rII Mutations Research Articles

Infrequent mutations of the transforming growth factor beta-type II receptor gene at chromosome 3p22 in human lung cancers with chromosome 3p deletions.

Mutations in the transforming growth factor beta-type II receptor (TGFbeta RII) gene have been detected in several types of human cancers that represent the phenotype of genomic instability. The TGFbeta RII gene has been mapped to chromosome 3p, on which loss of heterozygosity (LOH) was frequently detected in both small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). To investigate whether the TGFbeta RII gene on 3p22 is inactivated in lung cancers, we examined 35 sporadic lung cancers (15 SCLC and 20 NSCLC) with LOH on 3p for mutations of the TGFbeta RII gene. We previously produced eight intron based primer pairs for mutational analysis of the entire coding region of the TGFbeta RII gene. Using these primers, we screened for mutations of the TGFbeta RII gene by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis. A mutation was detected in a case of SCLC: one base insertion in the polyadenine tract of exon 3. This tumor showed the replication error (RER) phenotype. There were no mutations in exons 1, 2, 4, 5, 6 and 7. These results indicate that the polyadenine tract is a mutational hot spot in the TGFbeta RII gene in RER positive tumors, and that TGFbeta RII mutations occur rarely in lung cancers with LOH on chromosome 3p.

Mutation analysis of coding sequences of the entire transforming growth factor beta type II receptor gene in sporadic human colon cancer using genomic DNA and intron primers.

Mutations in the transforming growth factor beta type II receptor (TGFbeta RII) gene have been detected in several human cancers exhibiting microsatellite instability. To extend analyses of this gene, we previously investigated the exon-intron organization of the TGFbeta RII gene and defined seven exons and flanking intron sequences. In this study, we further determined the nucleotide sequences surrounding these seven exons and designed eight sets of intron-based primers to examine the entire coding region of the TGFbeta RII gene. Using these primers, we screened genomic DNA sequences from 30 sporadic colorectal cancers for mutations of the TGFbeta RII gene. TGFbeta RII mutations were detected in two of 30 tumors and both displayed microsatellite instability. One had a deletion in a polyadenine tract in exon 3 and the other had a point mutation in the kinase domain located in exon 7. There were no mutations in exons 1, 2, 4, 5 and 6. These results further implicate the polyadenine tract and kinase domain as mutational hotspots in the TGFbeta RII gene in cells with genomic instability and suggest that TGFbeta RII gene mutations occur rarely in cells lacking genomic instability.

Mutations of the transforming growth factor-beta type II receptor gene are strongly related to sporadic proximal colon carcinomas with microsatellite instability.

Mutations of the transforming growth factor-beta type II receptor gene (TGF-beta RII) have been found in several replication error-positive sporadic colorectal carcinomas and hereditary nonpolyposis colorectal carcinoma cell lines. The aim of this study was to clarify the role of TGF-beta RII in sporadic colorectal carcinogenesis. The authors screened for mutations at simple repeated sequences in the TGF-beta RII gene by polymerase chain reaction-single strand conformation polymorphism. They also examined genomic instability, using five microsatellite DNA markers in 69 sporadic colorectal carcinomas. When the carcinomas exhibited the TGF-beta RII mutations, the authors screened further for mutations in two DNA mismatch repair genes, hMSH2 and hMLH1. Seven of the 69 cancers (10%) showed one or two A deletions in TGF-beta RII and resultant frameshift mutations in nucleotide positions 709-718 containing a (A) 10 repeated sequence; but none of these appeared in the corresponding normal DNA, indicating a somatic mutation. All of the seven cancers were located in the proximal colon; there were none in the distal colon (P < 0.01). On the other hand, 22 of the 69 carcinomas (32%) showed the replication error-positive phenotype. The frequency of replication errors in proximal colon carcinomas was higher than that in distal colon carcinomas (P < 0.05). All 7 cancers with TGF-beta RII mutations showed replication errors. One of them revealed a nonsense mutation at codon 413, and 1 revealed a loss of heterozygosity in hMSH2. These data indicate that mutations of TGF-beta RII are strongly related to proximal colon carcinomas with microsatellite instability and that the mechanism of carcinogenesis in some proximal colon carcinomas is similar to that in hereditary nonpolyposis colorectal carcinoma.

Microsatellite instability and mutations of p53 and TGF-beta RII genes in gastric cancer.

To investigate the molecular mechanism of gastric carcinogenesis, we analyzed genetic instability and p53 gene mutations in 40 primary gastric carcinomas. Tumor samples were from untreated patients with no family history suggestive of genetic predisposition to cancer. We screened six microsatellite loci by the polymerase chain reaction (PCR) method, and exons 5-8 of the p53 gene by the PCR-based denaturing gradient gel electrophoresis and sequencing techniques. Microsatellite instability was detected in 32.5% (13/40), and gene mutations in 40% (16/40), of the tumors analyzed. No statistically significant associations were found between genetic alterations and clinico-pathological variables (with the exception of diffusion of lymph node metastases, which was inversely associated with the presence of microsatellite alterations; P < 0.01). Interestingly, a negative association was found between genetic instability and p53 gene mutations: 11 out of 13 tumors showing instability proved to carry a nonmutated p53 gene versus 2/13 carrying a mutated gene (P = 0.03). These observations suggest that genetic instability and p53 gene mutations play a crucial role in the gastric carcinogenic process, but likely act through distinct pathways during cancer development. However, genetic instability is not in and of itself neoplastic. Therefore, we investigated whether insertion/deletion mutations of the polyadenine tract within the transforming growth factor-beta type II receptor gene (TGF-beta RII) were frequently present in gastric tumors with an RER+ (replication error) phenotype. We found RII mutations in 8/40 (20%) samples: mutations were present in 7/13 (54%) RER+ tumors versus 1/27 (4%) RER- cases (P < 0.001).

Demonstration That Mutation of the Type II Transforming Growth Factor β Receptor Inactivates Its Tumor Suppressor Activity in Replication Error-positive Colon Carcinoma Cells

Escape from negative growth regulation by transforming growth factor beta (TGF-beta) as a result of the loss of TGF-beta type II receptor (RII) expression has been found to be associated with the replication error (RER) colorectal cancer genotype, which is characteristic of hereditary nonpolyposis colorectal cancers. The RER-positive HCT 116 colon carcinoma cell line was examined for RII mutations. A 1-base deletion was found within a sequence of 10 repeating adenines (nucleotides 709-718), which resulted in a frameshift mutation. Although it is reasonable to predict that the loss of RII function would be an important determinant of malignancy, the large number of potential mutations in cells of this phenotype raises the possibility that an RII mutation may not be a key event in the tumorigenic phenotype of these cells. One way to test directly the importance of RII mutations in determining the malignant phenotype would be to restore its expression. If restoration of expression leads to diminished tumorigenicity, it would indicate that RII mutation is an important determinant of malignancy in the RER phenotype. To determine whether restoration of RII would lead to reversal of malignancy in RER colon cancers, an RII expression vector was transfected into the HCT 116 cell line. RII stable clones showed mRNA and protein expression of transfected RII. The fibronectin mRNA level was increased by exogenous TGF-beta 1 treatment in a dose-dependent manner in RII-positive clones, whereas the control cells remained insensitive. The RII transfectants showed reduced clonogenicity in both monolayer culture and soft agarose. They were growth arrested at a lower saturation density than control cells. TGF-beta 1-neutralizing antibody stimulated the proliferation of RII-transfected but not control cells, indicating that the alterations in the growth parameters of the transfected cells were due to the acquisition of autocrine-negative activity. Tumorigenicity in athymic mice was reduced and delayed in RII transfectants. These results indicate that reconstitution of TGF-beta autocrine activity by reexpression of RII can reverse malignancy in RER colon cancers, thus verifying that the malignancy of hereditary nonpolyposis colorectal cancer can be directly associated with the loss of RII expression.

Analysis of mutagenesis induced by a thermolabile T4 phage deoxycytidylate hydroxymethylase suggests localized deoxyribonucleotide pool imbalance

To understand the molecular basis of mutation stimulated by deoxyribonucleotide pool imbalance, we studied a temperature-sensitive T4 phage gene 42 mutant (LB3), which specifies a thermolabile deoxycytidylate hydroxymethylase. Analysis of rII mutations, revertible to wild type along either GC-to-AT or AT-to-GC transition pathways, showed 8- to 80-fold stimulation of GC-to-AT mutations at a semi-permissive temperature (34 degrees C). One such marker, rII SN103, which showed the highest stimulation at 34 degrees C, was sequenced after amplification of the template by polymerase chain reaction. The mutant site in rII SN103 was identified at nucleotide position 265 from the rII B translational start as an AT-to-GC transition, which changes TCA to CCA. Sequence analysis of revertants and pseudorevertants generated at 34 degrees C showed that both cytosines within this triplet can undergo change to either thymine or adenine, consistent with the hypothesis that hydroxymethyldeoxycytidine triphosphate pools are depleted at replication sites. However, dNTP pool measurements in extracts of 34 degrees C cultures showed no significant deviations from values obtained at 30 degrees C, suggesting that pool imbalances occur only locally, close to replication forks. Our studies support the hypothesis that the mutator phenotype displayed by ts LB3 at semi-permissive temperature is a consequence of perturbation of the flow of nucleotide precursors into the DNA replication machinery. A putative localized depletion of hm-dCTP presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations.

Imbalanced deoxyribonucleoside triphosphate pools and spontaneous mutation rates determined during dCMP deaminase-defective bacteriophage T4 infections.

DNA precursor imbalances are known to be mutagenic in both eukaryotic and prokaryotic systems. Almost certainly, such mutagenesis involves competition between correctly and incorrectly base-paired precursors at replication sites. Since other factors may be involved, it is important to identify specific mutations induced by specific pool imbalances. Using bacteriophage T4, we have developed a system for such analysis. We prepare double mutants of T4; one mutation affects a phage-coded enzyme of deoxyribonucleoside triphosphate (dNTP) metabolism, while the second is an rII mutation known to revert along a specific pathway. We determine dNTP pools in infection by such a mutant and measure both the spontaneous reversion rate of the rII mutation and, in some cases, the nucleotide sequence at the mutant site. In this paper we analyze mutations induced by a deficiency of T4-encoded deoxycytidylate deaminase. This causes pools of 5-hydroxymethyl-dCTP to expand some 30-fold, while dTTP pools contract. This specifically stimulates AT-to-GC reversion. One of the four AT-to-GC reverters tested, rIIUV215, increases its reversion rate at least 1000-fold under these pool-imbalance conditions, while the other mutants tested show increases of only about 10-fold. Therefore, factors other than dNTP competition, including local DNA sequence environment, must be invoked to fully explain mechanisms of dNTP pool imbalance-induced mutagenesis. We discuss models for this, and we also report unexpected effects of the dCMP deaminase deficiency upon pools of ribonucleoside triphosphates.

Methyl methanesulfonate mutagenesis in bacteriophage T4.

MMS induces diverse rII mutations from a wild-type background in bacteriophage T4. About 56% are base pair substitutions, about 30% are frameshift mutations, and the remainder is a miscellaneous set of rapidly reverting or leaky mutants of unknown composition; but deletions were not detected. MMS-induced forward mutation is sharply reduced by the mutations px and y, which also reduce ultraviolet, photodynamic and gamma-ray mutagenesis and increase killing by all of these agents. Thus, many of the mutations arise via the T4 WXY system. The induction of G:C leads to A:T transitions was detected even in a px or y background using sensitive reversion tests, and the few forward rII mutations that were induced from this background also behaved like transition mutations. Thus, some MMS-induced mutations arise independently of the WXY system, perhaps as a result of the (rather weak) ability of MMS to alkylate the O6 position of guanine.

Genetic analysis of bacteriophage T4 transducing bacteriophages.

Mutations in the genes for nuclear disruption (ndd), endonuclease IV (denB), and the D1 region of the T4 genome are essential for converting bacteriophage T4 into a generalized transducing phage. These mutations gave rise to a very low frequency of transduction, about 10(-8) per infected bacterium. The addition of an rII mutation raised the transduction frequency about 20-fold. An additional 100-fold increase in the transduction frequency was observed with mutations in genes 42, 56, and alc. High-frequency generalized transduction by T4 results from the cumulative effect of these mutations.

Mutator effects of alleles of phage T4 genes 32, 41, 44, and 45 in the presence of an antimutator polymerase

When eight phage T4 rII base substitution mutations were each combined with an antimutator allele of gene 43 (DNA polymerase), reversion frequencies of the rII mutations were reduced from 3- to 90-fold. When these eight rII-gene 43 allele combinations were then combined with an allele of gene 32 (helix-destabilizing protein), the low reversion index was maintained in five cases. In the remaining three cases, strong mutator effects by the gene 32 allele were observed. One of these three rII alleles was then used as a tester in combination with an allele from each of genes 30, 37, 39, 41, 44, 45, 47, and 56. The corresponding triple mutants, consisting of the rII allele, the gene 43 antimutator allele, and an allele from each of the above genes was also constructed. The alleles of genes 41, 44, and 45 displayed strong mutator effects in the presence of the gene 43 antimutator polymerase and much weaker mutator effects in the presence of wild-type polymerase. The tested alleles of genes 30, 37, 39, 47, and 56 did not have such mutator effects. The genes whose alleles showed strong mutator effects (32, 41, 44, and 45) have been identified previously as being essential for DNA synthesis in vivo, and for a rapid rate of replication in vitro. It has recently been hypothesized that these gene products and the products of genes 61 and 62 may interact with the gene 43 DNA polymerase to form a multienzyme replicative complex which governs the accuracy of replication. The data presented here, together with previously reported results on mutator effects, support the hypothesis that all seven gene products of the replicative complex have a strong influence on the accuracy of replication.

Mutants of T7 bacteriophage inhibited by lambda prophage.

Mutants in gene 20, a new T7 gene, cannot grow on rex+ lambda lysogens. Gene 20-- mutants suppress in double mutants the phenotype of T7 ligase negative mutations, but not vice versa. Amber 20- mutants have been obtained. There are differences between these T7 mutations and the similar T4 rII mutations. There are host mutations which permit T7 20- mutants to grow on lambda+ lysogens. T7 DNA synthesis on normal lambda+ lysogens infected with 20- mutants is essentially normal, but the DNA is not packaged. The gene 20 protein is active in in vitro complementation and probably used late in infection for DNA packaging into phage heads.

Genetic evidence for an additional function of phage T4 gene 32 protein: interaction with ligase.

Gene 32 of bacteriophage T4 is essential for DNA replication, recombination, and repair. In an attempt to clarify the role of the corresponding gene product, we have looked for mutations that specifically inactivate one but not all of its functions and for compensating suppressor mutations in other genes. Here we describe a gene 32 ts mutant that does not produce progeny, but in contrast to an am mutant investigated by others, is capable of some primary and secondary DNA replication and of forming "joint" recombinational intermediates after infection of Escherichia coli B at the restrictive temperature. However, parental and progeny DNA strands are not ligated to covalently linked "recombinant" molecules, and single strands of vegetative DNA do not exceed unit length. Progeny production as well as capacity for covalent linkage in this gene 32 ts mutant are partially restored by additional rII mutations. Suppression by rII depends on functioning host ligase [EC 6.5.1.2; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming, NMN-forming)]. This gene 32 ts mutation (unlike some others) in turn suppresses the characteristic plaque morphology of rII mutants. We conclude that gene 32 protein, in addition to its role in DNA replication and in the formation of "joint" recombinational intermediates, interacts with T4 ligase [EC 6.5.1.1; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming)] when recombining DNA strands are covalently linked. The protein of the mutant that we describe here is mainly defective in this interaction, thus inactivating T4 ligase in recombination. Suppressing rII mutations facilitate substitution of host ligase. There is suggestive evidence that these interactions occur at the membrane.

Effect mutagèné de l'acide nalidixique sur le bactériophage T4

Nalidixic acid can efficiently induce the reversion of some T4 rII mutations. The great majority of the strains whose reversion can be induced by this antibiotic are also sensitive to the mutagenic action of proflavin, indicating that mutagenicity of nalidixic acid results in base pair addition or deletion. With bacterial host strains resistant to nalidixic acid, the mutagenic effect is greatly reduced but not the effects on phage multiplication. This fact shows that the mutagenic agent is not the nalidixic acid itself but a derivative synthesized in sensitive bacterial strains.

N-methyl- N′-nitro- N-nitrosoguanidine and hydroxylamine induced mutants of the rII region of phage T 4

rII mutations of bacteriophage T 4 were induced by in vivo treatment with N-methyl- N′-nitro- N-nitrosoguanidine (NG) and in vitro treatment with hydroxylamine (HA). All the NG induced mutations were mappable to small segments of the rIIA cistron and all except one were also highly revertible by 2-aminopurine (AP) treatment. From these observations, it is concluded that treatment of T 4 with NG induces only transitions and contrary to its effects on E. coli, in T 4, NG does not induce any deletions. Spectra of HA and NG induced mutants of the rIIA cistron were compared. Both mutagens seem to be more effective in inducing mutations nearer the two extremities of this cistron and very few in the middle. This asymmetric effect has been seen to be more pronounced in case of NG than in the case of HA.

N-methyl-N′-nitro-N-nitrosoguanidine and hydroxylamine induced mutants of the rll region of phace T4

rII mutations of bacteriophage T 4 were induced by in vivo treatment with N -methyl- N ′-nitro- N -nitrosoguanidine (NG) and in vitro treatment with hydroxylamine (HA). All the NG induced mutations were mappable to small segments of the rIIA cistron and all except one were also highly revertible by 2-aminopurine (AP) treatment. From these observations, it is concluded that treatment of T 4 with NG induces only transitions and contrary to its effects on E. coli , in T 4 , NG does not induce any deletions. Spectra of HA and NG induced mutants of the rIIA cistron were compared. Both mutagens seem to be more effective in inducing mutations nearer the two extremities of this cistron and very few in the middle. This asymmetric effect has been seen to be more pronounced in case of NG than in the case of HA.

Ligase-defective bacteriophage T4. II. Physiological studies.

The timing of the suppression of gene 30 (deoxyribonucleic acid ligase) mutations by rII mutations was studied by temperature shift-down experiments with a temperature-sensitive rII mutation. The rII function must remain inactivated for about 5 to 8 min at 37 C for suppression to occur, thus making suppression an early function. This result is in agreement with the timing of expression of other rII functions. A gene 30 defect can also be overcome by replacing the Na(+) cation in the growth medium with the Mg(2+) cation, a result similar to the relief of the lethality of rII mutations in lambda lysogens. Prior infection with bacteriophages T3 or T7, which produce their own deoxyribonucleic acid ligases, can also partially overcome the lethality of gene 30 mutations.

A mutation of T4B phage, which enhances suppression of ligase mutants with rII mutations

A mutation of T4B phage, which enhances suppression of ligase mutants with rII mutations

Enhanced recovery of rII mutations in the absence of selection in a ligase-negative mutant of bacteriophage T 4

Mutation in a temperature-sensitive, polynucleotide ligase-defective mutant of bacteriophage T 4 was investigated by means of temperature shift-down experiments. The principal results are: ( I) Brief intracellular exposure to the restrictive temperature of cells singly infected by ligase-defective r + phages results in a marked increase in the recovery of rII phages among the few progeny released. ( 2) The distribution of these mutations within the rII cistrons differs significantly from the standard spontaneous distribution. ( 3) Selection of rII mutations by virtue of their ability to suppress the ligase defect cannot account for the observed increase.

Suppression of DNA-arrested synthesis in mutants defective in gene 59 of bacteriophage T4

Amber nonsense mutants of gene 59 ( amC5, amHL628) in bacteriophage T4 will cause an arrest of DNA synthesis in a nonpermissive host, Escherichia coli B. The arrested synthesis of DNA is rapidly suppressed by the addition of chloramphenicol at 5–12 min after infection. To be effective, chloramphenicol must be added at a specific time after infection, and suppression requires continuous presence of chloramphenicol for at least 30 min after infection. Extragenic mutations in either gene 55 or gene 33 also restored DNA synthesis of the gene 59 mutants to the normal level. The physicochemical characteristics of intracellular DNA obtained by alkaline sucrose gradient centrifugation of a double mutant ( amBL292- amC5) is strikingly similar to that obtained in amC5-infected cells treated with chloramphenicol. Since both gene 55 and 33 control the expression of late genomes, it is likely that at least one of the late proteins is involved in the phenotypic expression of arrested DNA synthesis, a conclusion consistent with the chloramphenicol results. We have compared the suppression of amC5 (gene 59) with the suppression of amHX39 which affects polynucleotide ligase (gene 30) and which in its mutant form causes DNA arrested synthesis. Unlike its effect on the polynucleotide ligase mutant, an extragenic rII mutation introduced into amC5 did not affect the arrest of DNA synthesis of amC5. However, the plating efficiency in E. coli B was greatly increased.

Enzymatic Breakage and Joining of Deoxyribonucleic Acid: VIII. HYBRIDS OF RIBO- AND DEOXYRIBONUCLEOTIDE HOMOPOLYMERS AS SUBSTRATES FOR POLYNUCLEOTIDE LIGASE OF BACTERIOPHAGE T4

Abstract Polynucleotide ligase of bacteriophage T4, in contrast to the DNA ligase of Escherichia coli, catalyzes the joining of oligomers of deoxyribonucleotides hydrogen-bonded to complementary strands of either polydeoxyribonucleotides or polyribonucleotides. Hybrid molecules in which oligomers of ribonucleotides are hydrogen-bonded to complementary strands of polydeoxyribonucleotides can also serve as substrates for the T4 enzyme. The rate of joining of oligomers in hybrid molecules of this type is only 1 to 2% of that observed when both strands are polydeoxyribonucleotides. No activity has been detected when both strands are polyribonucleotides. The products of these reactions have been characterized by sedimentation and end group analysis; in all cases joining occurs through phosphodiester bond formation. The activities toward hybrid molecules and toward DNA containing single strand breaks appear to be identical as indicated by: (a) identical ratio of the activities during a 1000-fold purification, (b) similar reaction requirements, and (c) identical thermosensitivity of both activities purified from cells infected with a mutant bacteriophage containing a temperature-sensitive ligase. It had been shown previously that the introduction of an rII mutation into phage T4 could suppress an amber mutation in the ligase gene. In these studies it is shown that the double mutant, T4rIIamH39X, carrying an amber mutation in the ligase gene, does not have detectable levels of T4 ligase in the nonpermissive host. Similarly, T4rIItsA80, carrying a temperature-sensitive mutation in the ligase gene, can grow at the nonpermissive temperature, despite retaining its temperature-sensitive ligase.