Second Base Of Codon Research Articles

Lack of BCL10 Mutations in Germ Cell Tumors and B Cell Lymphomas

Lack of BCL10 Mutations in Germ Cell Tumors and B Cell Lymphomas

A new amyloidogenic transthyretin variant, [D38A], detected by electrospray ionization / mass spectrometry

A new variant of transthyretin (TTR) was detected by mass spectrometry (MS) in a 63-year-old Japanese female patient suffering from amyloidosis. TTR was analyzed by 2-dimensional liquid chromatography coupled with electrospray ionization MS. Variant TTR showed extra peaks in addition to normal TTR peaks. The extra peaks were about 44 Da smaller than normal TTR peaks, and the abundance of variant peaks showed about 80% of the corresponding normal free and adduct peaks. Direct genomic DNA sequencing of TTR exon 2 showed both adenine and cytosine in the position corresponding to the second base of codon 38. This codes for a variant alanine (GCT) as well as the normal aspartic acid (GA T), indicating that the case is heterozygous for the substitution, [D38A].

Point mutations of the c-H-ras gene in spontaneous liver tumors of transgenic mice carrying the human c-H-ras gene.

Spontaneous proliferative liver lesions were found in 15 (13 males and 2 females) of 244 (122 of each sex) transgenic (Tg) mice carrying the human prototype c-H-ras gene (rasH2). The liver lesions included 3 foci of cellular alteration, 1 hepatocellular adenoma, 5 hepatocellular carcinomas, and 4 hepatic hemangiosarcomas in the males and 1 focus of cellular alteration and 1 hepatocellular carcinoma in the females. The mutation patterns of the human and endogenous mouse c-H-ras codon 61 in these proliferative liver lesions were analyzed by DNA amplification using polymerase chain reaction, single-strand conformation polymorphism (PCR-SSCP), and oligonucleotide dot blot hybridization. The hepatocellular carcinomas in 4 males and 1 female contained a point mutation in the mouse c-H-ras gene: 3, 1, and 1 carcinomas had a CAA to AAA transversion at the first base of codon 61, a CAA to CTA transversions, and a CAA to CGA transition at the second base of codon 61, respectively. No point mutations in the human c-H-ras transgene were detected in any hepatocellular carcinoma. All 4 hepatic hemangiosarcomas had a CAG to CTG transversion at codon 61 of the human c-H-ras gene, but no point mutations were detected in codon 61 of the mouse c-H-ras gene. No mutations in human or mouse c-H-ras codon 61 were detected in altered cell foci or hepatocellular adenoma. These results indicate that spontaneous liver tumors in rasH2 Tg mice contain different mutation patterns depending on the histologic type or cell origin of the tumors (i.e., hepatocellular carcinomas or hepatic hemangiosacomas). The absence of similar mutations in foci of cellular alteration and the hepatocellular adenoma suggests that the occurrence of codon 61 point mutations is a late event in the progression of hepatocellular neoplasia in rasH2 Tg mice.

Ras and p53 genes are infrequently involved in N-nitroso- N-butylurea (NBU)-induced rat leukemia

Chemically-induced rodent tumor models help us to understand a series of genetic changes during carcinogenesis. In this study, we present N-nitroso- N-butylurea (NBU)-induced rat leukemia and compare it with the genetic alterations found in 7,12-dimethylbenz[ a]anthracene (DMBA)-induced erythroblastic leukemias which consistently have an A to T transversion at the second base of codon 61 in N- ras. By continuous NBU treatment for 120–150 days, 14 primary leukemias were induced in Long–Evans rats. Myeloblastic leukemia cells predominantly increased in all rats except in one case which predominantly had erythroblastic leukemia cells. Point mutations of Ha-, Ki-, N- ras and p53 were determined after RNA was transcribed into cDNA and this cDNA was used as a substrate for polymerase chain reaction (PCR) which was eventually sequenced. No abnormalities in exons 1 and 2 of Ha-, Ki- and N- ras were detected in all leukemias. In the p53 gene, an A to C transition was found at the second base of codon 198 (Asn-Thr) in one leukemia, but others had no mutation. These results suggest that ras and p53 genes are infrequently involved in NBU-induced leukemias. The genetic target of NBU during leukemogenesis seemed to be different from that of DMBA.

Fibrinogen Aa Chain Leu 554: an African-American kindred with late onset renal amyloidosis

An African-American kindred with renal amyloidosis is described. Four members in two generations developed nephropathy in the sixth to eighth decade of life. Kidney biopsy and subcutaneous fat aspirate biopsy of one patient revealed amyloid deposits. DNA analysis showed that patients were heterozygous for a mutation in the fibrinogen Aa chain gene with a guanine to thymine transversion at the second base of codon 554, predicting a leucinefor arginine substitution. Peptide sequence analysis 05 isolated plasma fibrinogen showed normal peptide as well as variant peptide with leucine replacing arginine at position 554, as predicted by the DNA sequence. The ratio between normal and variant peptides was approximately 1:1 in one patient and 3:2 in another. Although this African-American kindred has the exact same mutation as a previously described Peruvian-Mexican kindred, the onset age in this kindred is much later than in the Peruvian-Mexican kindred. This finding may indicate the existence of additional factor(s) beside the primary causative genetic mutation, which affect the expression of the disease.

Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity, and Ki-ras oncogene mutations in strain A/J mouse lung

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental polycyclic aromatic hydrocarbon, is the most potent carcinogen ever tested in mouse skin and rat mammary gland. In this study, DB[a,l]P was examined for DNA adduction, tumorigenicity, and induction of Ki-ras oncogene mutations in tumor DNA in strain A/J mouse lung. Groups of mice received a single i.p. injection of 0.3, 1.5, 3.0, or 6.0 mg/kg DB[a,l]P in tricaprylin. Following treatment, DNA adducts were measured at times between 1 and 28 days, while tumors were counted at 250 days and analyzed for the occurrence of point mutations in codons 12 and 61 of the Ki-ras oncogene. DB[a,l]P in strain A/J mouse lung induced six major and four minor DNA adducts. Maximal levels of adduction occurred between 5 and 10 days after injection followed by a gradual decrease. DB[a,l]P-DNA adducts in lung tissue were derived from both anti- and syn-11,12-dihydroxy-13,14-epoxy- 11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) and both deoxyadenosine (dAdo) and deoxyguanosine (dGuo) residues in DNA as revealed by cochromatography. The major adduct was identified as a product of the reaction of an anti-DB[a,l]PDE with dAdo in DNA. DB[a,l]P induced significant numbers of lung adenomas in a dose-dependent manner, with the highest dose (6.0 mg/kg) yielding 16.1 adenomas/mouse. In tricaprylin-treated control animals, there were 0.67 adenomas/mouse. Based on the administered dose, DB[a,l]P was more active than other environmental carcinogens including benzo[a]pyrene. As a function of time-integrated DNA adduct levels, DB[a,l]P induced lung adenomas with about the same potency as other PAHs, suggesting that the adducts formed by DB[a,l]P are similar in carcinogenic potency to other PAHs in the strain A/J mouse lung model. Analysis of the Ki-ras mutation spectrum in DB[a,l]P-induced lung tumors revealed the predominant mutations to be G-->T transversions in the first base of codon 12, A-->G transitions in the second base of codon 12, and A-->T transversions in the second or third base of codon 61, concordant with the DNA adduct profile.

Hot-spot mutations in the p53 gene of liver nodules induced in rats fed DL-ethionine with a methyl-deficient diet.

Male F-344 rats were fed for 15 weeks a methyl-deficient L-amino acid defined diet containing 0.05% DL-ethionine. Nodules protruding from the surface of the liver were dissected free of surrounding tissue, and polyadenylated RNA isolated from the nodules was reverse transcribed. The region of the p53 gene comprising codons 120-290 was amplified by the polymerase chain reaction, and cDNAs were sequenced. Mutations were detected in nodules obtained from 7 of 12 rats. In all seven cases, the same two point mutations were present. The first was at the first base of codon 246 and consisted of a C-->T transition (C:G-->T:A, Arg-->Cys), while the second was at the second base of codon 247 and consisted of a G-->T transversion (G:C-->T:A, Arg-->Leu). It is concluded that the hepatocarcinogen ethionine induces specific hot-spot p53 gene mutations; this is in contrast to the mutations at various sites previously observed to occur in rats fed a hepatocarcinogenic methyl-deficient diet alone. The results also provide the first evidence that ethionine is mutagenic in the rat.

Ki-ras mutations are an early event and correlate with tumor stage in transplacentally-induced murine lung tumors.

A previous study from this laboratory demonstrated that treatment of pregnant mice with 3-methylcholanthrene (MC) caused lung tumors in the offspring at 1 year after birth, the incidence of which correlated with fetal inducibility of Cyp1a1. Analysis by PCR amplification and allele-specific hybridization (ASO) of paraffin-embedded tumors generated from that study revealed the presence of point mutations in exon 1 of the Ki-ras gene. This work has now been expanded by PCR amplification and ASO analysis of 31 additional lesions. Point mutations were found in 37 of the 47 (79%) lesions analyzed in this and the previous study, the majority of which were G-->T transversions in the first or second base of codon 12. The mutational spectrum appeared to be dependent on the relative stage of differentiation of the lesion, as both the incidence of mutation and type of mutation produced correlated with malignant progression. Mutations occurred in 60% of the hyperplasias, 80% of the adenomas and 100% of the adenocarcinomas. In the lesions with mutations, GLY12-->CYS12 transversions occurred in 100% of the hyperplasias, 42% of the adenomas and 14% of the adenocarcinomas. The GLY12-->VAL12 transversions occurred in none of the hyperplasias, 42% of the adenomas and 57% of the adenocarcinomas. The remaining mutations, which consisted of ASP12 transitions and ARG13 transversions, occurred only in adenomas (17%) and adenocarcinomas (29%). Between this study and our previous analyses, the identity of the mutations obtained by ASO were confirmed by sequence analysis of eight of the 37 lesions that harbored mutations at the Ki-ras gene locus. There were no differences in the type or incidence of mutations relative to the metabolic phenotype or sex of the mice. These data suggest that mutational activation of the Ki-ras gene locus is an early event in transplacental lung tumorigenesis, and that the type of mutations produced by exposure to chemical carcinogens can influence the carcinogenic potential of the tumor. This may have prognostic significance in determining the malignant progression of the neoplasm.

Tissue-specific expression of the K- ras allele from the A/J parent in (A/J×TSG- p53) F 1 mice

Tissue-specific expression of parental K- ras allele(s) was investigated by single-strand conformation polymorphism analysis of the 3′ untranslated region of the K- ras gene in normal lung, spleen, liver and kidney from (A/J×TSG- p53) F 1 mice. The expression of A/J K- ras allele was equal to that of C57BL/6J allele in normal spleen, liver and kidney. However, transcripts from A/J K- ras allele were found to be 2–12-times greater than those from C57BL/6J allele in lung tissues harvested over a 20-week period. Similar to our previous observation with dimethylnitrosamine- and benzo[ a]pyrene-induced lung tumors, K- ras mRNA transcribed from A/J allele was 10–40-times more abundant than those from C57BL/6J allele in all of 40 (A/J×TSG- p53) F 1 mouse lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. In addition, K- ras mutations (G to A transitions at the second base of codon 12) were detected in 38 of 40 (95%) lung tumors and all of the mutations were found on the allele inherited from the A/J parent. These data demonstrate tissue-specific allele-specific transcription of the K- ras gene and provide further support to the thesis that K- ras allele itself is a primary mouse lung tumor susceptibility gene.

Significance of Cyclin D1 Overexpression and K-ras Point Mutations in Lung Tumors Induced by N-methyl-N-nitrosourethane in Hamsters.

Female Syrian golden hamsters were given a subcutaneous injection of 0.6 mg of N-methyl-N-nitrosourethane (MNUR) every 2 weeks for 8 weeks (a total of 5 doses) and then maintained without any treatment for the next 26 weeks. The MNUR-induced bronchiolo-alveolar cell tumors were all adenomas composed of basophilic or clear cells in a papillary growth pattern or papillary/solid tumors consisting of pleomorphic cells. Ultrastructurally, most of the tumor cells had the lamellar bodies characteristic of alveolar type II cells in their cytoplasm. Positive cyclin D1 immunostaining was associated with pleomorphic cell type adenomas, whereas no overexpression of p53 protein, hsp70 or mdm2 gene protein was detected in any of the tumors. The lung tumor samples were examined for the presence of mutations in the K-ras gene in codon 12, 13, and 61 by a non-isotopic method for selective oligonucleotide hybridization after PCR amplification of DNA from formalin fixed tissue embedded in paraffin. Lung tumors from 6 of 18 (33%) tumor bearers contained a point mutation in codon 13 or 61, 3 involving GGC→GAC transitions at the second base of codon 13 and 3 affecting the 61st codon, one CAA→CTA, and two CAA→CGA. No mutations were detected in codon 12. These findings indicate that, at least, a point mutation of the K-ras gene was related to the development of bronchiolo-alveolar cell adenomas induced by MNUR and may suggest that cyclin DI overexpression in pleomorphic cell type tumors plays a role in the process of malignant transformation.

The molecular basis of renal amyloidosis in Irish-American and Polish-Canadian kindreds.

Hereditary amyloidosis of an unusual form has been reported in two separate kindreds; one was Polish-Canadian and the other was Irish-American (Am J Med 1975; 59:121 and Trans Assoc Am Physicians 1981; 94:211). In both kindreds, affected members developed hypertension and nephrotic syndrome due to amyloidosis in their forties or fifties, but the genetic background responsible for the condition has been left undetermined. To identify the genetic defect in these kindreds, a portion of exon 5 of the fibrinogen alpha-chain gene in members of these kindreds was examined for a mutation by single-strand conformation polymorphism analysis and direct DNA sequencing. DNA analyses revealed an A-->T transversion at the second base of codon 526 of the fibrinogen alpha-chain gene in both of these kindreds. Analysis of DNA polymorphisms in the fibrinogen alpha-chain gene locus (TCTT repeat in intron 3, Rsal site in exon 5, and Taql site in the 3' flanking region of the gene) showed the haplotype B5-Rsal(+)-Taql(-) for the Val 526 mutant gene in both kindreds studied here, as well as in two kindreds previously described (J Clin Invest 1994; 93:731). The fibrinogen alpha-chain gene mutation (Val 526) is the genetic defect responsible for hereditary renal amyloidosis in these two kindreds, and the mutant genes in the Val 526 kindreds may have been derived from a single founder.

Specific N-ras mutation in bone marrow within 48 H of 7,12-dimethylbenz[a]anthracene treatment in Huggins-Sugiyama rat leukemogenesis

7,12-Dimethylbenz[a]anthracene (DMBA)-induced leukemias in Long-Evans rats consistently have an A --> T transversion at the second base of codon 61 in the N-ras gene. This mutation is also detected in the preleukemic stage. To determine when this specific N-ras mutation occurs in the early stages of leukemogenesis, we designed the mutant allele-specific amplification method, which was sensitive enough to detect one mutant cell among 10(6) normal cells. In the study reported here, N-ras mutation was found in bone-marrow cells 2 d after a single DMBA injection and thereafter throughout the preleukemic stage. These results show that DMBA induces a specific N-ras mutation soon after one DMBA injection and that this mutation is probably the first event in DMBA leukemogenesis.

Two novel missense mutations in the LDL receptor gene causing familial hypercholesterolemia.

We have employed analysis of single-strand conformation polymorphisms to identify mutations in the low density lipoprotein receptor gene causing familial hypercholesterolemia. Two familial hypercholesterolemia heterozygotes had abnormal single-strand conformation polymorphism patterns of exons 4 and 8. DNA sequencing revealed that the abnormal pattern of exon 4 was due to heterozygosity (G/T) at nucleotide 502. Nucleotide 502 is the first base of codon 147, and the G->T mutation (D147Y) changes this codon from AspGAC to TyrUAC. The abnormal pattern of exon 8 was due to heterozygosity (A/G) at nucleotide 1097. Nucleotide 1097 is the second base of codon 345, and the A->G mutation (Q345R) changes this codon from GlnCAG to ArgCGG. Based upon screening of 437 unrelated familial hypercholesterolemia heterozygotes, both D147Y and Q345R account for about 0.5% of the mutations causing familial hypercholesterolemia in Norway.

Effects of K-ras gene mutations in the development of lung lesions induced by 4-(N-methyl-n-nitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice.

The relationship between the development of peripheral lung lesions induced by tobacco‐specific 4‐(N‐methyl‐N‐nitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and K‐ras gene mutation in A/J mice, and the correlations between histological alterations and the course of lung lesion development after NNK treatment and K‐ras gene mutation were investigated. The acquisition of a selective growth advantage by the lung lesions with mutations was also examined using immunohistochemical labeling with bromodeoxyuridine. Thirty female 5 weeks old A/J mice were each injected intraperitoneally with a single dose of NNK (100 mg/kg body weight) and subdivided into 6 groups according to the time after NNK treatment. The lung lesions were characterized histologically as alveolar/bronchiolar hyperplasia, adenoma and adenocarcinoma, and point mutations in codons 12 and 61 of the K‐ras gene were detected by polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP) and dideoxy sequencing methods. K‐ras gene mutations were identified in 7 (58.3%) of 12 hyperplasias, 42 (75.0%) of 56 adenomas and 3 (75.0%) of 4 adenocarcinomas. The most frequent K‐ras gene mutation was a G‐to‐A transition at the second base of codon 12 and this accounted for 86.5% of all the mutations detected. Neither the frequency of activation of this gene nor the specific mutation was affected by the time after NNK treatment and there was no positive correlation between the proliferative activity of lung lesions and the presence of K‐ras gene mutations. Thus, K‐ras gene mutation is closely associated with the development of NNK‐induced peripheral lung lesions in A/J mice, but it plays no role in the selective growth advantage of these lesions.

Benzo[b]fluoranthene: tumorigenicity in strain A/J mouse lungs, DNA adducts and mutations in the Ki-ras oncogene.

The polycyclic aromatic hydrocarbon benzo[b]fluoranthene (B[b]F) is a pervasive constituent of environmental combustion products. We sought to examine the lung tumorigenic activity of B[b]F in strain A/J mice, to study the relationship between formation and decay of B[b]F-DNA adducts and to examine mutations in the Ki-ras proto-oncogene in DNA from B[b]F-induced tumors. Mice were given i.p. injections of 0, 10, 50, 100 or 200 mg/kg body wt and lung adenomas were scored after 8 months. B[b]F induced significant numbers of mouse lung adenomas in a dose-related fashion, with the highest dose (200 mg/kg) yielding 6.95 adenomas/ mouse, with 100% of the mice exhibiting an adenoma. In mice given tricaprylin, the vehicle control, there were 0.60 adenomas/mouse, with 55% of the mice exhibiting an adenoma. Based on dose, B[b]F was less active than benzo[a]pyrene. DNA adducts were analyzed qualitatively and quantitatively by 32P-post-labeling in lungs of strain A/J mice 1, 3, 5, 7, 14 and 21 days after i.p. injection. Maximal levels of adduction occurred 5 days after treatment with the 200 mg/kg dose group, producing 1230 amol B[b]F-DNA adducts/microgram DNA. The major B[b]F-DNA adduct was identified by co-chromatography as trans-9, 10-dihydroxy-anti-11, 12-epoxy-5-hydroxy-9, 10, 11, 12-tetra-hydro-B[b]F-deoxyguanosine. Approximately 86% of the tumors had a mutation in codon 12 of the Ki-ras oncogene, as determined by direct DNA sequencing of PCR-amplified exon 1 and single-stranded conformation polymorphism analysis. Analysis of the Ki-ras mutation spectrum in 25 of 29 B[b]F-induced tumors revealed the predominant mutation to be a G-->T transversion in the first or second base of codon 12, congruous with the DNA adduct data. Our data are consistent with previous reports in mouse skin implicating a phenolic diol epoxide as the proximate carcinogenic form of B[b]F that binds to guanine.

High yields of K-ras mutations in intraductal papillary mucinous tumors and invasive adenocarcinomas induced by N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine in the pancreas of female Syrian hamsters

Ductal adenocarcinoma, the most common form of pancreatic cancer in humans, is associated with activation of the K-ras oncogene in approximately 90% of cases. In contrast, K-ras mutations are found in < 50% of the relatively rare intraductal papillary mucinous tumor (IPMT), which arises in the main pancreatic ducts. Since both adenocarcinomas and IPMTs are believed to arise from ductal cells and progress through similar sequences of morphological changes (i.e. flat hyperplasia, papillary hyperplasia, atypia and carcinoma in situ), it is clear that such progression may not always necessitate activation of the ras oncogene. Experimentally ductal adenocarcinomas of the pancreas can be induced in the hamster model by a brief treatment with N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), while IPMTs can be induced by a combined treatment with HPOP and orotic acid (OA) in an initiation/promotion schedule. Since animals are exposed to the carcinogen only once, initiated normal epithelium is expected to give rise to a wide spectrum of neoplastic and preneoplastic lesions, progression of which will depend on the extent of mutagenesis induced at initiation in the targeted cells. In order to investigate the role of K-ras in progression of IPMTs as compared with adenocarcinomas we have examined the presence of K-ras mutations in the above two types of experimentally induced pancreatic cancers, as well as in associated and preneoplastic lesions. K-ras mutations at codons 12, 13 and 61 were determined by a designed restriction fragment length polymorphism method using mismatched nested primers in 77 neoplastic and preneoplastic foci microdissected from 20 pancreases. Mutations were found in all foci of atypical hyperplasia, in carcinomas in situ and invasive cancer, whether such lesions originated in lobular tissue or in the main pancreatic duct. Mutations were also found in papillary hyperplasia and flat hyperplasia in small ducts and also in the main duct at high frequency. With one exception, all ras mutations were G-->A transitions at the second base of codon 12. Mutations were occasionally accompanied by excessive presence of the mutant ras allele or loss of the wild-type ras allele, events that were more frequent in atypical hyperplasia (5/17), carcinomas in situ (5/14), IPMTs (2/5) and invasive adenocarcinomas (2/5) than in flat hyperplasia (0/6) or papillary hyperplasia (2/18). Our results demonstrate that: (i) K-ras mutations, predominantly G-->A transitions, are present in all experimentally induced hamster tumors; (ii) the incidence of K-ras mutations in IPMTs is lower in humans than in the hamster model; (iii) advanced lesions in both adenocarcinomas and IPMTs were frequently associated with an excess of the mutant over the wild-type K-ras allele. It is likely that both adenocarcinomas and IPMTs induced chemically in the hamster model arise by mechanisms which involve early activation of K-ras. Such a mechanism seems to be applicable only in a fraction of human IPMTs.

Role of Point Mutation of the K‐ras Gene in Tumorigenesis of B6C3F1 Mouse Lung Lesions Induced by Urethane

In order to elucidate the role of point mutation of the K‐ras gene in the tumorigenetic process of lung tumors, an experimental model of lung lesions in mice induced by the administration of urethane was used. A total of 135 B6C3F1 male mice, 6 weeks old, were given urethane in the drinking water at 0, 6, 60, 600 or 1200 ppm, and were then killed after varying periods of time. The lung lesions were histologically characterized as hyperplasia, adenoma and adenocarcinoma. Point mutations in codons 12 and 61 of the K‐ras gene were detected by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) and confirmed by using dideoxy sequencing analysis. K‐ras gene mutation was identified in 9 (23.7%) of 38 lesions classified as hyperplasia, 31 (46.3%) of 67 adenomas, and 3 (50%) of 6 adenocarcinomas. The most frequent mutation was an AT‐to‐TA transversion at the second base of codon 61 and this pattern accounted for 65% of the three mutant forms observed. These results suggest that the point mutation of K‐ras gene is involved in all stages of mouse lung tumorigenesis, i.e., activation of this gene can also influence the later stages of lung lesions.

Geographic sequence variation of latent membrane protein 1 gene of Epstein-Barr virus in Hodgkin's lymphomas.

To assess the role of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene in the development of Hodgkin's lymphoma (HL), the polymorphism of this gene in EBV isolates from different geographic locations was analyzed. A 497 bp fragment spanning LMP1 gene exons 1 and 2 was amplified by polymerase chain reaction (PCR), using a primer pair bracketing a Xhol restriction site. PCR products were subjected to Xhol digestion and to DNA sequencing analysis. Twenty-five HL biopsy specimens from the United States and five HL and four non-Hodgkin's lymphoma (NHL) biopsy specimens from Italy were examined. Eighty percent of LMP1-positive samples (12 of 15) from the United States maintained the Xhol restriction site and the remaining 20% partially lost the Xhol site. One of four EBV-positive HL and one of the three EBV-positive NHL specimens from Italy lost the restriction site. The other three EBV-positive HL DNAs were partially cut by Xhol. Direct DNA sequencing analysis revealed that those Italian samples not digested by Xhol were due to a G to C transversion at the first base of codon 18, resulting in the change of glycine to arginine. Those DNA samples partially cut by Xhol were due to a mixture of G/C at the same location. In contrast, those partially digested American HL DNAs had a mixture of G/T at the second base of codon 17.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of activating mutations in the human c-Ha-ras-1 proto-oncogene by oxygen free radicals.

The mutagenicity of oxygen free radicals was studied in a forward mutation system. pEC plasmid containing the human c-Ha-ras-1 proto-oncogene was reacted with oxygen free radicals generated by Cu2+ and H2O2 and was then transfected into NIH/3T3 cells. Transformed foci were observed with oxygen free radical-modified DNA but not with unmodified DNA. The mutations responsible for the Ha-ras-1 gene activation in 11 transformed foci were characterized. G-->T mutations at the second base of codon 12 were found in two transformed foci, A-->T transversions at the second base of codon 61 in five foci, and G-->T mutations at the third position of codon 61 in four transformed foci. These observed mutations are identical to those commonly found in human skin carcinomas, suggesting that reactive oxygen species may play an important role in the carcinogenesis of these tumors. Interestingly, a significant proportion of mutations was found at the second and third base of codon 61 (CAG). In a previous study, the same oxygen free radical-generating system was found to cause an intrastrand cross-link between adjacent purine nucleotides at AG sites in DNA (Carmichael et al., Carcinogenesis 13:1127-1135, 1992). These data demonstrate that oxygen radicals can induce DNA damage that can result in a specific activation of a human proto-oncogene.

Hemolytically inactive C4B complement allotype caused by a proline to leucine mutation in the C5-binding site.

The fourth component of complement (C4) is encoded by two highly homologous genes, C4A and C4B. Only one hemolytically inactive C4A allotype (C4A6) has been reported. No hemolytically inactive C4B allotype has been described. We report the first hemolytically inactive (hi) allotype of C4B, C4B*1 (hi). This unique variant was first recognized by hemolytic overlay assays and confirmed to segregate in the affected pedigree with the major histocompatibility complex haplotype A28,B35,CW4,DR6, C4A3,C4B1(hi), BFF,C2C. By single strand conformational polymorphism, we detected only a migration variant in exon 12 caused by a C to T transition in the second base of codon 459. This mutation results in a leucine substitution for proline (P459L) 1 residue downstream of a residue known to contribute to the C5-binding site. Allele-specific oligonucleotide analysis of samples demonstrated cosegregation of the mutation with the hemolytically inactive allotype in the affected pedigree. Site-directed mutagenesis and expression studies showed that the P459L mutation causes loss of hemolytic function. C4B*1(hi) is the first example of a circulating C4B protein lacking detectable hemolytic activity and the P459L mutation expands our knowledge of the C5-binding site of C4.