Frameshift Mutation In Codon Research Articles

Analysis of the sphingomyelin phosphodiesterase 1 gene (SMPD1) in Turkish Niemann–Pick disease patients: Mutation profile and description of a novel mutation

Niemann–Pick disease (NPD) is a lysosomal storage disorder that results from the deficiency of a lysosomal enzyme, acid sphingomyelinase. Niemann–Pick disease type A and B is caused by mutations in the sphingomyelin phosphodiesterase gene (SMPD1) coding for ASM. The aim of this study was to evaluate the spectrum of SMPD1 gene mutations in Turkish NPD patients and to study genotype–phenotype associations. We present a molecular analysis of 10 Turkish NPD type A/B patients. Four of the patients had type A and six had type B NPD. All mutant SMPD1 alleles were identified, including 5 different mutations, 1 of which was novel. These mutations included three missense mutations: c.409T>C (p.L137P), c.1262 A>G (p.H421R) and c.1552T>C (p.L549P), a common frameshift mutation in codon 189, identified in three patients, is caused by the deletion of the 567T, introducing a stop codon 65 amino acids downstream (p.P189fsX65), and a novel frameshift mutation c.1755delC (p.P585PfsX24) which was not reported previously.

A Case of von Hippel-Lindau Disease Presenting with Pancreatic Neuroendocrine Tumor

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that results from a germline mutation of the VHL gene. The affected individuals might develop several benign or malignant tumors such as central nervous system or retinal haemangioblastomas, endolymphatic sac tumors, renal cell carcinomas, pheochromocytomas or pancreatic cysts and neuroendocrine tumors. We report here on a case of a 21 year old female with von Hippel-Lindau disease and she presented with only pancreatic neuroendocrine tumor and no evidence of haemangioblastomas or other visceral complications. Further, direct sequencing of the VHL gene reveals a novel germline frameshift mutation of codon 198 from the deletion of nucleotide 592 (cytosine), leading to truncation of the VHL protein. (Endocrinol Metab 26:89-91, 2011)

A C-terminal amino acid substitution in the γ-chain caused by a novel heterozygous frameshift mutation (Fibrinogen Matsumoto VII) results in hypofibrinogenaemia

We found a novel hypofibrinogenemia designated as Matsumoto VII (M-VII), which is caused by a heterozygous nucleotide deletion at position g.7651 in FGG and a subsequent frameshift mutation in codon 387 of the gamma-chain. This frameshift results in 25 amino acid substitutions, late termination of translation with elongation by 15 amino acids, and the introduction of a canonical glycosylation site. Western blot analysis of the patient's plasma fibrinogen visualised with anti-gamma-chain antibody revealed the presence of two extra bands. To identify the extra bands and determine which of the above-mentioned alterations caused the assembly and/or secretion defects in the patient, 11 variant vectors that introduced mutations into the cDNA of the gamma-chain or gamma'-chain were transfected into Chinese hamster ovary cells. In vitro expression of transfectants containing gammaDelta7651A and gammaDelta7651A/399T (gammaDelta7651A with an amino acid substitution of 399Asn by Thr and a variant lacking the canonical glycosylation site) demonstrated a reduction in secretion to approximately 20% of the level seen in the transfectants carrying the normal gamma-chain. Furthermore, results from other transfectants demonstrated that eight aberrant residues between 391 and 398 of the M-VII variant, rather than the 15 amino acid extension or the additional glycosylation, are responsible for the reduced levels of assembly and secretion of M-VII variant fibrinogen. Finally, the results of this study and our previous reports demonstrate that the fibrinogen gamma-chain C-terminal tail (388-411) is not necessary for protein assembly or secretion, but the aberrant amino acid sequence observed in the M-VII variant (especially 391-398) disturbs these functions.

Compound heterozygosity for HbD Punjab and polyadenylation signal mutation causes clinically asymptomatic mild hypochromia and microcytosis

Dear Editor, A 30-year-old man was ascertained while evaluating his daughter for transfusion-dependent beta thalassemia. The child was diagnosed to have beta thalassemia major at the age of 1 year and was on regular transfusion. The couple wanted prenatal diagnosis for their second pregnancy. On evaluation, the child was found to inherit IVS1-5 (G>C) mutation (NG_000007.3:g.70691G>C) from her mother and poly A signal (NG_000007.3:g.72128 T>C) mutation from her father. The father was also found to have hemoglobin D Punjab on electrophoresis. Sequencing of his beta-globin genes confirmed him to be compound heterozygote for HbD Punjab mutation (NG_000007.3:g.71938G>C) and poly A signal mutation (Fig. 1). The chorionic villus sample showed only poly A mutation and reported as a carrier for beta thalassemia. The clinical details of the father were reviewed. He did not have anemia, jaundice, and blood transfusion in the past. There was no pallor, hepatomegaly, or splenomegaly. His investigations were as follows: Hemoglobin, 12.9 g% (12.5-18); total leukocyte count, 5,900/mm (4,000– 11,000); erythrocyte count, 5.70million/mm (4.5–6.5); mean corpuscular volume, 69.1 fl (76–96); mean corpuscular hemoglobin, 22.6 pg (27–32); mean corpuscular hemoglobin concentration, 32.7 g/dL (32–36); red cell distribution width, 16.3% (11.5–14.5); platelet count, 179,000/mm (150,000– 400,000). In addition to microcytes, anisocytosis and mild polychromasia were observed on peripheral smear examination. Capillary hemoglobin electrophoresis showed HbA, 22.7%; HbD, 72.3%; and HbA2, 5.0%. No HbF peak was noted (Fig. 1). Total serum bilirubin was 0.7 mg% and the conjugated bilirubin level was 0.2 mg%. Alanine transaminase was 25 U/L (5–40); aspartate transaminase, 22 U/L (5–40); and lactate dehydrogenase, 255 U/L (200–500). The mutation in poly A signal has been shown to extend the transcription of beta globin by at least 900 nucleotides 3′ of the normal polyadenylation site and thus is expected to produce reduction in normal beta chain production [1]. It is a rare mutation in beta-globin gene, but in association with IVS1-5 (G>C), the child had significant anemia requiring regular transfusions. Hb D Punjab (or Hb D-Los Angeles, NG_000007.3: g.71938G>C) is characterized by the substitution of glutamine for glutamic acid at position 121 of the betaglobin polypeptide. It is common in several parts of the world, including India. Carriers of HbD even in homozygous state are asymptomatic. It is known to produce a mild anemia when another beta-globin mutation is present in the same individual. Compound heterozygosity for a beta zero mutation (IVS II.1 G>A) in two individuals produced a minor thalassemia phenotype with moderate anemia and significantly elevated HbF [2, 3]. In another report, a child with HbD and frameshift mutation in codon 8/9 (+G; a beta zero mutation), had 94.5% of Hb D, without Hb A, the hematologic picture was that of thalassemia trait with moderate hemolytic anemia, intense microcytosis and hypochromia and numerous target cells [4]. Perea et al have observed that Hb S/Hb D patients had severe K. M. Girisha (*) Genetics Clinic, Department of Pediatrics, Kasturba Medical College, Manipal 576104, India e-mail: girishkatta@gmail.com

Protein Kinase C Phosphorylation Modulates N- and C-Terminal Regulatory Activities of the PITX2 Homeodomain Protein

PKC phosphorylation regulates PITX2 DNA binding and transcriptional activity. Mutation of individual PKC sites demonstrates the functional regulation of PITX2 through phosphorylation. Immunoprecipitation of PITX2 and a PITX2 PKC mutant protein reveal specific in vivo phosphorylation by PKC in transfected cells. The transcriptional activity of PITX2 is negatively regulated by N-terminal phosphorylation and positively regulated by C-terminal phosphorylation. We demonstrate a mechanism of increased PITX2 transcriptional activation through protein interactions facilitated by phosphorylation of the PITX2 C-terminal tail. Phosphorylation of the PITX2 C terminus enhances the interaction with cellular factors. In corroboration with the PITX2 PKC functional studies, a newly identified C-terminal PITX2 mutation associated with Axenfeld-Rieger syndrome (ARS) demonstrates reduced phosphorylation. This mutation (PITX2 DeltaT1261) creates a frameshift mutation in codon 227 resulting in 11 novel amino acids downstream followed by premature truncation of the protein. Three PKC sites in the C-terminal tail and OAR domain are deleted, which results in decreased transcriptional activation. PITX2 DeltaT1261 is unable to interact with a cellular factor to synergistically activate transcription and demonstrates the first link of ARS with defective PITX2 protein interactions. Gene expression profiling of homozygous Pitx2 mutant mouse tissue reveals decreased Dlx2 expression as a potential molecular basis for developmental defects associated with ARS patients. Overall, phosphorylation imparts another level of regulation to the activity of the PITX2 homeodomain protein during development.

A novel mutation in the PITX2 gene in a family with Axenfeld-Rieger syndrome

Purpose: To determine the underlying genetic cause of Axenfeld-Rieger syndrome (ARS) in a three-generation family. Introduction: ARS is a multisystem, autosomal dominant disorder characterized by specific ocular and non-ocular anomalies sometimes caused by mutations in the transcription factor gene, PITX2 . Methods: The three coding exons of the PITX2 gene, i.e., exons 2, 3, and 4, in affected and unaffected subjects were amplified by polymerase chain reaction (PCR) and sequenced. The PCR products of exon 4 were subcloned and sequenced to confirm the nature of the mutation. Results: A deletion of thymine (T) 1261 was identified, creating a frameshift mutation in codon 227. This change is predicted to create 11 novel amino acids downstream, followed by premature truncation of the protein. Conclusions: This mutation highlights the functional importance of a conserved 14-amino acid sequence at the C-terminus of the protein thought to be important in repressing DNA binding and in protein-protein interactions.

Null mutation in the human 11-cis retinol dehydrogenase gene associated with fundus albipunctatus.

Recent studies show that mutations in the gene encoding 11-cis retinol dehydrogenase are associated with fundus albipunctatus. The authors wanted to investigate whether additional, more severe, mutations in the 11-cis retinol dehydrogenase gene might be responsible for more severe forms of hereditary retinal diseases. Case-control molecular genetics study. Two index patients, 7 relatives, and 50 control individuals. The authors screened two index patients diagnosed with fundus albipunctatus for mutations in exons 2 to 5 and exon/intron boundaries of the 11-cis retinol dehydrogenase gene by direct sequencing. Control individuals were screened for the presence of the mutations using allele-specific oligonucleotide hybridization. Mutations in exons 2 to 5 and exon/intron boundaries of the 11-cis retinol dehydrogenase gene. In a compound heterozygote, two novel mutations were found: a 4 bp insertion in exon 2 and a missense mutation Cys267Trp in exon 5. In a second pedigree, a homozygous frameshift mutation in codon 43 (Arg42ct[1-bpdel]) was detected. In both families, the mutations segregate with the disease. The mutations were not found in 50 control individuals. On the basis of our observations, it is unlikely that mutations in the 11-cis retinol dehydrogenase gene are associated with other, possibly more severe, retinal pathologic conditions/dystrophies or syndromic diseases in which the retina is also affected.

Surfactant Protein B Deficiency: Insights into Surfactant Function through Clinical Surfactant Protein Deficiency

Surfactant protein B (SP-B) deficiency is a disorder of surfactant function with complete or transient absence of SP-B in term neonates. SP-B, 1 of 4 described surfactant-associated proteins, plays a key role in surfactant metabolism, particularly in intracellular packaging of surfactant components, formation of tubular myelin, and the presentation of the surfactant phospholipid monolayer to the air-fluid interface within the alveolus. Neonates with clinical SP-B deficiency best demonstrate the key role of SP-B in surfactant function. "Classic" deficiency results in severe respiratory failure in term infants and death unless lung transplantation is performed. Because the initial description of complete deficiency secondary to a homozygous frameshift mutation in codon 121 of the SP-B cDNA, partial deficiencies with differing genetic backgrounds and less severe clinical courses have been reported. These partial deficiency states may provide a clearer picture of genotype/phenotype relationships in SP-B function and surfactant metabolism. SP-B deficiency or dysfunction may be more common than once thought and may play a significant role in neonatal lung disease.

Mutation Analysis of Core Binding Factor A1 in Patients with Cleidocranial Dysplasia

Cleidocranial dysplasia (CCD) is a dominantly inherited disorder characterized by patent fontanelles, wide cranial sutures, hypoplasia of clavicles, short stature, supernumerary teeth, and other skeletal anomalies. We recently demonstrated that mutations in the transcription factor CBFA1, on chromosome 6p21, are associated with CCD. We have now analyzed the CBFA1 gene in 42 unrelated patients with CCD. In 18 patients, mutations were detected in the coding region of the CBFA1 gene, including 8 frameshift, 2 nonsense, and 9 missense mutations, as well as 2 novel polymorphisms. A cluster of missense mutations at arginine 225 (R225) identifies this residue as crucial for CBFA1 function. In vitro green fluorescent protein fusion studies show that R225 mutations interfere with nuclear accumulation of CBFA1 protein. There is no phenotypic difference between patients with deletions or frameshifts and those with other intragenic mutations, suggesting that CCD is generally caused by haploinsufficiency. However, we were able to extend the CCD phenotypic spectrum. A missense mutation identified in one family with supernumerary teeth and a radiologically normal skeleton indicates that mutations in CBFA1 can be associated exclusively with a dental phenotype. In addition, one patient with severe CCD and a frameshift mutation in codon 402 had osteoporosis leading to recurrent bone fractures and scoliosis, providing first evidence that CBFA1 may help maintain adult bone, in addition to its function in bone development.

Mutation of the mismatch repair gene hMSH2 and hMSH6 in a human T‐cell leukemia line tolerant to methylating agents

Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6-alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6-methylguanine (O6meG) induces cell death in OGAT-deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T-cell leukemic Jurkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSα, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15–21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA→TGA). In addition, a (C)8→(C)7 frameshift mutation in codon 1085–1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSα. Genes Chromosomes Cancer 23:159–166, 1998. © 1998 Wiley-Liss, Inc.

Mutation of the mismatch repair genehMSH2 andhMSH6 in a human T-cell leukemia line tolerant to methylating agents

Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6-alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6-methylguanine (O6meG) induces cell death in OGAT-deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T-cell leukemic urkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSalpha, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15-21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA-->TGA). In addition, a (C)8-->(C)7 frameshift mutation in codon 1085-1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSalpha.

Prenatal diagnosis of congenital alveolar proteinosis (surfactant protein B deficiency)

We report on the DNA-based prenatal diagnosis of congenital pulmonary alveolar proteinosis in a family in which alveolar proteinosis was associated with surfactant protein B (SP-B) deficiency. The parents had lost an eight-week-old female child due to this fatal disorder. The affected child was homozygous and both parents were heterozygous for a frame-shift mutation in codon 121 of the surfactant protein B gene (SFTP3-gene). Chorionic villus sampling (CVS) was performed in two subsequent pregnancies. DNA analysis revealed homozygosity for the codon 121 mutation in the first fetus, and the pregnancy was terminated. Homozygosity for the parental wild-type alleles was detected in the following prenatal diagnosis, and a healthy child has been born. DNA-based prenatal diagnosis of congenital alveolar proteinosis is simple, fast and reliable, and can be performed much earlier in pregnancy than any other method, e.g. the direct measurement of SP-B in amniotic fluid. In families with a term infant who dies of unexplained respiratory failure, genetic testing of the parents should be evaluated, since the presence of the codon 121 mutation enables prenatal diagnosis in later pregnancies.

MOLECULAR ANALYSIS OF P16 Ink4/CDKN2 AND P15 Ink4B/MTS2 GENES IN PRIMARY HUMAN TESTICULAR GERM CELL TUMORS

Purpose p16 (MTS 1) and p15 (MTS 2) are negative regulators of cell cycle progression at the G1 cell cycle checkpoint and function as tumor suppressor genes (TSG). Both p15 and p16 are located on chromosome 9p21 and alterations have been demonstrated in a variety of human malignancies and human cancer cell lines. In testicular germ cell tumors (TGCT) loss of heterozygosity of 9p21 to 23 has been observed in 41% to 72% of informative cases. The aim of our study was to examine TGCT and testicular cancer cell lines for deletions and mutations of the p15 and p16 genes. Materials and Methods 24 testicular germ cell tumors, 4 testicular cancer cell lines (TERA I, TERA II, HTE, HTH), 8 benign testicular tumors and 9 samples of normal testicular parenchyma were examined. Examinations for loss of heterozygosity (LOH) on 9p21 were performed for IFNA, D9S171, D9S126, D9S161, D9S1748 and PKY9 locus using Southern Blot analysis. Southern Blot analysis of the p16 gene to examine gross alterations was done using random primer labeled p16 cDNA. In addition all samples were examined for mutations of exons 1 to 3 of p16 and exons 1 to 2 of p15 by polymerase chain reaction (PCR) followed by single strand conformation polymorphism (SSCP) analysis on MDE-gels. All observed band shifts on SSCP were further evaluated by direct DNA sequencing techniques. Results No major deletions were detected for p16 and p15.42% of all TGCT demonstrated LOH of the IFNA locus, 16% of the D9S161 locus and 5.5% of the D9S171 locus. No LOH was seen for the D9S126 locus. 5/24 TGCT (21%) and 1 testicular cancer cell line (TERA II) demonstrated band shifts for exon 2, 8/24 TGCT (33%) and 1 testicular cancer cell line (TERA I) showed band shifts for exon 3; 4 TGCT had an allelic loss for exon 3 on SSCP. DNA sequencing analysis for exon 2 demonstrated a single base substitution (G->A) in codon 140 resulting in an amino acid exchange (ala->thre) in 4 cases and a single base deletion with frame shift mutation in codon 142 in 2 cases. DNA sequencing analysis for exon 3 demonstrated a polymorphism in intron 2 in all cases. For p15 no band shifts were observed for exons 1 to 2 in TGCT or testicular cancer cell lines; none of benign testicular tumors or normal control tissues demonstrated any band shifts for p15 or p16. Discussion We demonstrated a high frequency of mutations for exon 2 in specimens of primary testicular germ cell tumors, but not in benign tumors or normal controls. We therefore believe that p16 might be involved in the pathogenesis of human TGCT. The polymorphisms described for exon 3 might be utilized for further LOH studies. The absence of mutations in p15 gene in TGCT specimens suggests that p15 might not play an important role in the pathogenesis of testicular germ cell tumors.

P53 alterations in chemically induced hamster cheek-pouch lesions.

To confirm that the hamster cheek-pouch carcinogenesis model reflects development of human squamous cell carcinoma (SCC), we determined if and when p53 mutations occur in the development of SCC in this model by using immunohistochemical staining and polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis plus direct DNA sequencing. Twenty-four hamster cheek-pouches were treated with a solution of 0.5% 7,12-dimethylbenz[a]anthracene in mineral oil three times a week for 16 wk. The malignant endophytic and exophytic tumors induced with this protocol are preceded by a sequence of premalignant lesions such as hyperplasia with or without dysplasia and carcinoma in situ, similar to the development of this cancer in humans. For this study, p53 protein accumulation was evaluated by immunostaining of various hamster cheek-pouch exophytic and endophytic SCCs as well as flat dysplastic hyperplasia and carcinomas in situ. A moderate percentage (33.3%) of exophytic lesions and most endophytic carcinomas (90%) showed positive p53 staining. In addition we also found p53-positive staining in a number of preneoplastic lesions, including areas of focal hyperplasia, dysplastic hyperplasia, and carcinomas in situ. To determine whether the alterations in p53 staining were due to p53 gene mutation, we used PCR-SSCP analysis and direct sequencing. PCR products corresponding to exons 5a, 6, 7, and 8 from 40 tumors with the highest percentage of p53-stained cells were analyzed. We detected shifted bands in 17 lesions. Direct sequencing of eight selected shifted bands revealed four mutations, including two G-->T transversions in codons 216 (tumor #1) and 252 (tumor #2) and one G-->C transversion in codon 282 (tumor #3). Tumor #4 contained a frameshift mutation in codon 251. These mutations are consistent with those reported in many human cancers. Therefore, we concluded that in the hamster cheek-pouch model, p53 protein accumulation occurs frequently and early in carcinogenesis, as it does in human SCCs, and some of these p53 alterations are due to p53 gene mutations. These findings may help us better define the mechanisms of carcinogenesis in the hamster cheek-pouch model, and p53 alterations may be an early biomarker of progression for chemoprevention studies.

Suppression of cell growth by wild-type p53 in tumor-derived rat esophageal epithelial cells

Our laboratory previously identified a frameshift mutation in codon 176 of the p53 gene in the cell line RE-149DHD derived from transformation of rat esophageal epithelial cells by benzo(a)pyrene dihydrodiol. This mutation correlated with tumorigenic potential of the cells. To investigate the effect of rat wt-p53 expression on cell growth we constructed a recombinant expression vector, pcDNA3-RP53, in which wt-p53 cDNA is under transcriptional control of a cytomegalovirus promoter, and introduced the vector into RE-149DHD cells. A stable clonal cell line was isolated from each of four colonies transfected by pcDNA3-RP53 vector. Exogenous p53 DNA fragments were successfully amplified from three of the four cell lines with the sense primer complementary to T7 promoter sequences downstream of the CMV promoter. Restriction digestion analysis of these amplified PCR fragments showed fragments of the expected size for exogenous p53 DNA, indicating successful introduction of the pcDNA3-RP53 vector into RE-149DHD cells. However, expression of exogenous p53 mRNA was not detected by RT-PCR in these cell lines. To further confirm absence of exogenous p53 expression, a different set of primers, spanning codon 176 of the p53 gene, was used to amplify a 183-bp fragment of p53 mRNA from all transfected cell lines, the parental RE-149DHD cell line and normal esophageal tissue. Subsequent SSCP analysis of the RT-PCR products from all transfected cell lines and the parental cell line RE-149DHD showed the same mobility shift, indicating expression of endogenous mutant p53 alone, and no exogenous expression of p53 mRNA in all four stable clonal cell transfected with the pcDNA3-RP53 vector. This data suggests that wt-p53 may play a negative role in growth regulation of tumor-derived rat esophageal epithelial cells.

Mutation in the p53 tumor suppressor gene in rat esophageal papillomas induced by N-nitrosomethylbenzylamine.

Mutations in the p53 tumor suppressor gene have been implicated in the pathogenesis of a wide variety of human neoplasms. The location and types of p53 gene mutation can reflect exposure of humans to certain types carcinogenic agents. Much less is known about the role of p53 mutational inactivation in rodent tumors. Using both 'Hot' (radioactive) and 'Cold' (non-radioactive) single strand conformation polymorphism (SSCP) analyses, the present study of analyzed exons 5-8 and the exon-intron junction of the p53 gene from rat esophageal papillomas induces by N- nitrosomethylbenzylamine (NMBA) for mutations. Nine of 30 (30%) esophageal papillomas contained SSCP mobility shifts, principally within exons 5 and 7. These positive SSCP findings were further validated by direct DNA sequencing analysis. Eight of the nine mutations were G:C-->A:T transitions in codons 131, 149, 153, 242 (2), 243, 248, and the 5 end of intron 7. None of these G:C-->A:T mutations occurred at the CpG sites. The other mutation was a frameshift mutation in codon 176. The G:C-A:T transitions observed in this study are consistent with the documented formation of O(6)-methylguanine adducts in DNA N-nitroso compounds. These results suggest that point mutations of the p53 gene are involved in the development of approximately one-third of NMBA-induced rat esophageal papillomas. 'Hot' and 'Cold' SSCP methods were equally sensitive for the identification of mutations in the rat p53 gene.

Frameshift mutation in codon 176 of the p53 gene in rat esophageal epithelial cells transformed by benzo[a]pyrene dihydrodiol

Mutations in the p53 tumor suppressor gene have been associated with exposure to environmental chemical carcinogens. Cultured rat esophageal epithelial cells were transformed in vitro by treatment with benzo[a]pyrene dihydrodiol (BP-DHD). A BP-DHD-transformed cell line and control cell lines were analyzed for mutations in the p53 gene and in the Ha-ras gene by single-strand conformation polymorphism analysis of polymerase chain reaction-amplified products and direct DNA sequencing. The deletion of one cytosine in codons 174-176 (TGCCCCCAC-->TGCCCCAC) of the p53 gene was found only in the BP-DHD-transformed cell line. The BP-DHD-transformed cells were highly invasive and tumorigenic when transplanted into syngeneic rats, whereas control lines either were nontumorigenic or formed epithelial cysts. BP-DHD-transformed cells and control lines were negative for mutations in the Ha-ras gene. Our results suggest that the tumorigenic potential of the BP-DHD-transformed cell line is associated with a frameshift mutation in codon 176 of the p53 gene but not with mutations in the Ha-ras gene. The G/C-rich codons 174-176 in the rat p53 gene may be specific targets for BP-DHD.

Reversion analysis of dynein intermediate chain function.

The ODA6 locus of Chlamydomonas reinhardtii encodes a 70 kDa intermediate chain protein of the flagellar outer row dynein ATPase, and mutations at this locus prevent assembly of the entire outer row dynein arm complex. To initiate a structure-function analysis of the 70 kDa protein, we used transformation with chimeric mutant/wild-type genes to localize the defect in one assembly mutation, oda6-95. Sequence analysis revealed a frame-shift mutation in codon 53, which is followed by a stop codon after 13 amino acids in the new reading frame. By selecting intragenic pseudorevertants of this mutation we obtained 11 new oda6 alleles. Many of these pseudorevertants encode intermediate chain proteins that permit assembly of outer row arms but do not restore full wild-type motility. Revertant strains fall into two phenotypic classes, one with average beat frequencies of 54 Hz (similar to wild type) and one with average frequencies of 27 Hz (compared with 24 Hz for oda6-95) during normal forward swimming. Low beat frequency strains also display abnormalities during photophobic reversal (symmetric waveform). Amplification and sequence analysis of revertant alleles indicated that each reversion caused a second frame-shift, within a 115 nt interval, which restored the original reading frame, and that phenotypic severity was related to both direction (5' or 3') and distance between the original mutation and the reversion event. On the basis of immunoblot analysis of outer arm proteins, we conclude that revertant motility defects do not correlate with deficits in assembly of a specific dynein heavy chain or intermediate chain polypeptide, and electron microscopy confirms that revertants have normal outer arm structures. These results suggest that the 70 kDa intermediate chain plays a direct role in outer arm function distinct from its role in the assembly process.

The Polyadenylation Site Mutation in the α-Globin Gene Cluster

In a previous study, we described a form of nondeletion alpha-thalassemia (alpha T Saudi alpha) found in subjects of Saudi Arabian origin. In the current study, using synthetic oligoprobe hybridization and restriction enzyme analysis, we have demonstrated that the molecular basis of alpha T Saudi alpha is due solely to a single base mutation (AATAAA----AATAAG) in the polyadenylation signal of the alpha 2 gene and that the frameshift mutation in codon 14 of the linked alpha 1 gene is the result of a cloning artefact. The alpha 2 polyadenylation signal mutation occurs in other Middle Eastern and the Mediterranean populations and is responsible for the clinical phenotype of Hb H disease in some Saudi Arabian individuals with five alpha genes (alpha T Saudi alpha/(alpha alpha alpha)T Saudi). Evidence suggests that the (alpha alpha alpha)T Saudi haplotype has arisen as a result of a recombination between two misaligned chromosomes bearing the alpha T Saudi alpha defect.

The polyadenylation site mutation in the alpha-globin gene cluster

In a previous study, we described a form of nondeletion alpha- thalassemia (alpha T Saudi alpha) found in subjects of Saudi Arabian origin. In the current study, using synthetic oligoprobe hybridization and restriction enzyme analysis, we have demonstrated that the molecular basis of alpha T Saudi alpha is due solely to a single base mutation (AATAAA----AATAAG) in the polyadenylation signal of the alpha 2 gene and that the frameshift mutation in codon 14 of the linked alpha 1 gene is the result of a cloning artefact. The alpha 2 polyadenylation signal mutation occurs in other Middle Eastern and the Mediterranean populations and is responsible for the clinical phenotype of Hb H disease in some Saudi Arabian individuals with five alpha genes (alpha T Saudi alpha/(alpha alpha alpha)T Saudi). Evidence suggests that the (alpha alpha alpha)T Saudi haplotype has arisen as a result of a recombination between two misaligned chromosomes bearing the alpha T Saudi alpha defect.