Mutations activating human c-Ha-ras1 protooncogene (HRAS1) induced by chemical carcinogens and depurination.

In vitro modification of plasmids containing the human c-Ha-ras1 protooncogene (HRAS1) with the ultimate carcinogens N-acetoxy-2-acetylaminofluorene and r-7, t-8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (anti-BPDE) generated a transforming oncogene when the modified DNA was transfected into NIH 3T3 cells. The protooncogene was also activated by heating the plasmid at 70 degrees C, pH 4, to generate apurinic/apyrimidinic sites in the DNA. DNA isolated from transformed foci was analyzed by hybridization with 20-mer oligonucleotides designed to detect single point mutations within two regions of the gene commonly found to be mutated in tumor DNA. Of 23 transformants studied, 7 contained a mutation in the region of the 12th codon, whereas the remaining 16 were mutated in the 61st codon. Of the codon-61 mutants, 6 were mutated at the first base position (C X G), 5 at the second (A X T), and 5 at the third (G X C). The point mutations induced by anti-BPDE were predominantly G X C----T X A and A X T----T X A base substitutions, whereas four N-acetoxy-2-acetylaminofluorene-induced mutations were all G X C----T X A, and a single depurination-induced activation that was analyzed contained an A X T----T X A transversion. Together, these methods provide a useful means of determining point mutations produced by DNA-damaging agents in mammalian cells.     

Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons.

The importance of mutational activation of the Ha-ras protooncogene in polycyclic aromatic hydrocarbon-induced mouse skin tumors was investigated in a complete carcinogenesis model using repetitive applications of 7,12-dimethylbenz[a]anthracene (DMBA), or in an initiation-promotion model using a single application of dibenz[c,h]acridine (DB[c,h]ACR) or benzo[a]pyrene (B[a]BP) followed by chronic treatment with phorbol 12-myristate 13-acetate. DNA isolated from carcinomas induced by DMBA or DB[c,h]ACR, but not by B[a]P, efficiently transformed NIH 3T3 cells, and a high percentage of the transformed foci had an amplified Ha-ras gene. Restriction enzyme Southern blot analysis and DNA sequencing revealed that the amplified Ha-ras genes of the transformants had an A----T transversion in the second position of the 61st codon. The same mutation was also detected in primary tumor DNA in a high percentage of the DMBA- or DB[c,h]ACR-induced carcinomas. Identification of the mutation in NIH 3T3 cells transformed with DNA from DB[c,h]ACR-induced benign skin papillomas suggests that it is an early event in skin carcinogenesis. Thus, mutation of the 61st codon of the Ha-ras-1 gene appears to be a critical step in the formation of mouse skin tumors induced in both of the two models tested. Our analyses also delineate two other classes of hydrocarbon-induced carcinomas--namely, tumors whose DNAs efficiently transform 3T3 cells but do not contain mutated ras genes and tumors whose DNAs do not transform 3T3 cells.     

Identification and expression of human c-Ha-ras and c-sis sequences in NIH3T3 transformants

High-molecular-weight DNAs from 5 bladder carcinomas were used in transfection of mouse NIH3T3 cells. The manifestation of heterologous oncogene(s) expression in NIH3T3 cells was morphological transformation very often accompanied by changes in growth characteristics of recipient cells. In DNA samples from secondary NIH3T3 transformants human c-Ha-ras and c-sis sequences were identified. In some secondary transformants these sequences were expressed. On the basis of change of the growth characteristics of some secondary transformants we could expect the integration and expression of another human gene(s) for growth factor or growth factor receptor or even activation of mouse genes. We did not manage to identify any Alu sequences in some secondary transformants carrying human c-Ha-ras sequences. On the other hand, it has not been revealed yet that BamHI DNA fragments carrying c-Ha-ras gene contained any Alu sequence. So, the identification of Alu sequences does not have to be the first step in investigation of DNA samples from NIH3T3 transformants.     

Identification of an activated c-Ki-ras oncogene in rat liver tumors induced by aflatoxin B1.

Weanling male Fischer rats were administered 40 intraperitoneal injections of aflatoxin B1 (25 micrograms per animal per day) over a 2-month period. This chronic dosing regimen resulted in the sequential formation of hyperplastic foci, preneoplastic nodules, and hepatocellular carcinomas in all of the animals treated. The presence of transforming DNA sequences was detected by formation of anchorage-independent foci after transfection of tumor-derived DNA in NIH 3T3 mouse fibroblasts. Transfection of genomic DNA isolated from individual tumors from eight animals resulted in specific transforming activities ranging from 0.05 to 0.2 foci per micrograms of DNA. Primary transfectant DNAs were analyzed by Southern blot hybridization with DNA probes homologous to c-Ha-ras, c-Ki-ras, and N-ras oncogenes. A highly amplified c-Ki-ras oncogene of rat origin was detected in transformants derived from tumors in two of the eight animals tested. There was no evidence to suggest the presence of c-Ha-ras or N-ras sequences in any of the transformants. Analysis of primary liver tumor DNA showed no Ki-ras DNA amplification when compared to control liver DNA samples. Increased levels of c-Ki-ras p21 proteins were detected in 3T3 transformants containing activated rat c-Ki-ras genes. The presence of c-Ki-ras sequences of rat origin capable of inducing transformed foci can be taken as evidence that the c-Ki-ras gene has been activated in the primary liver tumors.     

Mutational activation of the c-Ha-ras gene in liver tumors of different rodent strains: correlation with susceptibility to hepatocarcinogenesis.

The frequency and pattern of mutations at codon 61 of the c-Ha-ras gene have been analyzed in 195 liver tumors and 132 precancerous liver lesions from various rodent strains with differing susceptibility to hepatocarcinogenesis. By using the polymerase chain reaction and allele-specific oligonucleotide hybridization, C----A transversions at the first base and A----T transversions or A----G transitions at the second base of c-Ha-ras codon 61 were detected in 20-60% of spontaneous or carcinogen-induced liver tumors of the C3H/He, CBA, CF1, and B6C3F1 mouse strains, which are highly susceptible to hepatocarcinogenesis. No such mutations, however, could be found in any of the 31 liver tumors of the insensitive C57BL/6J and BALB/c mouse strains or in any of the 35 liver tumors of the comparatively resistant Wistar rat. Further analyses of c-Ha-ras codon 12 mutations in liver tumors from the three insensitive rodent strains also failed to give any positive results. In early precancerous liver lesions, c-Ha-ras codon 61 mutations were found in 13-14% of lesions of the sensitive C3H/He and B6C3F1 mouse strains but not in any of the 34 lesions of the insensitive C57BL/6J mouse. Taken together, our results indicate a close correlation between the mutational activation of the c-Ha-ras gene in liver tumors of the different rodent strains and their susceptibility to hepatocarcinogenesis, whereby the mutations appear to provide a selective growth advantage, leading to a clonal expansion of the mutated liver cell population, only in livers of sensitive but not of insensitive strains.     

Hamster cell line suitable for transfection assay of transforming genes.

We established a subclone, SHOK, from the GHE-L cell line, an immortal line derived from a primary culture of Syrian hamster embryo cells, as a recipient cell line useful for the detection of oncogenes by transfection. SHOK cells were almost as susceptible as NIH 3T3 cells to focus formation by many oncogenes, including v-raf, v-Ha-ras, v-Ki-ras, or activated c-Ha-ras. The susceptibility of SHOK to focus formation was higher than that of NIH 3T3 for v-mos but was lower for v-fps, v-fgr, v-src, v-sis, and v-abl. When DNAs extracted from 27 human and murine tumors were tested for focus formation, 5 DNAs were positive in NIH 3T3 cells, whereas 9 were positive in SHOK cells at the primary transfection. Using SHOK cells as recipients of tumor cellular DNA, we isolated another oncogene and a c-Ki-ras2 gene mutated at codon 146 that were difficult to detect in NIH 3T3 cells. SHOK cells have a low rate of spontaneous transformation, produce easily distinguishable foci, and maintain a stable karyotype in transformed cells. In addition to being useful for the screening of human tumor DNAs, SHOK cells will be useful for the isolation of oncogenes from murine tumors because of their hamster origin.     

Transfection of BLV containing DNA into NIH 3T3 cells

Cell DNA isolated from bovine leukosis virus (BLV) productive cell clones was transfected into the NIH3T3 cells. DNA from some cell clones was able to transform NIH3T3 cells. The transformed cells were cloned, and in 4 cell clones out of 33 bovine leukosis virus specific sequences were detected by hybridization with labeled BLV probe. According to the restriction analysis the BLV sequences were incomplete, they were rearranged, deleted, or both. The DNA from NIH3T3 transformants with BLV sequences was able to transform in the second round transfection experiments NIH3T3 cells again, but in these transformants BLV specific sequences were not detected. Cell DNA from sheep tumors induced by BLV was able to transform the NIH3T3 cells too, but BLV specific sequences were not present in the transformants. It appears that BLV specific sequences are not required for NIH3T3 cell transformation.     

Sequential expression and cooperative interaction of c-Ha-ras and c-erbB genes in in vivo chemical carcinogenesis.

The level of expression of several cellular protooncogenes is examined at different stages of 7,12-dimethylbenzanthracene (DMBA)-induced tumor development in hamster buccal pouch epithelium (HBPE). Results presented demonstrate overexpression of c-Ha-ras gene at a very early stage of tumor development, and this elevated level of expression of the gene persists throughout the tumorigenesis process. The expression of the cellular protooncogene c-erbB, on the other hand, can be detected only after 8-10 weeks of DMBA treatment of the tissue and increases with the progression of the disease. The overexpression of c-erbB gene can be correlated with the stage of extensive proliferation and subsequent invasion of the HBPE cells into the underlying connective tissue. This sequential pattern of stage-specific expression of the two cellular protooncogenes can be observed in (i) treated tissues, (ii) stage-representative cultured cells, and (iii) NIH 3T3 transformants derived with DNA from HBPE cells. The low-level expression of c-myc and c-sis genes detected in control tissues remains unaffected, while c-fos gene activity cannot be detected at any stage of tumor development. The overexpression of c-Ha-ras gene alone in HBPE cells derived from tissues treated for 5 weeks (DM5) is not sufficient to induce tumors in athymic mice, whereas expression of c-Ha-ras and c-erbB genes at later stages of tumor development (DM10 and HCPC cells) induce histopathologically defined epithelial cell carcinoma in athymic mice within 2-3 weeks. The sequential overexpression of c-Ha-ras and c-erbB genes in a stage-specific manner and their cooperative interaction in the DMBA-induced in vivo oral carcinogenesis have been demonstrated.     

Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: sequence analysis of an enzymatically amplified mutant HRAS allele.

Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies (11 X 10(-4) consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo[a]pyrene (+/-)anti- 7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cell DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed i in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. Direct sequence analysis of the amplified DNA indicated equal presence of a wild-type (GGC) and mutant (GTC) allele of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC----TA codon 12 transversions that have been commonly observed in human tumors. This oncogene as well as yet to be identified oncogene are also shown to stably confer anchorage-independence to human cells.     

Characterization of c-Ki-ras oncogene alleles by direct sequencing of enzymatically amplified DNA from carcinogen-induced tumors.

Activated c-Ki-ras genes in liver tumors from rats exposed to the potent hepatocarcinogen aflatoxin B1 were analyzed to determine the nature of their activation by characterization of two c-Ki-ras alleles present in tumor-derived NIH 3T3 mouse transformants. Using selective hybridization of synthetic oligonucleotides to transformant DNA, we have determined that a single G X C to A X T base transition in either the first or second position of the 12th codon is associated with activation of the gene. Such mutations would lead to amino acid substitutions of aspartate or serine for glycine in the mutant proteins. To confirm these findings, we applied a technique for direct sequence analysis of a 90-base-pair region of the rat c-Ki-ras gene produced by primer-directed enzymatic amplification. Findings produced by this approach, which provides a convenient method to characterize mutations in multiple alleles without the necessity to clone individual genes, confirmed the presence and identity of the 12th codon mutations in the activated oncogene, as initially determined by the oligonucleotide hybridization technique.     

Activated neu oncogene sequences in primary tumors of the peripheral nervous system induced in rats by transplacental exposure to ethylnitrosourea.

Neurogenic tumors were selectively induced in high incidence in F344 rats by a single transplacental exposure to the direct-acting alkylating agent N-ethyl-N-nitrosourea (EtNU). We prepared DNA for transfection of NIH 3T3 cells from primary glial tumors of the brain and from schwannomas of the cranial and spinal nerves that developed in the transplacentally exposed offspring between 20 and 40 weeks after birth. DNA preparations from 6 of 13 schwannomas, but not from normal liver, kidney, or intestine of tumor-bearing rats, transformed NIH 3T3 cells. NIH 3T3 clones transformed by schwannoma DNA contained rat repetitive DNA sequences, and all isolates contained rat neu oncogene sequences. One schwannoma yielded a transformant with rat-specific sequences for both neu and N-ras. A point mutation in the transmembrane region of the putative protein product of neu was identified in all six transformants and in the primary tumors from which they were derived as well as in 5 of 6 schwannomas tested that did not transform NIH 3T3 cells. Of 59 gliomas, only one yielded transforming DNA, and an activated N-ras oncogene was identified. The normal cellular neu sequence for the transmembrane region, but not the mutated sequence, was identified in DNA from all 11 gliomas surveyed by oligonucleotide hybridization. Activation of the neu oncogene, originally identified [Schechter, A.L., Stern, D.F., Vaidyanathan, L., Decker, S.J., Drebin, J.A., Greene, M.I. & Weinberg, R.A. (1984) Nature (London) 312, 513-516] in cultured cell lines derived from EtNU-induced neurogenic tumors that by biochemical but not histologic criteria were thought to originate in the central nervous system in BD-IX rats, appears specifically associated with tumors of the peripheral nervous system in the F344 inbred strain.     

Isolation of transforming sequences of two human lung carcinomas: structural and functional analysis of the activated c-K-ras oncogenes.

Human lung tumors PR310 and PR371 maintained in nude mice contain activated c-K-ras oncogenes detectable by the ability of their DNAs to induce the morphological transformation of NIH 3T3 mouse fibroblasts. Using phage libraries constructed with DNA from NIH 3T3 mouse fibroblast transformants, we have isolated human sequences that span greater than 40 kilobase pairs of the c-K-ras oncogene. Based on the conservation of these human sequences in mouse fibroblast transformants, we conclude that the transforming ability of the oncogene activated in these tumors resides within a 43- to 46-kilobase-pair DNA region. No clear differences were observed between the structures of the PR310 and PR371 cloned oncogene sequences. Nucleotide sequence analysis in concert with DNA transfection experiments suggests that the PR371 oncogene has been activated by a single base change in the first exon, which results in the substitution of cysteine for glycine in position 12 of the predicted amino acid sequence. The genetic alteration responsible for the transforming activity of the PR310 oncogene, however, does not reside in the first exon. These results indicate that the activation of the c-K-ras oncogene in human lung cancer can occur by different mutational events.     

Mutations in c-Ki-ras oncogenes in diseased livers of winter flounder from Boston Harbor.

Livers of a natural population of winter flounder from a contaminated site in Boston Harbor were examined for the presence of oncogenes by transfection of DNA into NIH 3T3 mouse fibroblasts. Tissues analyzed contained histopathologic lesions including abnormal vacuolation, biliary proliferation, and, in many cases, hepatocellular and cholangiocellular carcinomas. Fibroblasts transfected with liver DNA samples from 7 of 13 diseased animals were effective in the induction of subcutaneous sarcomas in nude mice. Further analysis revealed the presence of flounder c-Ki-ras oncogenes in all 10 nude mouse subcutaneous tumors analyzed. Direct DNA sequencing and allele-specific oligonucleotide hybridization following amplification of the tumor DNA by the polymerase chain reaction showed mutations in the 12th codon in this gene. Analysis of DNA from all nude mouse tumors as well as the livers from which they were derived showed mutations at this codon. The mutations comprised G.C----A.T or G.C----T.A base changes resulting in substitution of serine, valine, or cysteine for glycine. Liver DNA samples from five histologically normal livers of animals from a less polluted site were ineffective in the transfection assay and showed only wild-type DNA sequences (GGT) at the 12th codon of c-Ki-ras. The prevalence of mutations in this gene region was associated with the presence of liver lesions and could signify DNA damage resulting from environmental chemical exposure.     

Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes.

In vitro DNA amplification followed by oligonucleotide dot blot analysis were used to study RAS gene mutations in acute myeloid leukemia (AML). Fifty-two presentation AML DNAs were screened for mutations in codons 12, 13, and 61 of NRAS and in codons 12 and 61 of KRAS and HRAS. Fourteen (27%) contained mutations--all in NRAS and predominantly in codon 12. The most common amino acid substitution identified was of glycine by aspartic acid at codon 12 (7/18), with a G----A transition being the most common base change (11/18). No particular correlation was observed between disease subtype and the incidence or type of NRAS mutation. In DNA samples from four patients, 2 NRAS mutations were found to coexist. NIH 3T3 focus-formation assays revealed that in each case the mutations were present in different NRAS alleles. We also report the absence of a mutated RAS gene in relapse DNAs of four patients in which a RAS oncogene had been detected at presentation. These observations suggest that RAS mutations arise as part of the evolution of neoplastic transformation.     

Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes.

These studies have compared the ability of NIH 3T3 cells containing different ras oncogenes to form tumor nodules in the lungs of nude mice after tail vein injection. The genes studied include the normal cellular and bladder tumor ras genes, recombinant viral/cellular ras genes, recombinant yeast/mammalian ras genes, and a constructed gene with yeast RAS1 sequences significantly modified by deletions and an oncogenic mutation. The results show that NIH 3T3 cells containing these genes readily form lethal tumor nodules in the lungs of nude mice after tail vein injection. No control NIH 3T3 cells formed lung tumors within 66 days. Although there were some quantitative differences in the potencies of the various lines, the striking conclusion is that NIH 3T3 cells transformed by either normal or activated mammalian ras genes form approximately equal numbers of experimental lung metastases. In addition, cells transformed by a significantly modified yeast RAS1 gene containing a purposefully introduced oncogenic mutation were also equally active in this assay. The amount of p21 (the 21-kDa protein encoded by ras), as measured by immunoprecipitation, was approximately the same in the parent lines before injection as in the tumors recovered after injection. This result indicates that there is no selection for metastatic sublines containing larger quantities of p21. Transfection of EJ bladder tumor ras DNA into NIH 3T3 cells followed by injection 3 days later into the tail veins of nude/beige mice indicated that the EJ ras gene can confer a metastatic phenotype within 3.5 cell generations without selection or clonal growth in vitro. Thus, the biochemical changes initiated after introduction of the c-Ha-ras gene into NIH 3T3 cells result in the almost immediate acquisition of phenotypes necessary for experimental metastasis.     

ras癌基因第12密码子点突变和胃癌患者预后关系的研究

对福尔马林固定，石蜡包埋的胃癌组织使用移聚酶链式反应－限制性片段长度多态性（简称ＰＣＲ－ＲＦＬＰ）技术同时进行ｃ－Ｈａ－ｒａｓ基因第１２位和６１位，Ｎ－ｒａｓ基因第１２位，ｋ－ｒａｓ第１２位和１３位密码子点突变的研究，发现３３．３％（１４／４２），的胃癌有ｃ－Ｈａ－ｒａｓ基因第１２位密码子的点突变；４．８％（２／４２）的病例有ｋ－ｒａｓ基因第１２位密码子的点突变，点突变的发生与患者的预后，淋巴结转     

Oncogene activation in human benign tumors of the skin (keratoacanthomas): is HRAS involved in differentiation as well as proliferation?

In vitro DNA amplification followed by oligonucleotide mismatch hybridization was used to study the frequency of HRAS mutations in the benign self-regressing skin tumors keratoacanthomas and in squamous cell carcinomas. We used freshly obtained keratoacanthomas as well as Formalin-fixed paraffin-embedded tissues from both types of tumors. DNA from 50 samples of each tumor type was analyzed for activating mutations involving codons 12 and 61. A relatively high percentage (30%) of HRAS mutations was found in the keratoacanthomas compared with 13% in the squamous cell carcinomas. The most frequent mutation identified is the A-T-to-T.A transversion in the second position of codon 61. The present findings demonstrate the involvement of the HRAS oncogene in human benign tumors. Moreover, they indicate that an activated HRAS oncogene is not sufficient to maintain a neoplastic phenotype and argue against a role of HRAS in the progression of skin tumorigenesis.     

Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites.

Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.     

Activation of ras oncogene in aflatoxin-induced rat liver carcinogenesis.

The presence of activated transforming genes was investigated in four primary aflatoxin-induced rat liver tumors in male Fischer rats, in two cell lines generated from such tumors, in an epithelial liver-derived nontransformed cell line, and in the latter cell line after transformation by aflatoxin B1 in vitro. When DNA extracted from these sources was transfected into NIH 3T3 cells, negative results were obtained from focus assays. Cotransfection of these DNA samples with a gene for resistance to G418, followed by selection for resistance to that antibiotic, and tumorigenicity testing in nude mice demonstrated DNA-mediated transfer of the neoplastic phenotype in all cases except for DNA from the nontransformed cell line. DNA extracted from these primary nude mouse tumors used in a secondary round of transfection with NIH 3T3 cells gave positive results in focus assays, which were conserved through succeeding rounds of transfection. By use of appropriate radiolabeled probes, activated ras oncogenes were detected in all samples. N-ras activation was detected in three of the primary rat liver tumors and both hepatoma cell lines. Ki-ras activation was detected in one primary rat liver tumor, and Ha-ras activation was detected in the cell line transformed in vitro with activated aflatoxin B1. The activated Ki-ras oncogene was further characterized by use of synthetic oligonucleotide probes and was shown to contain a G----A transition at the second nucleotide in codon 12.