An actin gene-related polymerase chain reaction (PCR) test for identification of chicken in meat mixtures.

Using the polymerase chain reaction (PCR) and DNA extracted from muscle, a single pair of oligonucleotide primers can yield amplification products from several members of the actin multigene family simultaneously. These multiple PCR products form species-specific "fingerprints" on gel electrophoresis which may be useful for meat authentication. However, for analysis of meat mixtures, the presence of a single band unique to a species would have many advantages over a multi-component fingerprint. A procedure is described in which primers amplify at a single actin gene locus, giving a positive band with DNA extracted from chicken and turkey, but no reaction with duck, pheasant, porcine, bovine, ovine or equine DNA. The chicken signal was clearly detectable with DNA from meat admixtures containing 1% chicken/99% lamb and from meat heat-treated at 120°C. For further discrimination, the chicken PCR product could be differentiated from turkey by restriction enzyme digestion.

The actin multigene family and livestock speciation using the polymerase chain reaction.

Actins constitute a family of highly-conserved multifunctional intracellular proteins, best known as myofibrillar components in striated muscle fibres. Most vertebrate genomes contain numerous actin genes with high sequence homology in protein coding regions but considerable variability in intron number and sizes. This genetic diversity can be utilised for livestock speciation purposes. The high sequence conservation has enabled a single pair of oligonucleotides to be used to prime the polymerase chain reaction (PCR) with DNA extracted from all animals so far studied. Multiple amplification products were obtained which on gel electrophoresis constituted characteristic species-specific 'fingerprints'. The patterns were reproducible, did not vary between individuals of the same breed or between different breeds within a species, and could be generated even from heat-processed muscle held at 120 degrees C for one hour. Given the capacity of PCR to amplify relatively short sequences in highly-degraded DNA, this approach may be suitable for authentication of processed meat products.

Detection and characterization of novel polymorphisms in the CYP2E1 gene.

To investigate whether interindividual variation in CYP2E1 levels can be explained by genetic polymorphism, we analysed DNA samples from 40 healthy individuals by single-strand conformational polymorphism analysis for polymorphisms in the CYP2E1 coding sequence and promoter region. DNA sequencing of samples showing mobility shifts on single-strand conformational polymorphism detected polymorphisms at positions -316 (A to G), -297 (T to A), -35 (G to T), 1107 (G to C; intron 1), 4804 (G to A Val179Ile; exon 4) and 10157 (C to T; exon 8). All individuals positive for either A(-316)G, G(-35)T, G(4804)A or the previously described RsaI polymorphism at -1019 were also positive for T(-297)A, which had the highest allele frequency of the observed polymorphisms (0.20). A(-316)G, G(-35)T and G(4804)A were detected at allele frequencies of 0.022, 0.052 and 0.013, respectively. The functional significance of the upstream polymorphisms was examined by preparing constructs of positions -549 to +3 of CYP2E1 containing the observed combinations of the polymorphisms fused to luciferase reporter genes and transfecting HepG2 cells. For the G(-35)T/T(-297)A construct, a 1.8-fold increase in luciferase activity compared with the wild-type sequence (P = 0.06) and 2.5-fold compared with T(-297)A only (P = 0.025) was observed. No significant difference in activity was observed between the other constructs. The significance of the predicted Val179Ile base change from G(4804)A was determined by expression of the wild-type and mutated full length cDNAs in lymphoblastoid cells. No significant difference in kinetic constants for chlorzoxazone hydroxylation between mutant and wild-type was observed. In summary, this study demonstrated six novel CYP2E1 polymorphisms, including three upstream of the promoter, but with the possible exception of G(-35)T, none appeared to be of functional significance.

Meat speciation by restriction fragment length polymorphism analysis using an α-actin cDNA probe.

Classical DNA fingerprinting is based on separation of DNA restriction fragments by electrophoresis and hybridisation to nucleic acid probes containing repetitive nucleotide sequences. The use of such mini- or micro-satellite probes tends to yield patterns specific to an individual rather than to a species, hence their value in forensic analysis but general unsuitability for meat speciation. In the present study, a cDNA probe based on conserved sequences contained in members of the actin multigene family has been evaluated for potential application in meat speciation. Genomic DNA was extracted from muscle and digested with BamHI before electrophoresis and hybridisation to a murine α-actin cDNA probe. Beef, pork, lamb, horse, chicken and fish DNA restriction fragments formed characteristic 'fingerprints' which were reproducible and varied sufficiently to allow discrimination even between closely-related species. However no major differences were seen between individuals of the same breed or between different breeds within a species. When DNA obtained from fresh tissue and also from meat heated at 120 °C was analysed, the gel patterns were essentially the same. An attractive feature of this approach is that it employs a single cross-reacting probe and set of conditions, and gives different patterns with all species so far studied. This simplicity suggests applications in meat speciation or related areas of biology.

Recent advances in understanding the molecular basis of polymorphisms in genes encoding cytochrome P450 enzymes.

The cytochrome P450 superfamily is known to exhibit a high degree of genetic polymorphism and polymorphisms associated with absent or low enzyme activity in CYP2D6, CYP2C19 and CYP2C9 are particularly well studied. However, despite early reports of strong disease associations for particular CYP2D6 phenotypes, these have not been confirmed in recent, more detailed studies and it now appears that analysis of CYP2D6, CYP2C19 and CYP2C9 genotype is of most value in predicting metabolism of specific drugs. Polymorphisms in other cytochrome P450 genes are less well studied and appear not to be associated with complete absence of enzyme activity. We have recently carried out studies of polymorphism in both CYP1A1 and CYP2E1. The molecular basis of the apparent CYP1A1 'high inducibility' polymorphism was investigated by studying CYP1A1 and Ah receptor polymorphisms in a group of phenotyped individuals who were genotyped both for known and novel CYP1A1 and Ah receptor polymorphisms. Three novel polymorphisms in CYP1A1 (C(-459)T, G(-469)A and C(4151)T) and one in the Ah receptor (G(1768)A; V(570)I) were detected by single strand conformational polymorphism analysis and DNA sequencing. Among both novel and previously known polymorphisms, only the Ah receptor G(1721)A polymorphism, which has an allele frequency of 0.12 in Caucasians and was detected previously in a Japanese population, was significantly associated with high induced CYP1A1 activity. In the case of CYP2E1, we have detected three polymorphisms in the promoter region (A(-316)G, T(-297)A and G(-35)T) and one in the coding sequence (G(4804)A; V(179)I) by screening Caucasian DNA samples. The significance of these alleles has been investigated but only G(-35)T combined with T(-297)A, which has an allele frequency of 0.05, appears to be of functional significance, with an apparent 1.8-fold increase in levels of transcriptional activity compared with the wild-type.

Characterization and PCR-based detection of two different hybrid CYP2D7P/CYP2D6 alleles associated with the poor metabolizer phenotype.

The majority of humans deficient in the cytochrome P450 CYP2D6 enzyme, so-called poor metabolizers (PMs), can now be identified by genotyping for several different PM-associated mutations. However, additional null alleles remain to be identified as demonstrated by subjects with the PM phenotype in the absence of a corresponding genotype. The rare 11 kb band on Xba I RFLP analysis, which is distinct from the 13 kb CYP2D6D (CYP2D6*5) allele, has been proposed to constitute such a unique non-functional allele. Here we demonstrate that the 11 kb band represents at least two different nine exon CYP2D7P/CYP2D6 hybrids generated by large deletions in the CYP2D gene cluster due to unequal cross-over or looping-out mechanisms. The total allele frequency was approximately 0.001-0.01 in European and North American Caucasians. The most common variant (CYP2D6*16) had breakpoints lying between the end of exon 7 and the start of exon 9 of the respective genes. The "CYP2D7-like' part of the gene was most homologous to the previously described CYP2D7AP and CYP2D7 (44/11.5) sequences. The other chimeric allele consisted of exon 1 of CYP2D7 and exons 2-9 from CYP2D6, and may be similar to a hybrid gene termed CYP2D6*13 recently described in a French individual. Two different routine PCR assays were developed for rapid and sensitive detection of these alleles, namely amplification of a 8 kb fragment from both CYP2D6*13 and CYP2D6*16, together with a CYP2D6*16-specific method which gave a 1.4 kb PCR product. The 8 kb assay for the CYP2D6*13 and CYP2D6*16 alleles also produced a 9.5 kb fragment in samples positive for the 13 kb CYP2D6*5 allele. Therefore, it is now possible to screen for the large CYP2D gene deletions by a single long PCR method.

Lung cancer risk in African-Americans in relation to a race-specific CYP1A1 polymorphism.

The possible association between lung cancer and a polymorphism of the CYP1A1 gene specific to African-Americans was examined using peripheral blood DNA from 144 incident cases of lung cancer and 230 population controls with detailed data on smoking and other risk factors for the disease. The CYP1A1 variant allele was present in 15.2% of controls and 16.7% of cases. The smoking-adjusted odds ratio for the presence of the variant allele in relation to lung cancer risk overall was 1.3 (95% confidence interval, 0.7-2.4). According to histological type, the strongest association was observed for squamous cell carcinoma (odds ratio, 2.1), but this result was compatible with chance (95% confidence interval, 0.8-5.9). Adenocarcinoma was not materially associated with the presence of the variant allele (odds ratio, 1.3; 95% confidence interval, 0.5-3.2). No important associations were observed upon stratification by several risk factors for lung cancer, including smoking history, occupational exposures to asbestos and motor vehicle exhaust, or low intake of the micronutrient antioxidants beta-carotene, vitamin E, or vitamin C. These results do not confirm an earlier report that this CYP1A1 polymorphism may be an important risk factor for adenocarcinoma of the lung in African-Americans.