Galactose-␣1,3-galactose (␣Gal) epitopes, the synthesis of which requires the enzyme product of ␣1,3-galactosyltransferase (␣1,3GT), are sugar chains on the cell surface of most mammalian species. Notable exceptions are higher primates including Old World monkeys, apes, and humans. The ␣Gal-negative species as well as mice with deletion of the ␣1,3GT gene produce abundant anti␣Gal antibodies. The evolutionary loss of ␣Gal epitopes has been attributed to point mutations in the coding region of the gene. Because no transcripts could be found in the higher primate species with Northern blot analysis, a potential alternative explanation has been loss of upstream regulation of the gene. Here, we have demonstrated that the rhesus promoter is functional. More importantly, a variety of full-length transcripts were detected with sensitive PCR-based methods in the tissues of rhesus monkeys, orangutans, and humans. Five crucial mutations were delineated in the coding region of the human and rhesus and three in the orangutan, any one of which could be responsible for inactivation of the ␣1,3GT gene. Two of the mutations were shared by all three higher primates. These findings, which elucidate the molecular basis for the evolutionary loss of ␣Gal expression, may have implications in medical research.Most mammals express the cell surface carbohydrate epitope galactose-␣1,3-galactose (␣Gal), 1 with notable exceptions that include Old World monkeys, apes, and humans (1). With the loss of the ␣Gal epitopes, the synthesis of which is dependent on enzyme product of the ␣1,3-galactosyltransferase (␣1,3GT) gene, higher primates produce anti-␣Gal antibodies (2) that are responsible for the hyperacute rejection of organs transplanted from ␣Gal-positive donors (3).In 1989, Joziasse et al. (4) reported the sequence of fulllength cDNA clone of the bovine ␣1,3GT gene and demonstrated the presence of this gene in the DNA of human cell lines. Using an 804-bp fragment derived from bovine fulllength cDNA as a probe, they also detected mRNA transcripts in bovine and marmoset (New World monkey) but not in human or African green monkey cell lines (4). The absence of ␣1,3GT mRNA has since been widely viewed as a feature of all Old World monkeys, apes, and humans (5, 6). The molecular basis for the inactivation of the ␣1,3GT gene in the ␣Gal-negative species has been attributed to a mutation(s) localized to a partial sequence of exon 9 (4, 5, 7).Stimulated by the current interest in producing transgenic pigs for clinical use as tissue and organ xenograft donors, the cDNA for the ␣1,3GT gene of the ␣Gal-positive pig was isolated (8), and the coding regions were characterized (9 -12). Further, the full genomic organization of the porcine ␣1,3GT, gene including its upstream regulatory region, was completed (12). A CpG island (i.e. a C connected by a 3Ј-5Ј phosphodiester bond to a G) characteristic of housekeeping genes was observed around exon 1 of the pig ␣1,3GT gene (12). It is of interest that a CpG island was not present in the mouse gene (13-15...

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A PCR-based assay for Bordetella pertussis was inhibited by using a calcium alginate fiber-tipped swab with an aluminum shaft but not by using a Dacron fiber-tipped swab with a plastic shaft. The calcium alginate fiber component inhibited the assay following storage for less than 1 min in a suspension of 103 CFU of B. pertussis per ml, whereas the aluminum shaft component required storage for at least 48 h in order to cause inhibition. We recommend the Dacron swab over the calcium alginate swab for collecting specimens for testing in PCR-based assays. Recent studies (5, 6, 9) suggest that PCR-based assays are more sensitive than culture for detection of Bordetella pertussis in nasopharyngeal specimens. Primers used for the amplification of target regions within the chromosome of B. pertussis and probes used for the detection of the amplified products (5, 6, 9, 14) have been developed from the DNA sequence information that is available for a repeated chromosomal element (5) and the pertussis toxin gene (10). The PCR-based assays have the potential for same-day turnaround time, whereas final isolation and identification with culture generally take 3 to 5 days. Since antibiotic susceptibility testing is not routinely performed with isolates of B. pertussis, the PCR may eliminate or reduce the need for culture. Nasopharyngeal swab and aspirate specimens are used with culture and PCR-based assays for detection of B. pertussis. For isolation of B. pertussis by culture, calcium alginate swabs are superior to Dacron, rayon, and cotton swabs (4, 7, 8). However, the effect of different swab types on PCR-based detection of B. pertussis is unknown. Suspensions of bordetellae were used to assess the effects of calcium alginate and Dacron swabs on a PCR-based assay for B. pertussis. Calcium alginateand Dacron fiber-tipped nasopharyngeal swabs and swab components were obtained from Medical Packaging Corp. (Camarillo, Calif.). The calcium alginate fiber was attached to an aluminum shaft, and the Dacron fiber was attached to a plastic (polystyrene) shaft. A recent clinical isolate of B. pertussis (strain BP5) was cultivated for 72 h on charcoal agar (Oxoid Unipath Ltd., Basingstoke, Hampshire, England) containing 10% defibrinated sheep blood. The bordetellae were harvested, washed three times by centrifugation at 2,000 x g, and suspended in sterile saline to provide suspensions with desired concentrations of the bordetellae. Template DNA was obtained from suspensions of bordetellae by digestion with lysozyme and proteinase K, and the chromosomal DNA was purified by extraction with phenol

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A multiplex PCR-based assay was developed for the detection of Bordetella pertussis in nasopharyngeal swab specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a B. pertussis repetitive element and a 438-bp target within the ␤-actin gene of human DNA (PCR amplification control). PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30 (6%) of the specimens inhibited the amplification of the ␤-actin target, in all 29 specimens studied, the inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest. Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the PCR-based assay, whereas 13.9% were positive by culture (P < 0.0001). After resolving the PCR-positive, culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based assay, the PCR-based assay had a sensitivity and specificity of 98.9 and 99.7%, respectively, compared with values of 73.4 and 100%, respectively, for culture. In comparison with patients with culture-confirmed pertussis, those with PCR-positive, culture-negative results were older and more likely to have had prolonged cough, immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.