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At least 19 recordsLinked to original sources

PubMed2001

Structure, expression and activation of fish ras genes.

Ras genes encode proteins that play a central role in cell growth signaling cascades. The fish ras genes characterized to date, have a high degree of nucleotide sequence and deduced amino acid similarity with the mammalian ras gene counterparts. A large proportion and wide variety of mammalian tumors possess mutant forms of ras. In such cases, the localization of ras mutations has been restricted to exons I and II, and to codons 12, 13 and 61. Experimental exposure of fish to a range of genotoxic compounds has similarly led to the production of a ras mutational profile for selected species. The inducing compound, tissue investigated and the fish species studied affect the ras mutational spectrum and incidence observed, despite the apparent conserved sequence homology. Furthermore, the fish ras mutational profile differs from that observed in rodent models, including a novel codon (16) mutation. The role of ras genes in tumor formation in feral fish has been investigated using several species collected from areas of high hydrocarbon contamination. Tomcod (Microgadus tomcod), winter flounder (Pseudopleuronectes americanus) and dragonet (Callionymus lyra) liver samples display evidence of ras gene mutations, though for the latter species the codon affected is not characteristic of ras gene mutational profiles. English sole (Pleuronectes vetulus) and European flounder (Platichthys flesus) liver tumor samples so far examined, on the other hand, do not display ras gene mutations. Thus, the pattern and incidence of ras gene mutations in environmentally-induced tumors also appear to be species specific. In determining the basis of both the species susceptibility observed in the field and species differences in effects of laboratory controlled exposures, the interaction of fish ras genes with other components of the cell growth signaling cascade (such as protein kinase C, additional oncogenes and tumor suppressor genes) are discussed. The effect of promoting agents following contaminant-induced initiation could similarly provide answers in unraveling the question of species susceptibility.

Amino Acid Sequence↗

PubMed1991

Multiplex polymerase chain reaction amplification and direct sequencing of homologous sequences: point mutation analysis of the ras genes.

ras proto-oncogenes are activated by point mutation in a wide variety of human and animal tumors, making ras gene analysis a major area of clinical and basic cancer research. Activating point mutations, in each of the three ras genes (Ha-, Ki-, or N-ras), usually occur in one of three specific codons (12, 13, or 61). Thus, an adequate assessment of activating ras gene mutations should include the analysis of at least nine codons. We have developed a rapid method for point mutation analysis of the ras genes, which involves simultaneous (multiplex) PCR amplification of all three homologous ras genes (in the regions surrounding codons 12-13 and codon 61) in a single reaction starting with only 1 microgram of genomic DNA. Although multiplex PCR has been previously used for unrelated sequences, we demonstrate here that multiplex PCR can also be used for highly homologous sequences. Importantly, after coamplification, each of the homologous ras genes can be individually and specifically sequenced even though the other two closely related genes are present in the same template mixture, by using high-stringency conditions permitted by Taq DNA polymerase. An automated multicycle DNA sequencing procedure is used to allow the double-stranded PCR products to be sequenced directly without the need to generate single-stranded templates, further simplifying the protocol. Our multiplex PCR amplification and direct DNA sequencing procedures should greatly facilitate more complete analyses of activating ras gene point mutations, particularly in studies involving many tumor samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

What's new in ras genes? Physiological role of ras genes in signal transduction and significance of ras gene activation in tumorigenesis.

Ras gene mutations have been found with variable prevalence in different tumor types. While during the past decade a lot of information has been accumulated on the frequency of ras oncogene activation in tumors, the last two years brought considerable progress in elucidating molecular mechanisms of signal transduction for which cellular ras proteins are key elements. They transmit signals from upstream tyrosine kinases to downstream serine/threonine kinases ultimately leading to changes of gene expression cytoskeletal architecture, cell-to-cell interactions and metabolism. These signalling pathways are of interest for the physiological regulation of proliferation and differentiation in normal, as well as in cancer tissue. Mutational activation of cellular ras genes to transforming oncogenes is thought to promote cell growth even in the absence of extracellular stimuli, and may thereby contribute to the initiation and/or progression of tumors.

Animals↗

PubMed1986

Extensive sequence homology of the goldfish ras gene to mammalian ras genes.

We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1999

H-ras and K-ras gene mutations in primary human soft tissue sarcoma: concomitant mutations of the ras genes.

ras gene mutations have been described with varying frequency in several types of human malignancies. To determine the incidence and type of ras mutations in human soft tissue tumors, we studied 45 sarcomas, including 27 malignant fibrous histiocytomas (MFHs), 10 liposarcomas, 2 rhabdomyosarcomas, and 6 leiomyosarcomas. Al of the tumors were investigated by direct sequence analysis with the automated DNA sequencing of polymerase chain reaction-amplified ras sequences. Twenty (44%) of the sarcomas examined harbored K-ras mutations, 18 (90%) of which were MFHs. All of the K-ras mutations were G-to-A transition mutations in the second position of codon 13 (glycine --> aspartic acid). Of the samples with K-ras activation, 7 (16% of the total of 45 tumors), including 6 MFHs and 1 leiomyosarcoma, also contained H-ras mutation. All of the tumors that showed H-ras alteration had G-to-T transversion mutations in the second base of codon 12 (glycine --> valine). These data possibly implicate that ras gene activation may be a relatively uncommon event in soft tissue tumors, with the exception of MFH. It is suggested that the oncogenic process underlying the development of tumors between these groups may be different and that ras gene mutations may play a role in the etiology and/or progression of MFH. It is noteworthy that when ras gene activation occurs in sarcoma, it predominantly affects the K-ras gene, particularly codon 13. Moreover, H-ras mutations in our samples were detected only in association with tumors that also displayed K-ras-mutated genes. This study demonstrates for the first time concomitant mutations in two different members of the ras gene family in sarcoma

Adolescent↗

PubMed1989

In vitro growth properties and PCR ras gene analysis of a murine embryonal carcinoma cell line harboring an amplified c-Ki-ras gene.

Ras proto-oncogenes are thought to be involved in both proliferation and malignant processes. We examined some growth properties of a murine embryonal carcinoma cell line (ECC), PCC4, that have been shown previously to be amplified for the c-KI-ras gene. Our results show that doubling time and plating efficiency are not significantly affected by such amplification. To examine the possible link between malignant behavior and c-Ki-ras alteration, we investigated activating mutations in this PCC4 cell line as well as in other ECC. Analysis of the in vitro amplified Ki-ras gene by PCR technology has not revealed point mutations in any of the ECC examined.

Animals↗

PubMed1984

Saccharomyces cerevisiae synthesizes proteins related to the p21 gene product of ras genes found in mammals.

A family of normal vertebrate genes and oncogenes has been called the ras gene family. The name ras was assigned to this gene family based on the species of origin of the viral oncogenes of the rat-derived Harvey and Kirsten murine sarcoma viruses. There are now three known functional members of the ras gene family, and genes homologous to ras genes have been detected in the DNA of a wide variety of mammals and in Drosophila melanogaster. Prior experiments have detected proteins coded for by ras genes in a large number of normal cells, cell lines, and tumors. We report here the detection of ras-related proteins in D. melanogaster, a result predicted by the earlier detection of ras-related genes in the Drosophila genome. We also report for the first time the detection of ras-related proteins in a single-cell eucaryocyte, Saccharomyces cerevisiae. These proteins, approximately 30K in size, are recognized by both a monoclonal antibody which binds to the p21 coded for by mammalian ras genes and a polyclonal rat serum made by transplanting a v-Ha-ras-induced tumor in Osborne-Mendel rats. The p21 of v-Ha-ras and the 30K proteins from S. cerevisiae share methionine-labeled peptides as detected by two-dimensional tryptic peptide maps. The results indicate that S. cerevisiae synthesizes ras-related proteins. A genetic analysis of the function of these proteins for yeast cells may now be possible.

Animals↗

PubMed1989

Detection of ras gene alterations and ras proteins in colorectal cancer.

DNA extracted from 31 primary colorectal carcinomas was analyzed for the presence of ras gene amplification and mutations. Nine carcinomas had Ha-ras amplification and seven Ki-ras amplification. Nine carcinomas had codon 12 Ki-ras mutations. Immunohistochemical staining for ras proteins revealed a normal membrane association in normal mucosa and benign polyps but an abnormal cytoplasmic distribution in carcinomas. Amplification, mutations, and immunohistochemical staining were independent of histologic differentiation, Dukes' stage, or DNA ploidy status. This study demonstrates that abnormalities of ras genes are a common finding in colorectal carcinomas. They are potentially important biologic changes associated with malignancy, although they do not appear to be related to clinical behavior.

Adult↗

PubMed1995

Analysis of mutations of neurofibromatosis type 1 gene and N-ras gene in acute myelogenous leukemia.

Neurofibromatosis type 1 (NF1) gene is a tumor suppressor gene, and the NF1 gene product, neurofibromin, can downregulate the N-ras gene. Because the N-ras gene is often mutated in acute myelogenous leukemia (AML), we wondered if the NF1 gene might be mutated in those AML samples not having N-ras mutations. We investigated the mutational status of the N-ras gene and the FLR exon of codons 1371-1423 of the open reading frame of the full-length NF1 cDNA, which has a strong homology with the mammalian ras GTPase-activating protein (GAP), especially for a stretch of three consecutive amino acids (F, L, R), by single-strand conformation polymorphism analysis and direct sequencing in samples from patients with AML. Of 48 AML patients, 10 (21%) had point (missense) mutations of the N-ras gene involving codons 12, 13 and 61. However, mutations in the FLR exon of the NF1 gene were not detected in any of the AML samples. We also examined the difference of clinical response to induction therapy between AML patients with and without N-ras mutation. A significantly lower rate of complete remission was noted in individuals with N-ras gene mutations. These results suggest that mutation of the NF1 gene, at least in the FLR exon, is very rare in AML and the NF1 gene probably is not a functional complement of the N-ras gene mutation. The presence of N-ras gene mutation may be associated with a lower clinical response to antileukemic therapy.

Adult↗

PubMedDate not supplied

Immortalization by truncated myc or ras genes and synergism between myc and ras genes in cell transformation.

Human c-myc and normal or mutant T24 H-ras1 genes either linked to the regulatory sequences of the human metallothionein IIa gene or unlinked have been employed individually or in combination to transfect early passage Chinese hamster lung cells. Since the c-myc and H-ras1 genes are present in recombinant plasmids carrying the aminoglycoside phosphotransferase (aph), gene transfectants were selected in the presence of geneticin. A synergistic effect has been observed between myc and H-ras1 genes in measurement of both the number of geneticin resistant colonies obtained and the parameters of morphological transformation and anchorage independence. Out study also demonstrates the ability of truncated myc and ras genes to induce immortalization of hamster cells.

Animals↗

PubMed1998

Alterations of p53 gene and Ha-ras gene are independent events in solar keratosis and squamous cell carcinoma.

In order to clarity the multiple-step progression from solar keratosis to squamous cell carcinoma, aberrations of the p53 gene (exons 2-11) and ras genes (exons 1 and 2) in solar keratosis and squamous cell carcinoma were investigated by polymerase chain reaction and single-strand conformation polymorphism analysis. In a series of Japanese patients, eight of 27 (30%) samples of solar keratosis and three of six (50%) samples of squamous cell carcinoma showed structural abnormalities in the p53 gene. Only one solar keratosis (4%) showed a point mutation in the Ha-ras gene but not in the p53 gene. Among these cases, no mutation of ras genes could be detected in squamous cell carcinoma. Simultaneous mutation of ras genes and the p53 gene was not detected in any cases of either solar keratosis or squamous cell carcinoma. It is concluded that aberrations of the p53 gene and ras genes are induced through independent processes of ultraviolet irradiation in the course of carcinogenic change from solar keratosis to squamous cell carcinoma.

Carcinoma, Squamous Cell↗

PubMed1989

Spontaneous Ha-ras gene activation in cultured primary murine keratinocytes: consequences of Ha-ras gene activation in malignant conversion and malignant progression.

The activation of the c-Ha-ras gene and its contribution to the tumorigenic phenotype were examined in cultured mouse keratinocytes and squamous tumors using transfection into NIH 3T3 cells and nucleic acid hybridization. When normal keratinocytes were cultured in medium with 0.05 mM Ca2+ (low Ca2+ medium), many cells died within 2-3 wk, while others formed rapidly growing foci that could be subcultured. These rapidly growing cells produced benign tumors when grafted to nude mice and possessed a heterozygous mutation in the c-Ha-ras gene with an A----T transversion in codon 61. Fibroblast-conditioned low Ca2+ medium prevented cell death, focus formation, c-Ha-ras gene mutation, and tumorigenicity. Thus, suboptimal culture conditions favored a spontaneous mutation in codon 61 of the c-Ha-ras gene of keratinocytes. When a v-Ha-ras gene was introduced into normal keratinocytes by a replication-defective retrovirus, the recipient cells produced papillomas in vivo, and after 2 mo, 60% of the tumors converted to squamos cell carcinomas. None of the 22 converted tumors had an endogenous c-Ha-ras gene mutation at codon 61. However, the A----T transversion mutation developed when these carcinoma cells were cultured in low Ca2+ medium but not in fibroblast-conditioned medium. Cells with both an exogenous v-Ha-ras and an activated c-Ha61-ras gene produced undifferentiated, rapidly lethal carcinomas, while cells with only v-Ha-ras maintained the squamous carcinoma phenotype. Undifferentiated carcinomas also developed when the v-Ha-ras gene was introduced into papilloma cells with a chemically induced endogenous c-Ha61-ras gene mutation. These results suggest that mutation in the c-Ha-ras gene can contribute to initiation, malignant conversion, and malignant progression in skin carcinogenesis, and gene dosage may determine the phenotype expressed.

Amino Acids↗

PubMed1994

Isolation of two novel ras genes in Dictyostelium discoideum; evidence for a complex, developmentally regulated ras gene subfamily.

In Dictyostelium discoideum, three ras genes (rasD, rasG and rasB) and one ras-related gene (rap1) have been previously isolated and characterized, and the deduced amino acid sequence of their predicted protein products share at least 50% sequence identity with the human H-Ras protein. We have now cloned and characterized two additional members of the ras gene subfamily in Dictyostelium, rasC and rasS. These genes are developmentally regulated and unlike the previously isolated Dictyostelium ras genes, maximum levels of their transcripts were detected during aggregation, suggesting that the encoded proteins have distinct functions during aggregation. The rasC cDNA encodes a 189 amino acid protein that is 65% identical to the Dictyostelium RasD and RasG proteins and 56% identical to the human H-Ras protein. The predicted 194 amino acid gene product encoded by rasS is 60% identical to the Dictyostelium RasD and RasG proteins and 54% identical to the human H-Ras protein. Whereas RasD, RasG, RasB and Rap1 are totally conserved in their putative effector domains relative to H-Ras, RasC and RasS have single amino acid substitutions in their effector domains, consistent with the idea that they have unique functions. In RasC, aspartic acid-38 has been replaced by asparagine (D38N), and in RasS, isoleucine-36 has been replaced by leucine (I36L). In addition, both proteins have several differences in the effector-proximal domain, a domain which is believed to play a role in Ras target activation. In RasC, there is a single conservative amino acid change in the canonical sequence of the binding site for the Ras-specific monoclonal antibody Y13-259, and consequently, RasC is less immunoreactive with the antibody than either of the Dictyostelium RasD or RasG proteins. In contrast, RasS, which has three substitutions in the Y13-259 binding site, does not react with the Y13-259 antibody.

Amino Acid Sequence↗

PubMed1993

Mutations of the p53 tumor suppressor gene and the ras gene family in intrahepatic cholangiocellular carcinomas in Japan and Thailand.

The incidence and pattern of mutations of the ras oncogenes and the p53 tumor suppressor gene have been shown to differ among different cancer types and even among the same cancer types with different etiological backgrounds. For example, in a previous study we showed that not only the etiology but also the incidence of point mutation of the c-Ki-ras oncogene in cholangiocellular carcinomas (CCCs) differ between Japanese and Thai patients. In the study presented here, we examined the incidence of mutations in the ras gene family and the p53 gene in CCCs of both Japanese and Thai patients by single-strand conformation polymorphism and direct sequencing analyses and compared the pattern of p53 mutation between these two CCC groups. Although the incidence of ras mutation differed markedly between Japanese (seven of 12, 58%) and Thai (two of 26, 8%) cases, the incidence of p53 mutation was similar: four of 12 (33%) and nine of 26 (35%), respectively. Except for one case in which deletion-insertion was detected in the second exon of the N-ras gene, all ras mutations occurred at codon 12 or 13 of the c-Ki-ras gene. All p53 mutations but one were detected in a highly conserved region, and the predominant form of the mutations was G:C-->A:T transition at CpG sites in both Japanese and Thai cases, similar to that reported for colorectal cancers. Therefore, in contrast to the ras oncogenes, mutation of the p53 gene was frequently involved in the development of CCCs in both Japanese and Thai patients, irrespective of any difference in etiology.

Adult↗

PubMed1987

Structure of the human and murine R-ras genes, novel genes closely related to ras proto-oncogenes.

The human R-ras gene was isolated by low-stringency hybridization with a v-H-ras probe. The predicted 218 amino acid R-ras protein has an amino-terminal extension of 26 residues compared with H-ras p21, and shows 55% amino acid identity; conserved domains include the p21 GTP-binding site and the carboxy-terminal membrane localization sequence. R-ras has at least six exons, with the position of the first intron conserved relative to the Drosophila ras64B and Dictyostelium ras genes; there is no similarity in the exon-intron structure of the R-ras gene and of the mammalian H-, K-, and N-ras proto-oncogenes. Cloned mouse R-ras cDNAs exhibit 88% nucleotide and 94.5% predicted amino acid identity to human R-ras. Human R-ras was localized to chromosome 19, a site different from ras p21 genes. Mouse R-ras is syntenic with c-H-ras on chromosome 7.

Animals↗

PubMedDate not supplied

Inhibition of the membrane localization of p21 ras proteins by lovastatin in tumor cells possessing a mutated N-ras gene.

Mutated ras genes are found in a variety of human tumors. For biological activity the gene product p21 ras needs to be bound to the cell membrane by a farnesyl residue. Treatment of tumor cells with lovastatin reduces the availability of farnesyl pyrophosphate for the modification of the ras proteins. The membrane localization of p21 ras has been reduced by 30-36% after the tumor cells have grown in the presence of 10 microM lovastatin for 7 days. The extent of the inhibition depends on the growth kinetics of the cell lines.

Acute Disease↗

PubMed1997

Susceptibility of transgenic mice carrying human prototype c-Ha-ras gene in a short-term carcinogenicity study of vinyl carbamate and ras gene analyses of the induced tumors.

To determine if hemizygous transgenic mice carrying the human c-Ha-ras gene (CB6F1-Tg Hras2 mice (Hras2 mice)) are susceptible to the carcinogenic potential of known murine carcinogens, male and female Hras2 mice and their non-transgenic CB6F1 littermates (non-Tg mice) were each given a single intraperitoneal injection of 60 mg of vinyl carbamate (VC)/kg body weight or saline (vehicle control) and monitored for 16 wk without further treatment. At necropsy, grossly visible tumors were fixed for histopathologic diagnosis and, when of sufficient size, portions were frozen for subsequent molecular analysis. Nine of 31 male and nine of 29 female Hras2 mice treated with VC died within 16 wk as a result of lung tumor burden. At the termination of the study, lung tumors (alveolar-bronchiolar epithelial neoplasms and hemangiosarcomas) and focal alveolar-bronchiolar hyperplasias were present in both sexes of Hras2 and non-Tg mice treated with VC; there were significantly more proliferative lung lesions in Hras2 than non-Tg mice. Splenic hemangiosarcomas and squamous cell tumors of the forestomach were induced in male and female VC-treated Hras2 mice but not in VC-treated non-Tg mice. Polymerase chain reaction-single-strand conformation polymorphism analysis and DNA sequencing of the induced lung tumors revealed point mutations at codon 61 of the transgene in two of 29 lung tumors (one of 16 in males and one of 13 in females) from VC-treated Hras2 mice; no mutations in murine Ki-ras were found in these tumors. Point mutations at codons 12 and 61 of the murine Ki-ras gene were observed, however, in one of 10 and six of 10 lung tumors respectively, from VC-treated non-Tg mice. These findings indicate that Hras2 mice are highly sensitive to pulmonary neoplasms and splenic and lung hemangiosarcomas after treatment with VC. The molecular analyses suggest that point mutations of the transgene and the murine Ki-ras gene do not play a major role in VC induction of pulmonary neoplasms in these transgenic mice.

Adenocarcinoma, Bronchiolo-Alveolar↗

PubMedDate not supplied

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.

Animals↗