/- 2 microM, turnover = 2.3 min(-1)). This metabolite formation was also supported both in vitro and in vivo by fatty acid hydroperoxides described in the accompanying report (p. 1044). Together, these data indicate that CYP2S1 contributes to the metabolism of environmental carcinogens via an NADPH independent activity.

Peripheral and mesencephalic transfer of a synthetic gene for the thymic peptide thymulin.

Thymulin is a thymic peptide with antiinflammatory activity in the brain. We constructed a recombinant adenoviral vector, RAd-FTS, expressing a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin and used it for peripheral and central gene transfer in rats. Thymulin concentration in serum and brain tissue was determined by bioassay. Reporter gene expression in the substantia nigra (SN) was quantitated by enzymohistochemistry or fluorescence microscopy using an appropriate image analysis software. A single intramuscular injection (10(8) plaque forming units (pfu)/animal) of RAd-FTS in thymectomized rats (nondetectable serum thymulin) induced supraphysiologic serum thymulin levels for at least 110 days (123+/-22 fg/ml versus 598+/-144 fg/ml in intact and vector-injected rats, respectively). Stereotaxic intranigral injection of RAd-FTS induced steady expression levels of met-FTS for at least 90 days, whereas expression of adenovirally transferred reporter genes coding for green fluorescent protein fused to HSV thymidine kinase (GFP-TK)(fus) or E.coli beta-galactosidase (beta-gal), declined drastically within a month (% transgene expression in the SN on post-injection day 30 relative to day 2 was: 18, <1 and 125%, for beta-gal, (GFP-TK)(fus) and met-FTS, respectively). We conclude that RAd-FTS constitutes a suitable biotechnological tool for the assessment of peripheral and central thymulin gene therapy in animal models of nigral dopaminergic neurodegeneration induced by pro-inflammatory agents.

Secretion of human epidermal growth factor from Saccharomyces cerevisiae using synthetic leader sequences.

We have investigated different leader sequences for their ability to direct the efficient secretion of human epidermal growth factor (hEGF) from Saccharomyces cerevisiae. We designed a consensus signal sequence which directs secretion of hEGF from yeast as efficiently as the yeast invertase signal sequence. However, secretion is increased over fivefold by the introduction, after the signal sequence, of a synthetic 19-amino acid (aa) pro-sequence containing a cleavage recognition site for the KEX2 protease. Even in the absence of an Asn-linked glycosylation site, secretion of hEGF using the synthetic prepro-leader was as efficient as that directed by the alpha-factor leader. The role of the KEX2 protease cleavage site was investigated by mutation of the yeast alpha-factor KEX2 site (cleavage after Lys-Arg). Cleavage was obtained with the following order of efficiency, Lys-Arg greater than Pro-Arg greater than Asp-Arg, although the sequence context was also found to affect efficiency.