The utility of N5 for gene therapy of human cancer

Cancer is a result of defects in the coordination of cell proliferation and programmed cell death. The extent of cell death is physiologically controlled by the activation of a programmed suicide pathway that results in a morphologically recognizable form of death termed apoptosis. Inducing apoptosis in tumor cells by gene therapy provides a potentially effective means to treat human cancers. The p84N5 is a novel nuclear death domain containing protein that has been shown to bind an amino terminal domain of retinoblastoma tumor suppressor gene product (pRb). Expression of N5 can induce apoptosis that is dependent upon its intact death domain and is inhibited by pRb. In many human cancer cells the functions of pRb are either lost through gene mutation or inactivated by different mechanisms. N5 based gene therapy may induce cell death preferentially in tumor cells relative to normal cells. We have demonstrated that N5 gene therapy is less toxic to normal cells than to tumor cells. To test the possibility that N5 could be used in gene therapy of cancer, we have generated a recombinant adenovirus engineered to express N5 and test the effects of viral infection on growth and tumorigenicity of human cancer cells. Adenovirus N5 infection significantly reduced the proliferation and tumorigenicity of breast, ovarian, and osteosarcoma tumor cell lines. Reduced proliferation and tumorigenicity were mediated by an induction of apoptosis as indicated by DNA fragmentation in infected cells. We also test the potential utility of N5 for gene therapy of pancreatic carcinoma that typically respond poorly to conventional treatment. Adenoviral mediated N5 gene transfer inhibits the growth of pancreatic cancer cell lines in vitro. N5 gene transfer also reduces the growth and metastasis of human pancreatic adenocarcinoma in subcutaneous and orthotopic mouse model. Interestingly, the pancreatic adenocarcinoma cells are more sensitive to N5 than they are to p53, suggesting that N5 gene therapy may be effective in tumors resistant to p53. We also test the possibilities of the use of N5 and p53 together on the inhibition of pancreatic cancer cell growth in vitro and vivo. Simultaneous use of N5 and RbΔCDK has been found to exert a greater extent on the inhibition of pancreatic cancer cell growth in vitro and in vivo. ^

Alterations in the ras oncogene and the p53 tumor suppressor gene in ultraviolet radiation-induced skin carcinogenesis

A rapid increase of the ultraviolet radiation (UVR)-related skin cancer incidence has attracted more and more public attention during the last few decades. Prevention and treatment of UVR-related skin cancer has become an important public health issue in the United States. Recent studies indicate that mutations in ras and/or p53 genes may be involved in UVR-induced skin tumor development but the precise molecular mechanism remains unclear. In this study, alterations of H-ras and p53 genes were investigated in different stages of carcinogenesis in a chronic UVR (solar simulator) exposure-induced Sencar mouse skin carcinogenesis model in order to clarify the role of the alterations of these genes during the skin carcinogenesis process and to further understand the mechanisms by which UVR causes skin cancer.^ Positive ras-p21 staining in cell membranes and cytosol were detected in 18/33 (55%) of squamous cell carcinomas (SCCs), but were not detected in UV-exposed skin, papillomas, or spindle cell tumors (SCTs). Positive staining of the malignant progression marker K13 was found in 17/33 (52%) of SCCs only. A significant positive correlation was observed between the K13 and the ras-p21 expression. Polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis and gene sequencing analysis revealed three point mutations, one (codon 56) in UV-exposed non-tumor bearing skin and the other two (codons 21 and 13) in SCCs. No UV-specific mutation patterns were found.^ Positive p53 nuclear staining was found in 10/37 (27%) of SCCs and 12/24 (50%) of SCTs, but was not detected in normal skin or papillomas. PCR-based SSCP and sequencing analysis revealed eight point mutations in exons 5 and 6 (four in SCTs, two in SCCs, and two in UV-exposed skin) including six C-T or C-A transitions. Four of the mutations occurred at a dipyrimidine (CC) sequence. The pattern of the mutations indicated that the mutagenic lesions were induced by UVR.^ These results indicate that overexpression of ras-p21 in conjunction with aberrant expression of K13 occurred frequently in UVR-induced SCCs in Sencar mouse skin. The point mutation in the H-ras gene appeared to be a rare event in UVR skin carcinogenesis and may not be responsible for overexpression of ras-p21. UVR-induced P53 gene alteration is a frequent event in UVR-induced SCCs and later stage SCT tumors in Sencar mice skin, suggesting the p53 gene mutation plays an important role in skin tumor malignant progression. ^

Genetic variation in genes for the xenobiotic-metabolizing enzymes CYP1A1, EPHX1, GSTM1, GSTT1, and GSTP1 and susceptibility to colorectal cancer in Lynch syndrome.

Individuals with Lynch syndrome are predisposed to cancer due to an inherited DNA mismatch repair gene mutation. However, there is significant variability observed in disease expression likely due to the influence of other environmental, lifestyle, or genetic factors. Polymorphisms in genes encoding xenobiotic-metabolizing enzymes may modify cancer risk by influencing the metabolism and clearance of potential carcinogens from the body. In this retrospective analysis, we examined key candidate gene polymorphisms in CYP1A1, EPHX1, GSTT1, GSTM1, and GSTP1 as modifiers of age at onset of colorectal cancer among 257 individuals with Lynch syndrome. We found that subjects heterozygous for CYP1A1 I462V (c.1384A>G) developed colorectal cancer 4 years earlier than those with the homozygous wild-type genotype (median ages, 39 and 43 years, respectively; log-rank test P = 0.018). Furthermore, being heterozygous for the CYP1A1 polymorphisms, I462V and Msp1 (g.6235T>C), was associated with an increased risk for developing colorectal cancer [adjusted hazard ratio for AG relative to AA, 1.78; 95% confidence interval, 1.16-2.74; P = 0.008; hazard ratio for TC relative to TT, 1.53; 95% confidence interval, 1.06-2.22; P = 0.02]. Because homozygous variants for both CYP1A1 polymorphisms were rare, risk estimates were imprecise. None of the other gene polymorphisms examined were associated with an earlier onset age for colorectal cancer. Our results suggest that the I462V and Msp1 polymorphisms in CYP1A1 may be an additional susceptibility factor for disease expression in Lynch syndrome because they modify the age of colorectal cancer onset by up to 4 years.

Predictors of participation in genetic testing for hereditary breast and ovarian cancer

Up to 10% of all breast and ovarian cancers are attributable to mutations in cancer susceptibility genes. Clinical genetic testing for deleterious gene mutations that predispose to hereditary breast and ovarian cancer (HBOC) syndrome is available. Mutation carriers may benefit from following high-risk guidelines for cancer prevention and early detection; however, few studies have reported the uptake of clinical genetic testing for HBOC. This study identified predictors of HBOC genetic testing uptake among a case series of 268 women who underwent genetic counseling at The University of Texas M. D. Anderson Cancer Center from October, 1996, through July, 2000. Women completed a baseline questionnaire that measured psychosocial and demographic variables. Additional medical characteristics were obtained from the medical charts. Logistic regression modeling identified predictors of participation in HBOC genetic testing. Psychological variables were hypothesized to be the strongest predictors of testing uptake—in particular, one's readiness (intention) to have testing. Testing uptake among all women in this study was 37% (n = 99). Contrary to the hypotheses, one's actual risk of carrying a BRCA1 or BRCA2 gene mutation was the strongest predictor of testing participation (OR = 15.37, CI = 5.15, 45.86). Other predictors included religious background, greater readiness to have testing, knowledge about HBOC and genetic testing, not having female children, and adherence to breast self-exam. Among the subgroup of women who were at ≥10% risk of carrying a mutation, 51% (n = 90) had genetic testing. Consistent with the hypotheses, predictors of testing participation in the high-risk subgroup included greater readiness to have testing, knowledge, and greater self-efficacy regarding one's ability to cope with test results. Women with CES-D scores ≥16, indicating the presence of depressive symptoms, were less likely to have genetic testing. Results indicate that among women with a wide range of risk for HBOC, actual risk of carrying an HBOC-predisposing mutation may be the strongest predictor of their decision to have genetic testing. Psychological variables (e.g., distress and self-efficacy) may influence testing participation only among women at highest risk of carrying a mutation, for whom genetic testing is most likely to be informative. ^

Mutation analysis of the PVRL1 gene in caucasians with nonsyndromic cleft lip/palate.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P, MIM 119530) is perhaps the most common major birth defect. Homozygous PVRL1 loss-of-function mutations result in an autosomal recessive CL/P syndrome, CLPED1, and a PVRL1 nonsense mutation is associated with sporadic nsCL/P in Northern Venezuela. To address the more general role of PVRL1 variation in risk of nsCL/P, we carried out mutation analysis of PVRL1 in North American and Australian nsCL/P cases and population-matched controls. We identified a total of 15 variants, 5 of which were seen in both populations and 1 of which, an in-frame insertion at Glu442, was more frequent in patients than in controls in both populations, though the difference was not statistically significant. Another variant, which is specific to the PVRL1 beta (HIgR) isoform, S447L, was marginally associated with nsCL/P in North American Caucasian patients, but not in Australian patients, and overall variants that affect the beta-isoform were significantly more frequent among North American patients. One Australian patient had a splice junction mutation of PVRL1. Our results suggest that PVRL1 may play a minor role in susceptibility to the occurrence of nsCL/P in some Caucasian populations, and that variation involving the beta (HIgR) isoform might have particular importance for risk of orofacial clefts. Nevertheless, these results underscore the need for studies that involve very large numbers when assessing the possible role of rare variants in risk of complex traits such as nsCL/P.

T-786C polymorphism of the endothelial nitric oxide synthase gene and neuralgia-inducing cavitational osteonecrosis of the jaws.

OBJECTIVE: We hypothesized that, similar to idiopathic hip osteonecrosis, the T-786C mutation of the endothelial nitric oxide synthase (eNOS) gene affecting nitric oxide (NO) production was associated with neuralgia-inducing cavitational osteonecrosis of the jaws (NICO). DESIGN: In 22 NICO patients, not having taken bisphosphonates, mutations affecting NO production (eNOS T-786C, stromelysin 5A6A) were measured by polymerase chain reaction. Two healthy normal control subjects were matched per case by race and gender. RESULTS: Homozygosity for the mutant eNOS allele (TT) was present in 6 out of 22 patients (27%) with NICO compared with 0 out of 44 (0%) race and gender-matched control subjects; heterozygosity (TC) was present in 8 patients (36%) versus 15 control subjects (34%); and the wild-type normal genotype (CC) was present in 9 patients (36%) versus 29 controls (66%) (P = .0008). The mutant eNOS T-786C allele was more common in cases (20 out of 44 [45%]) than in control subjects (15 out of 88 [17%]) (P = .0005). The distribution of the stromelysin 5A6A genotype in cases did not differ from control subjects (P = .13). CONCLUSIONS: The eNOS T-786C polymorphism affecting NO production is associated with NICO, may contribute to the pathogenesis of NICO, and may open therapeutic medical approaches to treatment of NICO through provision of L-arginine, the amino-acid precursor of NO.

Estimation of the incidence of a rare genetic disease through a two-tier mutation survey.

Recent attempts to detect mutations involving single base changes or small deletions that are specific to genetic diseases provide an opportunity to develop a two-tier mutation-screening program through which incidence of rare genetic disorders and gene carriers may be precisely estimated. A two-tier survey consists of mutation screening in a sample of patients with specific genetic disorders and in a second sample of newborns from the same population in which mutation frequency is evaluated. We provide the statistical basis for evaluating the incidence of affected and gene carriers in such two-tier mutation-screening surveys, from which the precision of the estimates is derived. Sample-size requirements of such two-tier mutation-screening surveys are evaluated. Considering examples of cystic fibrosis (CF) and medium-chain acyl-CoA dehydrogenase deficiency (MCAD), the two most frequent autosomal recessive disease in Caucasian populations and the two most frequent mutations (delta F508 and G985) that occur on these disease allele-bearing chromosomes, we show that, with 50-100 patients and a 20-fold larger sample of newborns screened for these mutations, the incidence of such diseases and their gene carriers in a population may be quite reliably estimated. The theory developed here is also applicable to rare autosomal dominant diseases for which disease-specific mutations are found.

Hepatic and renal cytochrome p450 gene regulation during citrobacter rodentium infection in wild-type and toll-like receptor 4 mutant mice.

Citrobacter rodentium is the rodent equivalent of human enteropathogenic Escherichia coli infection. This study investigated regulation of hepatic and renal cytochrome P450 (P450) mRNAs, hepatic P450 proteins, cytokines, and acute phase proteins during C. rodentium infection. Female C3H/HeOuJ (HeOu) and C3H/HeJ (HeJ) mice [which lack functional toll-like receptor 4 (TLR4)] were infected with C. rodentium by oral gavage and sacrificed 6 days later. Hepatic CYP4A10 and 4A14 mRNAs were decreased in HeOu mice (<4% of control). CYP3A11, 2C29, 4F14, and 4F15 mRNAs were reduced to 16 to 55% of control levels, whereas CYP2A5, 4F16, and 4F18 mRNAs were induced (180, 190, and 600% of control, respectively). The pattern of P450 regulation in HeJ mice was similar to that in HeOu mice for most P450s, with the exception of the TLR4 dependence of CYP4F15. Hepatic CYP2C, 3A, and 4A proteins in both groups were decreased, whereas CYP2E protein was not. Renal CYP4A10 and 4A14 mRNAs were significantly down-regulated in HeOu mice, whereas other P450s were unaffected. Most renal P450 mRNAs in infected HeJ mice were increased, notably CYP4A10, 4A14, 4F18, 2A5, and 3A13. Hepatic levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNFalpha) mRNAs were significantly increased in infected HeOu mice, whereas only TNFalpha mRNA was significantly increased in HeJ mice. Hepatic alpha1-acid glycoprotein was induced in both groups, whereas alpha-fibrinogen and angiotensinogen were unchanged. These data indicate that hepatic inflammation induced by C. rodentium infection is mainly TLR4-independent and suggest that hepatic P450 down-regulation in this model may be cytokine-mediated.

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

Translational regulation of nuclear gene COX4 expression by mitochondrial content of phosphatidylglycerol and cardiolipin in Saccharomyces cerevisiae.

Previous results indicated that translation of four mitochondrion-encoded genes and one nucleus-encoded gene (COX4) is repressed in mutants (pgs1Delta) of Saccharomyces cerevisiae lacking phosphatidylglycerol and cardiolipin. COX4 translation was studied here using a mitochondrially targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5' and 3' untranslated regions (UTRs). Lack of mtGFP expression independent of carbon source and strain background was established to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but was rather caused directly by the lack of phosphatidylglycerol and cardiolipin in mitochondrial membranes. Reintroduction of a functional PGS1 gene under control of the ADH1 promoter restored phosphatidylglycerol synthesis and expression of mtGFP. Deletion analysis of the 5' UTR(COX4) revealed the presence of a 50-nucleotide fragment with two stem-loops as a cis-element inhibiting COX4 translation. Binding of a protein factor(s) specifically to this sequence was observed with cytoplasm from pgs1Delta but not PGS1 cells. Using HIS3 and lacZ as reporters, extragenic spontaneous recessive mutations that allowed expression of His3p and beta-galactosidase were isolated, which appeared to be loss-of-function mutations, suggesting that the genes mutated may encode the trans factors that bind to the cis element in pgs1Delta cells.

ANKRD1, the gene encoding cardiac ankyrin repeat protein, is a novel dilated cardiomyopathy gene.

OBJECTIVES: We evaluated ankyrin repeat domain 1 (ANKRD1), the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients. BACKGROUND: CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction. METHODS: In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP. CONCLUSIONS: ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.

ABERRATIONS OF A PUTATIVE TUMOR SUPPRESSOR GENE SEL1L IN PANCREATIC DUCTAL ADENOCARCINOMA

Introduction: Pancreatic cancer is the fourth leading cause of cancer-related death among males and females in the United States. Sel-1-like (SEL1L) is a putative tumor suppressor gene that is downregulated in a significant proportion of human pancreatic ductal adenocarcinoma (PDAC). It was hypothesized that SEL1L expression could be down-modulated by somatic mutation, loss of heterozygosity (LOH), CpG island hypermethylation and/or aberrantly expressed microRNAs (miRNAs). Material and methods: In 42 PDAC tumors, the SEL1L coding region was amplified using reverse transcription polymerase chain reaction (RT-PCR), and analyzed by agarose gel electrophoresis and sequenced to search for mutations. Using fluorescent fragment analysis, two intragenic microsatellites in the SEL1L gene region were examined to detect LOH in a total of 73 pairs of PDAC tumors and normal-appearing adjacent tissues. Bisulfite DNA sequencing was performed to determine the methylation status of the SEL1L promoter in 41 PDAC tumors and 6 PDAC cell lines. Using real-time quantitative PCR, the expression levels of SEL1L mRNA and 7 aberrantly upregulated miRNAs that potentially target SEL1L were assessed in 42 PDAC tumor and normal pairs. Statistical methods were applied to evaluate the correlation between SEL1L mRNA and the miRNAs. Further the interaction was determined by functional analysis using a molecular biological approach. Results: No mutations were detected in the SEL1L coding region. More than 50% of the samples displayed abnormally alternate or aberrant spliced transcripts of SEL1L. About 14.5% of the tumors displayed LOH at the CAR/CAL microsatellite locus and 10.7% at the RepIN20 microsatellite locus. However, the presence of LOH did not show significant association with SEL1L downregulation. No methylation was observed in the SEL1L promoter. Statistical analysis showed that SEL1L mRNA expression levels significantly and inversely correlated with the expression of hsa-mir-143, hsa-mir-155, and hsa-mir-223. Functional analysis indicated that hsa-mir-155 acted as a suppressor of SEL1L in PL18 and MDAPanc3 PDAC cell lines. Discussion: Evidence from these studies suggested that SEL1L was possibly downregulated by aberrantly upregulated miRNAs in PDAC. Future studies should be directed towards developing a better understanding of the mechanisms for generation of aberrant SEL1L transcripts, and further analysis of miRNAs that may downregulate SEL1L.

DYSREGULATION OF MEOX2 FOLLOWING WT1 MUTATION IN KIDNEY DEVELOPMENT AND WILMS TUMORIGENESIS

Wilms tumor (WT) is a childhood tumor of the kidney and a productive model for understanding the role of genetic alteration and interactions in tumorigenesis. The Wilms tumor gene 1 (WT1) is a transcriptional factor and one of the few genes known to have genetic alterations in WT and has been shown be inactivated in 20% of WTs. However, the mechanisms of how WT1 mutations lead to Wilms tumorigenesis and its influence on downstream genes are unknown. Since it has been established that WT1 is a transcriptional regulator, it has been hypothesized that the loss of WT1 leads to the dysregulation of downstream genes, in turn result in the formation of WTs. To identify the dysregulated downstream genes following WT1 mutations, an Affymetrix GeneChip Human Genome Array was previously conducted to assess the differentially expressed genes in the WT1-wildtype human and WT1-mutant human WTs. Approximately 700 genes were identified as being significantly dysregulated. These genes were further prioritized based on their statistical significance, fold change, chromosomal region, spatial pattern of gene expression and known or putative cellular functions. Mesenchyme homeobox 2 (MEOX2) was one of the most significantly upregulated genes in WT1-mutant WT. MEOX2 is known to play a role in cell proliferation, apoptosis, and differentiation. In addition to its biological roles, it is expressed during early kidney development in the condensed mesenchyme similar to WT1. Furthermore, the use of the Match® web-based tool from the BIOBASE Biological Data base identified a significant predicted WT1 binding site within the first intron of MEOX2. The similarity in spatial gene expression in the developing kidney and the significant predicted WT1 binding site found in the first intron of MEOX2 lead to the development of my hypothesis that MEOX2 is upregulated via a WT1-dependent manner. Here as a part of my master’s work, I have validated the Affymetrix GeneChip Human Genome Array data using an independent set of Wilms tumors. MEOX2 remained upregulated in the mutant WT1 Wilms tumor by 41-fold. Wt1 and Meox2 gene expression were assessed in murine newborn kidney; both Wt1 and Meox2 were expressed in the condensed, undifferentiated metanephric mesenchyme. I have shown that the in vivo ablation of Wt1 during embryonic development at embryonic day (E) 13.5 resulted in the slight increase of Meox2 gene expression by two fold. In order to functionally demonstrate the effect of the loss of Wt1 on Meox2 gene expression in undifferentiated metanephric mesenchyme, I have generated a kidney mesenchymal cell line to genetically ablate Wt1 in vitro by adenoviral infection. The ablation of Wt1 in the kidney mesenchymal cell line resulted in the upregulation of Meox2 by 61-fold. Moreover, the upregulation of Meox2 resulted in the significant induction of p21 and Itgb5. In addition to the dysregulation of these genes the ablation of Wt1 in the kidney mesenchymal cells resulted in decrease in cell growth and loss of cellular adherence. However, it is uncertain whether the upregulation of Meox2 caused this particular cellular phenotype. Overall, I have demonstrated that the upregulation of Meox2 is Wt1-dependent during early kidney development.

Detection and sequence analysis of a nickel-induced mutation in a retroviral model system

We have developed a novel way to assess the mutagenicity of environmentally important metal carcinogens, such as nickel, by creating a positive selection system based upon the conditional expression of a retroviral transforming gene. The target gene is the v-mos gene in MuSVts110, a murine retrovirus possessing a growth temperature dependent defect in expression of the transforming gene due to viral RNA splicing. In normal rat kidney cells infected with MuSVts110 (6m2 cells), splicing of the MuSVts110 RNA to form the mRNA from which the transforming protein, p85$\sp{\rm gag-mos}$, is translated is growth-temperature dependent, occurring at 33 C and below but not at 39 C and above. This splicing "defect" is mediated by cis-acting viral sequences. Nickel chloride treatment of 6m2 cells followed by growth at 39 C, allowed the selection of "revertant" cells which constitutively express p85$\sp{\rm gag-mos}$ due to stable changes in the viral RNA splicing phenotype, suggesting that nickel, a carcinogen whose mutagenicity has not been well established, could induce mutations in mammalian genes. We also show by direct sequencing of PCR-amplified integrated MuSVts110 DNA from a 6m2 nickel-revertant cell line that the nickel-induced mutation affecting the splicing phenotype is a cis-acting 70-base duplication of a region of the viral DNA surrounding the 3$\sp\prime$ splice site. These findings provide the first example of the molecular basis for a nickel-induced DNA lesion and establish the mutagenicity of this potent carcinogen. ^

Molecular characterization of a gene required for entry into S phase of the mitotic cell cycle in the yeast {\it Saccharomyces cerevisiae\/}

A strain of Saccaromyces cerevisiae (SC3B) with a temperature sensitive defect in the synthesis of DNA has been isolated. This defect is due to a single recessive mutation in a gene named INS1 required for the initiation of S phase. Arrested cells carrying the ins1$\sp{ts}$ allele are defective in the completion of G1 to S phase transition events including SPB duplication or separation, initiation of DNA synthesis, normal control of budding, and bud neck stability. The mutation and a gene which complements the mutation were mapped to chromosome IV. The complementing gene was proved to be the wild type allele of the temperature sensitive mutation by genetic linkage of an integrated clone. A very low abundance 4.2 kb RNA message was observed in the strain SC3B which increased greatly in this strain transformed with a multiple copy plasmid carrying the complementing clone. The wild type gene was sequenced and found to encode a 1268 amino acid protein of with a molecular weight of 142,655 Daltons. Computer assisted searches for similar DNA sequences revealed no significant homology matches. However, searches for protein sequence homology revealed a protein (the DIS3 gene product of S. pombe) with a similar sequence over a 534 amino acid stretch to the predicted INS1 gene product. A later search revealed a near identical sequence for a gene (SRK1) also isolated from S. cerevisiae. ^