BCG vaccination early in life does not improve COVID-19 outcome of elderly populations, based on nationally reported data.

The reported numbers of Covid-19 cases and deaths were compared for 18 countries (14 in Western Europe, plus Australia, Brazil, Israel and the USA) to assess the effect of historic and current national BCG immunizations. In view of the high death rate for Covid-19 patients over 70 years of age, and given the fact that BCG vaccination is typically given early in life, we compared countries that had introduced BCG in the 1950s with those that had not. No effect on Covid-19 case fatality rate (CFR) or number of deaths per population could be demonstrated. Since some countries test for Covid-19 more than others, the effect of tests performed per million population on reported deaths per million was also assessed, but again did not demonstrate an effect of BCG vaccination in the 1950s. Whether countries had never used the vaccine, had historically used it but since ceased to do so, or were presently vaccinating with BCG did not correlate with national total number of deaths or CFR. We conclude that there is currently no evidence for a beneficial effect of BCG vaccination on Covid-19 reported cases or fatalities.

Renal pathology in hemizygous sickle cell mice.

Transgenic mice have been developed that express exclusively human sickle cell beta hemoglobin and have major pathological features found in humans with sickle cell disease. These mice provide a unique opportunity to investigate the fundamental mechanisms of this disease and to design new strategies to correct the associated genetic defect(s). We found that in breeding males expressing only adult human alpha-globin and sickle beta-globin (homozygous SS mice) with females containing these transgenes plus one copy of the mouse beta-globin gene (hemizygous SS mice) greater than expected numbers of hemizygous offspring were produced than homozygous mice (carrying no mouse beta-globin gene). These hemizygous mice, expressing the human alpha and sickle beta(s) transgenes in combination with mouse beta+/-, were used for our preliminary studies of their renal pathology. No kidney lesions were found in the control (129/Sv) mice, whereas about 50% of the hemizygous SS mice showed mild-to-severe kidney lesions, including glomerulonephritis, cystic atypical hyperplastic tubules, and general nephropathy. Kidneys of some hemizygous mice were normal or showed minimal nephropathy, yet those of the susceptible phenotype developed a mild-to-more-severe form of renal lesions. The tubular epithelium of kidneys of hemizygous mice of the more affected phenotype exhibited increased expression of inducible nitric oxide synthase with an increased 3-nitrotyrosine in close proximity. There was also a stronger immunostaining for vascular cell adhesion molecule-1 in the interstitial capillary cells as well as the tubular epithelial cells of the renal cortex, compared with normal control mice. The occurrence of a high incidence of renal abnormalities in our hemizygous SS mice suggests that these mice may provide a suitable model to study the pathogenesis of nephropathy resulting from altered blood flow and/or insufficient oxygen delivery.

Impaired FHIT expression characterizes serous ovarian carcinoma.

The FHIT (fragile histidine triad) gene on chromosome 3p14.2 is a candidate tumour suppressor gene. To define the role of the FHIT gene in the development of ovarian cancer, we have examined 33 ovarian carcinomas, 2 borderline tumours and 10 benign adenomas for the presence of FHIT gene alterations. FHIT transcripts were analysed by RT-PCR and sequencing. Aberrant FHIT transcripts were observed in 5/33 carcinomas (15%) and in 1 of 2 borderline tumours. Loss of normal FHIT transcript was observed in 5/33 carcinomas (15%) but not in 2 borderline tumours or 10 benign adenomas. Allelic losses at D3S1300 and D3S4103, both located within intron 5 of FHIT, were detected in 5/24 (21%) and 5/25 (20%) informative ovarian carcinomas, respectively. Expression of Fhit protein was analysed by immunohistochemistry in 44 carcinomas, 19 borderline tumours and 16 benign adenomas. Loss or significantly reduced expression of Fhit protein was observed in 6/44 (14%) ovarian carcinomas but not in any of 19 borderline tumours or 16 benign adenomas. The impaired Fhit protein expression was significantly correlated with the loss of normal FHIT transcription. Most notably, loss of normal FHIT transcript and impaired expression of Fhit protein occurred only in serous adenocarcinomas of grade 2 and 3 (5/15; 33% and 6/19; 32%, respectively). The present data suggest that inactivation of the FHIT gene by loss of expression is one of the important molecular events associated with the genesis of ovarian carcinoma, especially of high-grade serous carcinoma.

PCGEM1, a prostate-specific gene, is overexpressed in prostate cancer.

A prostate-specific gene, PCGEM1, was identified by differential display analysis of paired normal and prostate cancer tissues. Multiple tissue Northern blot analysis revealed that PCGEM1 was expressed exclusively in human prostate tissue. Analysis of PCGEM1 expression in matched normal and primary tumor specimens revealed tumor-associated overexpression in 84% of patients with prostate cancer by in situ hybridization assay and in 56% of patients by reverse transcription-PCR assay. Among various prostate cancer cell lines analyzed, PCGEM1 expression was detected only in the androgen receptor-positive cell line LNCaP. Extensive DNA sequence analysis of the PCGEM1 cDNA and genomic DNA revealed that PCGEM1 lacks protein-coding capacity and suggests that it may belong to an emerging class of noncoding RNAs, also called "riboregulators." The PCGEM1 locus was mapped to chromosome 2q32. Taken together, the remarkable prostate-tissue specificity and androgen-dependent expression of PCGEM1 as well as its elevated expression in a significant percentage of tumor tissues suggest specific functions of PCGEM1 in the biology and tumorigenesis of the prostate gland.

Possible roles of nitric oxide and redox cell signaling in metal-induced toxicity and carcinogenesis: a review.

Toxic doses of transition metals are capable of disturbing the natural oxidation/reduction balance in cells through various mechanisms stemming from their own complex redox reactions with endogenous oxidants and effects on cellular antioxidant systems. The resulting oxidative stress may damage redox-sensitive signaling molecules, such as NO, S-nitrosothiols, AP-1, NF-kappaB, IkappaB, p53, p21ras, and others, and thus derange the cell signaling and gene expression systems. This, in turn, may produce a variety of toxic effects, including carcinogenesis. Experimental support for the relevance of oxidative damage to the mechanisms of metal toxicity and carcinogenicity is particularly strong for two essential (but toxic when overdosed) metals--iron and copper-- and three well-established human metal carcinogens--nickel, chromium, and cadmium. However, along with more specific effects of toxic metals associated with their selective binding to particular cell constituents and affecting calcium signaling, oxidative damage seems to become important as well in explaining mechanisms of pathogenicity of other metals, such as lead, mercury, and arsenic.

Activation of neu by missense point mutation in the transmembrane domain in schwannomas induced in C3H/HeNCr mice by transplacental exposure to N-nitrosoethylurea.

Transplacentally initiated schwannomas in mice and rats arise preferentially in the Gasserian ganglion of the trigeminal nerve and spinal root ganglia, while those of the Syrian golden hamster most commonly occur subcutaneously. Rat and hamster schwannomas almost invariably contain a mutationally activated neu oncogene. In rat schwannomas, the mutant allele predominates, while the relative abundance of mutant alleles is very low in hamster nerve tumors. We investigated whether neu is mutated in mouse schwannomas and whether the pattern and allelic ratio of the mutation resemble those for the hamster or the rat. Pregnant C3H/HeNCr mice received 0.4 micromol N-nitrosoethylurea/g body weight on day 19 of gestation. Ten trigeminal and one peripheral nerve schwannomas developed in 11 of the 201 offspring. Missense T --> A transversion mutations were detected in the neu transmembrane domain in eight of ten schwannomas analyzed, as determined by MnlI digestion of polymerase chain reaction products. The mutant allele was predominantly detected in two tumors and was abundant in six others. Transfection of eight out of ten mouse tumor DNAs into hamster cells yielded transformed foci; seven out of eight contained mutant mouse neu. Mouse schwannomas closely resembled those of rats both in the preferred anatomical site and in the mutant/wild-type neu allele ratios.

neu mutation in schwannomas induced transplacentally in Syrian golden hamsters by N-nitrosoethylurea: high incidence but low allelic representation.

Peripheral nerve tumors (PNT) and melanomas induced transplacentally on day 14 of gestation in Syrian golden hamsters by N-nitrosoethylurea were analyzed for activated oncogenes by the NIH 3T3 transfection assay, and for mutations in the neu oncogene by direct sequencing, allele-specific oligonucleotide hybridization, MnlI restriction-fragment-length polymorphism, single-strand conformation polymorphism, and mismatch amplification mutation assays. All (67/67) of the PNT, but none of the melanomas, contained a somatic missense T --> A transversion within the neu oncogene transmembrane domain at a site corresponding to that which also occurs in rat schwannomas transplacentally induced by N-nitrosoethylurea. In only 2 of the 67 individual hamster PNT did the majority of tumor cells appear to carry the mutant neu allele, in contrast to comparable rat schwannomas in which it overwhelmingly predominates. The low fraction of hamster tumor cells carrying the mutation was stable through multiple transplantation passages. In the hamster, as in the rat, specific point-mutational activation of the neu oncogene thus constitutes the major pathway for induction of PNT by transplacental exposure to an alkylating agent, but the low allelic representation of mutant neu in hamster PNT suggests a significant difference in mechanism by which the mutant oncogene acts in this species.

Degradable dUMP outer primers in merged tandem (M/T)-nested PCR: low- and single-copy DNA target amplification.

PCR amplification of DNA from a single initiating genomic molecule or low-copy template often requires two sequential amplification reactions with nested primer pairs to achieve the necessary specificity and sensitivity. Residual outer primers can result in undesired primer activity during the inner nested cycles. To circumvent this problem, we have used dU-containing primers for first round amplification and then uracil N-glycosylase (UNG) to degrade them and the ends of their dU-primer-containing amplified DNA products. We have applied this method to the detection of an exon 11 mutation in the HEXA gene. We have merged the step of a single-tube PCR amplification with outer dU primers with a tandem amplification using non-dU-nested primers (hence, the term merged tandem-nested or M/T-nested PCR). Serial dilutions of genomic DNA showed that this method could amplify a specific target from as few as three haploid genome equivalents of template DNA. Specific products were obtained from the DNA of single cells in 19 of 20 replicates, using 12 outer and 28 inner nested PCR cycles, with an intervening UNG digestion step. When coupled with heteroduplex mutational analysis, this method reliably distinguished mutant versus wild-type HEXA gene fragments amplified from single cells without primer artifact.

Preparation of PCR products for DNA sequencing.

We demonstrate that routine PCR product analytical agarose gels can also serve as preparative gels for quick DNA template purification before sequencing. The band of interest is excised, placed into a Gel Nebulizer inside a Micropure separator and rapidly purified in a single centrifugation step. Gel-purified PCR product, suitable for manual and automated sequencing, is delivered within 10 min.

Implications of p53 mutation spectrum for cancer etiology in gastric cancers of various histologic types from a high-risk area of central Italy.

Examination of p53 mutation spectra may provide clues to molecular mechanisms involved in different histologic types of gastric cancer. A total of 105 gastric cancer cases classified according to the Laurén's system were selected from a high-risk area around Florence, Italy. Exons 5-8 of the p53 gene were examined for mutations by the polymerase chain reaction-single strand conformation polymorphism technique and DNA sequencing, using DNA from formalin-fixed paraffin-embedded tissues. Mutation frequency was similar in intestinal-type (12/28) and unclassified tumors (9/18), but was significantly lower in diffuse cancers (12/57, P < 0.05). A similar frequency of p53 mutations was observed among tumor stages in both intestinal-type and unclassified cancers, but in diffuse tumors mutations tended to be associated with invasion beyond the muscularis propria. When base changes were considered, G:C-->A:T transitions at CpG sites were the most common mutations for all the three tumor types with 6 of 11 (55%) in intestinal type, 8 of 12 (67%) in diffuse type, and 5 of 8 (63 %) in unclassified tumors. Frequent p53 mutations in both intestinal-type and unclassified tumors support the hypothesis that unclassified tumors represent variants of the intestinal type and suggest that unclassified tumors, like the intestinal type, may also associate with environmental exposures. The predominance of G:C-->A:T transitions at CpG sites, which are associated with methyltransferase-induced DNA methylation at carbon 5 of cytosine, in all three tumor types suggests that the status of DNA methylation may be the major determinant for p53 mutations and may be also equally important in gastric carcinogenesis regardless of histology.

Alteration of p16 and p15 genes in common epithelial ovarian tumors.

We have examined the roles of 2 putative tumor-suppressor genes, the p16 and p15 inhibitor-of-cyclin-dependent-kinase genes, in the most commonly occurring epithelial tumors of the human ovary. Expression of p16 mRNA, examined by RT-PCR, was significantly reduced in 15 of the 48 tumors. Aberrant expression of p16 protein, detected by immunohistochemistry, occurred in 22 of 60 tumors, more frequently in low-grade tumors, and had significant correlation with low p16 mRNA expression. Hypermethylation of a site within the 5'-CpG island of the p16 gene was significantly associated with loss of p16 mRNA and protein expression. Homozygous gene deletion, evaluated by differential PCR analysis, was found in 2 tumors for the p16 gene and in 1 tumor for the p15 gene among 70 ovarian tumors examined. PCR-SSCP analysis detected point mutations in p16 in 4 tumors and in p15 in 1 tumor. One was a 38-bp deletion, from codons 48 to 60, in a mucinous tumor of low malignant potential; another was a non-sense mutation in codon 60 in a mucinous adenocarcinoma. The remaining 2 mutations were mis-sense mutations, one in codon 58 and the other in codon 60, in 2 endometrioid adenocarcinomas. We conclude that inactivation of p16, by loss of p16 mRNA and protein expression as a consequence of hypermethylation of the 5'-CpG island, rather than by gene deletion or point mutation, may play an important role in the genesis of human ovarian epithelial tumors.

Lack of p53 and ras mutations in Helicobacter hepaticus-induced liver tumors in A/JCr mice.

Helicobacter hepaticus is a recently discovered bacterium that invades mouse liver causing chronic active hepatitis followed by development of preneoplastic hepatocellular foci, hepatocellular adenomas and carcinomas. This establishes a unique animal model for study of the mechanisms of cancer development due to a chronic bacterial infection. A possible mechanism of bacteria-associated tumorigenesis is mutation of oncogenes or tumor suppressor genes. Since mutations in ras oncogenes have been widely detected in a variety of chemically induced and spontaneous mouse liver tumors and specific mutations in the p53 tumor suppressor gene have been associated with human bladder cancers attributed to chronic schistosomal infection, we studied exons 1 and 2 of the N-, K- and H-ras genes and exons 5-8 of the p53 gene for the presence of point mutations in 25 liver tumors from 10 naturally infected A/JCr mice, ranging in age from 16 to 24 months. The 20 adenomas and five carcinomas varied in size from 0.1 to 2.3 cm and arose in livers characterized by a wide assortment of pathological profiles, including hepatitis, inflammation, hyperplasia, hypertrophy, leukocyte infiltration, necrosis and focal phenotypic alteration. DNA samples extracted from formalin-fixed paraffin-embedded tissues were screened by PCR/SSCP analysis and showed no mutations in the analyzed genes. Complete absence of mutations in ras genes in 25 mouse liver tumors is unusual. Other genes may be targeted or H. hepaticus infection causes liver cancer through other pathways than direct damage to DNA.

Transplacental mutagenicity of cisplatin: H-ras codon 12 and 13 mutations in skin tumors of SENCAR mice.

Cisplatin is an anticancer agent sometimes used in pregnant women. It is also a potent initiator of skin tumors in mice when administered transplacentally. For characterization of the transplacental mutagenicity of cisplatin, tumors initiated in fetal skin by cisplatin or 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by postnatal 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were analyzed for H-ras mutations by 'cold' single-strand conformation polymorphism analysis and direct sequencing. The expected high incidence of exon II codon 61 mutations (20/20) was found in transplacental DMBA-initiated tumors, with no exon I change. By contrast, 6/10 cisplatin tumors had seven mutations in codons 12 or 13 of exon I, all at GpG dinucleotides. Four of these were unique codon 13 GGC --> GTC changes, significantly different from the DMBA group and from historical TPA-only controls. The activation of codons 12 and 13 by cisplatin is in accord with the known in vitro preference of cisplatin for GpG sites for intrastrand cross-linking adduct formation. These results provide the first evidence that cisplatin can act transplacentally to cause specific mutations in fetal skin that are not seen in skin tumors caused by treatment of adult skin with this agent. This is evidence for unique molecular fetal carcinogenic pathways and underscores concern about human fetal risk due to maternal cisplatin treatment.

K-ras codon 12 and 61 point mutations in bromodeoxyuridine- and N-nitrosomethylurea-induced rat renal mesenchymal tumors.

The mutagenic thymidine analog bromodeoxyuridine (BrdUrd) may incorrectly incorporate opposite deoxyguanine in DNA, then pair with deoxyadenosine during subsequent replication. It appears to preferentially target the 3'-G of 5'-NGGN-3' sequences in mammalian cells in culture to induce G-->A transitions. Ras genes should therefore be vulnerable to activation by mutation at glycine codons 12 (GGT) and/or 13 (GGC) by misincorporation of BrdUrd. There is limited evidence that BrdUrd may be carcinogenic or co-carcinogenic in rats: three renal mesenchymal tumors, a tumor known to be associated with activating mutations in the c-K-ras-2 oncogene, were reported in 87 rats treated with BrdUrd alone, while N-nitrosomethylurea (NMU) alone or NMU + BrdUrd resulted in incidences of 12/52 and 26/76, respectively, against a zero incidence in untreated rats. We analyzed renal mesenchymal tumors from rats treated with BrdUrd for mutations in K-ras exons 1 and 2 and compared the prevalence and spectrum of mutations with those found in comparable tumors induced with NMU. DNAs from 22 paraffin-embedded renal mesenchymal tumors from rats treated 12-15 months earlier with BrdUrd (three specimens) or NMU (11 specimens) or both agents sequentially (eight specimens) were amplified by PCR. The base sequence of codons 12-13 and 59-63 of K-ras was determined by the dideoxynucleotide method. Sequencing results were confirmed by allele-specific oligonucleotide hybridization. Two of three tumors that appeared in rats given BrdUrd alone contained both a codon 12 GGT-->GAT transition and a codon 61 CAA-->CTA transversion. One tumor induced by NMU alone also showed a codon 12 GGT-->GAT mutation, while only wild type sequence could be demonstrated in the codon 12-13 region in the remaining ten such tumors. Three NMU-induced tumors also showed codon 61 CAA-->CTA mutations, while the remaining tumors had wild type sequence. While the GGT-->GAT transitions identified in tumors from BrdUrd-treated rats are consistent with BrdUrd mutagenesis by misincorporation, the co-occurrence of CAA-->CTA transversions, the overall low prevalence of mutations, and the lack of any difference in mutation spectrum between tumors induced by NMU and those that occurred in BrdUrd-treated rats suggests that in both groups the mutations that did occur did not result from a direct effect of either agent.

Studies of oncogene activation and tumor suppressor gene inactivation in normal and neoplastic rodent tissue.

Emerging short-term bioassays for chemically-induced carcinogenesis are dependent for their relevance to human risk assessment on the degree of coincidence of human and rodent tumor pathways. Since these pathways do not always converge, these new tests may have a number of unanticipated pitfalls. Models of liver and renal tumors are described. The results from Rb and p53 tumor suppressor gene transgenic animals are compared to human tumor syndromes. The question of mutagenic and epigenetic fingerprints of chemicals versus the cell-specific selection of spontaneous mutations is debated. Examples of specific pitfalls, such as the recently discovered Helicobacter hepaticus promoted liver tumors in mice are presented. The rat pseudogenes for p53 and the rare role of p53 in most important rodent tumor models other than epithelial tumors present experimental quandaries. The differential effects of carcinogens during various stages of rodent perinatal and adult development are also discussed. It is concluded that the pathways of both animal models and their human counterparts should be better identified so that realistic endpoint markers can be chosen for human carcinogenic risk assessment.

Induction of c-myc and c-jun proto-oncogene expression in rat L6 myoblasts by cadmium is inhibited by zinc preinduction of the metallothionein gene.

Certain proto-oncogenes transfer growth regulatory signals from the cell surface to the nucleus. These genes often show activation soon after cells are exposed to mitogenic stimulation but can also be activated as a nonmitogenic stress response. Cadmium (Cd) is a carcinogenic metal in humans and rodents and, though its mechanism of action is unknown, it could involve activation of such proto-oncogenes. Metallothionein (MT), a metal-inducible protein that binds Cd, can protect against many aspects of Cd toxicity, including genotoxicity and possibly carcinogenesis. Thus, the effects of Cd on expression of c-myc and c-jun in rat L6 myoblasts, and the effect of preactivation of the MT gene by Zn treatment on such oncogene expression, were studied. MT protein levels were determined by the Cd-heme assay, and MT, c-myc, and c-jun mRNA levels were measured using oligonucleotide hybridization and standardized to beta-actin levels. Cd (5 microM CdCl2, 0-30 h) stimulated both c-myc and c-jun mRNA expression. An initial peak of activation of c-myc expression occurred 2 h after initiation of Cd exposure, and levels remained elevated throughout the assessment period. Zn pretreatment markedly reduced the activation of c-myc expression by Cd compared to cells not receiving Zn pretreatment. Cd treatment increased c-jun mRNA levels by up to 3.5-fold. Again, Zn pretreatment markedly reduced Cd-induced activation of c-jun expression as minimal increases occurred with Cd exposures of < or = 1 h, but otherwise the Zn pretreatment prevented activation of c-jun. The Zn pretreatment elevated MT protein levels > 5-fold over control at the point of Cd exposure, but Cd exposure did not further elevate these Zn-induced MT levels. Similarly, Zn pretreatment did not result in increased relative MT mRNA levels above Cd exposure alone at various time points after Cd exposure. Therefore, Zn pretreatment, possibly by providing elevated MT protein levels at the point of Cd exposure, inhibited the Cd-induced c-myc and c-jun proto-oncogene expression. The extent of Cd-induced proto-oncogene activation thus may be limited by the presence of cellular MT.

Cloning and sequence of a processed p53 pseudogene from rat: a potential source of false 'mutations' in PCR fragments of tumor DNA.

We describe here the nucleotide (nt) sequence of a p53 processed pseudogene (psi-gene) from the normal F344 rat genome. Exon-derived primers were utilized to amplify and clone a 1447-bp polymerase chain reaction (PCR) product corresponding to the coding regions of exons 2-11 of the functional gene. This psi-gene is a cDNA-like sequence possessing 87% homology with the functional rat p53. We have also partially characterized two additional and distinctly different putative rat p53 psi-genes, focussing on the sequences surrounding the reported rat p53 mutational hot spots of codons 202R and 211R within exon 6/7. Each of these three psi-gene sequences contained various single- and/or double-nt substitutions, small deletions and insertions that distinguish them from p53. One substitution, 211R CGG-->CAG, found both in the cloned psi-gene and in one of the partially characterized, putative psi-genes, corresponded precisely with the sequence that has been reported as a mutation at one of the hot spots. Co-amplification of one or more of the p53 psi-genes with portions of the functional p53 is likely, if exon-based primers are utilized for PCR amplification of rat p53. Consequently, psi-gene sequences are potential sources of sequence variations that can be misidentified as somatic cell mutations by direct sequencing of inappropriately generated PCR products.

Microsatellite instability and alterations in the hMSH2 gene in human ovarian cancer.

The role of the replication error-positive (RER+) phenotype in the development of specific subtypes of sporadic ovarian carcinomas was examined by screening for the presence of microsatellite instability (MI) in 47 tumors. The overall frequency of ovarian MI was 17% only. However, MI occurred in 50% of the ovarian endometrioid-type tumors, which was significantly more often than in all the other histological subtypes combined (8%). Five of the 8 RER+ tumors exhibited most marked type I instability, possibly representing a different mechanism than for the remaining type 2 tumors. The cDNA of the mutation suppression gene hMSH2, the gene most often associated with MI, was screened for alterations in 8 MI-positive and 5 MI-negative ovarian tumors. Only 3 changes were found. Complete loss of hMSH2 mRNA expression was detected in I tumor, while another expressed only an abnormal transcript containing a deletion of exon 3. One additional RER+ serous adenocarcinoma contained a rare polymorphism with a non-conservative amino acid change. One of 8 RER+ tumors showed loss of heterozygosity at the hMSH2 loci. Genetic instability, caused in part by alterations in the hMSH2 gene, may play an important role in the sporadic endometrioid subtype of ovarian tumors. Other mutator-phenotype genes may be responsible for the remaining cases of RER+ ovarian tumors.

Thrombin receptor polymorphism in Chinese hamster.

We report the existence of a new Chinese hamster thrombin receptor allele, characterized by an in-frame insertion of three nucleotides at position 250 of the published sequence. As a consequence, an additional proline is inserted into a proline-rich region of the extracellular amino-terminal domain of the receptor. A corresponding proline at this position is also found in the rat thrombin receptor. A silent base-pair change is found in the cytoplasmic tail of the receptor gene. Single-strand conformation polymorphism and sequence analysis indicate this new receptor allele is present in several cell lines derived from different individual Chinese hamsters. Embryonic CHEF IIC9 cells and primary culture cells are homozygous for this new allele. In contrast, the CCL39 lung fibroblast cell line is heterozygous for both the new and old alleles. Both alleles are transcribed into mRNA and code for functional receptors. Given the allelic distribution and sequence alignment with thrombin receptors from other species, we propose that the new sequence represents the actual predominant allele in Chinese hamster.