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PubMed1996

[Increased promoter activity of human delta-globin gene with point mutation of C-T at site-64].

With PCR point mutation technique, the CCAAC box in the promoter region of delta-globin gene was changed into CCAAT which is required in the normal transcription of beta-globin gene at the corresponding site. The normal and mutant segments of the delta-globin promoter region obtained with each segment of about 300 bp (-280/+54 bp), a fragment sufficient for the requirement of its normal promoter function, were separately cloned into pUC19/Hinc II. After sequencing, it showed that the CCAAC box had been successfully changed into CCAAT without other mutations. The two segments were released by Nco I and BamH I, and were cloned into the corresponding site of plasmid pMG3 which has the firefly luciferase as its reporter gene. The positive clones were selected by Dot Blot and were comfirmed with restriction mapping. Then the two recombinant plasmids were transfected into HeLa cell line respectively and the promoter activities of the two fragments were identified by comparing the light intensity generated by the cell extracts when added with luciferase assay substrate. The results demonstrated that the luminescence of the mutant group is on average five times higher than that of the normal group, which indicate that the C-T mutation of the CCAAC box of delta-globin promoter region could increase its promoter activity. Our results have confirmed the hypothesis that the sequence of CCAAC box of human delta-globin gene is one of the main reasons which accounts for its low expression level. The author is now studying the specific DNA binding protein of the upstream promoter region of human delta-globin gene.

Globins↗
PubMed2001

Point mutation of neu oncogene in animal peripheral nerve sheath tumors.

Thirty-four peripheral nerve sheath tumors of four domesticated animal species were characterized and assayed for point mutation of the neu oncogene. Based on their morphoimmunophenotype, 32 tumors were classified as schwannomas. Schwannoma morphology was characterized by the presence of Antoni type A and B pattern and immunoreactivity for S-100 protein and vimentin. Two anaplastic and metastatic tumors originating from spinal cord root, immunonegative for S-100 protein and positive for vimentin, were classified as malignant peripheral nerve sheath tumors (MPNSTs). Four malignant schwannomas and two MPNSTs expressed a point mutation of the neu oncogene by the polymerase chain reaction-restriction fragment length polymorphism method. The finding of neu oncogene mutation could be a useful diagnostic genetic marker in the malignant form of peripheral nerve sheath tumors in animals.

Animal Diseases↗
PubMed1989

Oncogenic point mutations in the human retinoblastoma gene: their application to genetic counseling.

Mutations of the retinoblastoma gene, most of which cannot be detected by conventional Southern blotting, are known to cause both the nonhereditary and hereditary forms of retinoblastoma and have been implicated in the development of other cancers. Nonhereditary retinoblastoma is caused by a somatic mutation. Hereditary retinoblastoma is caused by a germ-cell mutation, most often a new one, and thus there is usually no family history of the disease. Unlike patients with the nonhereditary disease, those with the hereditary form are at risk for additional retinoblastomas, and their progeny are at risk for the tumors. We used a sensitive technique of primer-directed enzymatic amplification, followed by DNA sequence analysis, to identify mutations as small as a single nucleotide change in tumors from seven patients with simplex retinoblastoma (with no family history of the disease). In four patients the mutation involved only the tumor cells, and in three it involved normal somatic cells as well as tumor cells but was not found in either parent; thus, these mutations appeared to be new, germ-cell mutations. In addition, we found point mutations in cells from a bladder carcinoma, a small-cell carcinoma of the lung, and another retinoblastoma. We conclude that the technique that we have described can distinguish hereditary from nonhereditary retinoblastoma and that it is useful in risk estimation and genetic counseling.

Base Sequence↗
PubMed1994

Point mutations of the connexin32 (GJB1) gene in X-linked dominant Charcot-Marie-Tooth neuropathy.

Ten families with X-linked dominant CMT neuropathy (CMTX1) were screened for point mutations of the connexin32 (Cx32, GJB1) gene. Two families showed missense mutations, respectively an A-->G transition at amino acid 102 (glutamate to glycine) and a C-->T transition at amino acid 142 (arginine to tryptophan). Three families showed nonsense mutations, respectively a C-->T transition at amino acid 22 (arginine to stop) a G-->T transversion at amino acid 186 (glutamate to stop), and a T-->A transversion at amino acid 217 (cysteine to stop). Five CMTX1 neuropathy families showed no evidence of point mutations of the connexin32 coding sequence. These findings suggest that the CMTX1 neuropathy genotype is heterogeneous or the result of promoter mutations, 3'-untranslated region mutations or exon/intron splice site mutations. Four of the reported mutations created or destroyed restriction enzyme sites: a HaeIII restriction enzyme site was destroyed by the mutation at amino acid position 22, a HpaII site was eliminated at amino acid position 142, a Bfal restriction site was created by the mutation at amino acid 186 and a Ddel restriction site was created by the mutation at amino acid 217. These changes allowed us to test family members for the mutations and observe the segregation of the disease with the mutations.

Amino Acid Sequence↗
PubMed1997

Abnormal glycosylation of procathepsin L due to N-terminal point mutations correlates with failure to sort to lysosomes.

A single point mutation in the lysosomal proenzyme receptor-inhibiting sequence near the N terminus of mouse procathepsin L can result in glycosylation of a normally cryptic site near its C terminus. When alanine replaced His36, Arg38, or Tyr40, the nascent chain of the mutant protein cotranslationally acquired a high mannose oligosaccharide chain at Asn268. In contrast, when alanine replaced Ser34, Arg37, or Leu39, this second carbohydrate chain was not added. This alternating pattern of abnormal glycosylation suggested that propeptide residues 36-40 normally assume an extended conformation having the side chains of residues 36, 38, and 40 facing in the same direction. When tyrosine conservatively replaced His36 or lysine replaced Arg38, Asn268 was not glycosylated. But the procathepsin L mutant having phenylalanine in place of Tyr40 was glycosylated at Asn268, which indicates that the hydrogen bond between the hydroxyl group of Tyr40 and the carboxylate group of Asp82 is necessary for normal folding of the nascent proenzyme chain. Mutation of the adjacent alpha2p (ERININ) helix of the propeptide or addition of a C-terminal epitope tag sequence to procathepsin L also induced misfolding of the proenzyme, as indicated by addition of the second oligosaccharide chain. In contrast, the propeptide mutation KAKK99-102AAAA had no effect on carbohydrate modification even though it reduced the positive charge of the proenzyme. Misfolded mutant mouse procathepsin L was not efficiently targeted to lysosomes on expression in human HeLa cells, even though it acquired phosphate on mannose residues. The majority of the mutant protein was secreted after undergoing modification with complex sugars. Similarly, epitope-tagged mouse procathepsin L was not targeted to lysosomes in homologous mouse cells but was efficiently secreted. Since production of mature endogenous protease was not reduced in cells expressing the tagged protein, the tagged protein did not compete with endogenous procathepsin L for targeting to lysosomes.

3T3 Cells↗
PubMed2002

Clonally expanded mtDNA point mutations are abundant in individual cells of human tissues.

Using single-cell sequence analysis, we discovered that a high proportion of cells in tissues as diverse as buccal epithelium and heart muscle contain high proportions of clonal mutant mtDNA expanded from single initial mutant mtDNA molecules. We demonstrate that intracellular clonal expansion of somatic point mutations is a common event in normal human tissues. This finding implies efficient homogenization of mitochondrial genomes within individual cells. Significant qualitative differences observed between the spectra of clonally expanded mutations in proliferating epithelial cells and postmitotic cardiomyocytes suggest, however, that either the processes generating these mutations or mechanisms driving them to homoplasmy are likely to be fundamentally different between the two tissues. Furthermore, the ability of somatic mtDNA mutations to expand (required for their phenotypic expression), as well as their apparently high incidence, reinforces the possibility that these mutations may be involved actively in various physiological processes such as aging and degenerative disease. The abundance of clonally expanded point mutations in individual cells of normal tissues also suggests that the recently discovered accumulation of mtDNA mutations in tumors may be explained by processes that are similar or identical to those operating in the normal tissue.

Adult↗
PubMed1990

MNU-induced point mutation and activation of c-Ha-ras oncogene in cell lines derived from human pancreas explants.

Point mutation and activation of c-Ha-ras oncogene was studied at various stages of carcinogenesis in cell lines developed from MNU-treated human pancreas explants. DNAs from normal pancreas and nontumorigenic cell lines showed no transforming activity in NIH 3T3 cells whereas DNA from one of the tumorigenic cell lines transformed NIH 3T3 cells. In this cell line the point mutation was demonstrated to be at codon 12 of c-Ha-ras gene by the loss of an Msp I site. The mutation possibly affected the transcription of c-Ha-ras gene which in turn contributed to the transformation of these cells.

Blotting, Southern↗
PubMed2001

Point mutations of the RUNx1/AML1 gene in sporadic and familial myeloid leukemias.

The RUNX1/AML1 gene is known to be the most frequent target for chromosomal translocation in leukemia. In addition, recent studies have demonstrated point mutations in the RUNX1 gene as an another mode of genetic lesion resulting in leukemia. Of particular interest, sporadic point mutations of biallelic type are found in a tight association with either the acute myelogenous leukemia (AML) MO subtype or trisomy 21. Germline mutations give rise to a familial platelet disorder that results in a predisposition to acute myelogenous leukemia (FPD/AML). Most of the RUNX1 mutants were defective in DNA binding but still active in beta binding, a characteristic that is consistent with the 3-dimensional structural findings and may explain the dominant inhibitory effects. Although genuine haploinsufficiency of RUNX1 was observed in some cases, a greater majority of mutant RUNX1 proteins may also act in a dominant-negative manner, possibly creating a higher propensity for leukemia development. The stronger dominant-negative effect was also deduced to be the major mechanism of the chimeric genes created by chromosomal translocations. The decrement of RUNXI activity may be a common underlying cause for RUNX1-related leukemias. However, because these RUNX1 abnormalities per se are insufficient for leukemogenesis, cooperating genetic alteration(s) should be intensively sought for further mechanistic insights and future clinical applications.

Animals↗
PubMed2006

Heterogeneous expression of hydrocephalic phenotype in the hyh mice carrying a point mutation in alpha-SNAP.

The hyh mouse carrying a point mutation in the gene encoding for soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein alpha (alpha-SNAP) develops inherited hydrocephalus. The investigation was designed to study: (i) the clinical evolution of hyh mice; (ii) factors other than the alpha-SNAP mutation that may influence the expression of hydrocephalus; (iii) the neuropathological features underlying the different forms of clinical evolution. The study included 3017 mice, 22.4% of which were hydrocephalic. The neuropathological study was performed in 112 mice by use of light and electron microscopy. It was found that maternal- and sex-related factors are involved in the heterogeneous expression of hyh phenotype. The clinical evolution recorded throughout a 4-year period also revealed a heterogeneous expression of the hydrocephalic phenotype. Two subpopulations were distinguished: (i) 70% of mice underwent a rapidly progressive hydrocephalus and died during the first 2 months of life; they presented macrocephaly, extremely large expansion of the ventricles, equilibrium impairment and decreased motor activity. (ii) Mice with slowly progressive hydrocephalus (30%) survived for periods ranging between 2 months and 2 years. They had no or moderate macrocephaly; moderate ventricular dilatation and preserved general motor activity; they all presented spontaneous ventriculostomies communicating the ventricles with the subarachnoid space, indicating that such communications play a key role in the long survival of these mice. The hyh mutant represents an ideal animal model to investigate how do the brain "adapt" to a virtually life-lasting hydrocephalus.

Animals↗
PubMed1989

Osteogenesis imperfecta type IV. Detection of a point mutation in one alpha 1(I) collagen allele (COL1A1) by RNA/RNA hybrid analysis.

We have identified a point mutation in one alpha 1(I) collagen allele (COL1A1) of a child with the type IV osteogenesis imperfecta phenotype. When compared to parental and control samples, skin fibroblasts of the proband synthesized two populations of type I collagen molecules. One population was normal; the other was delayed in secretion and electrophoretic migration due to post-translational overmodification. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated a gradient of overmodification beginning near the carboxyl-terminal CB peptides. This predicts that the mutation delaying helix formation is near the carboxyl-terminal end of one of the component chains of type I collagen. The mRNA of the patient was probed with overlapping antisense riboprobes to type I collagen cDNA. Cleavage of a mismatch in RNA/RNA hybrids of RNase A allowed the location of the mutation to a 225-base pair region of alpha 1(I) cDNA. The mismatch was not present in RNA/RNA hybrids from either parent. This region of both alpha 1(I) alleles of the patient was isolated by screening a lambda ZAP cDNA library. Sequence determination of both alleles demonstrated a single nucleotide change, G----A, resulting in the substitution of a serine for a glycine at amino acid residue 832. This point mutation occurs in the coding region for alpha 1(I) CB6 and is concordant with the protein data. The finding of a glycine substitution in an alpha 1(I) chain of a patient with the milder type IV osteogenesis imperfecta phenotype requires modification of current molecular models for types II and IV osteogenesis imperfecta.

Alleles↗
PubMed1988

Point mutations and small substitution mutations in three different upstream elements inhibit the activity of the mouse alpha 2(I) collagen promoter.

Point mutations and small substitution mutations were introduced in three different promoter segments of the mouse alpha 2(I) collagen gene that are binding sites for specific DNA-binding proteins. The first of these promoter elements contains a CCAAT motif between -80 and -84 and binds a novel factor consisting of two different components which are both needed for DNA binding (Hatamochi, A., Golumbek, P.T., Van Schaftingen, E., and de Crombrugghe, B. (1988) J. Biol. Chem. 263, 5940-5947). Four different point mutations in the CCAAT motif, which strongly inhibit binding of this heterodimeric CCAAT-binding factor, decrease the activity of the promoter at least 8-fold when assayed by DNA transfection of NIH 3T3 fibroblasts. The second cis-acting element is located around -250. Two mutations in this element, one a 3-base pair (bp), another a 4-bp substitution mutation, decrease the activity of the promoter in DNA transfection experiments by about 8- and 12-fold, respectively. The third element is located between -315 and -295 and binds nuclear factor 1 (NF1). Both 1- and 2-bp mutations which strongly inhibit binding of NF1 to this site decrease the activity of the promoter 8-10-fold in DNA transfection assays whereas another mutation in this site shows less inhibition in the activity of the promoter. Transfer of this NF1-binding site 5' to the SV40 early promoter increases the activity of this promoter more than 10-fold. These results indicate that the integrity of each of these three cis-acting elements is required for optimal activity of the alpha 2(I) collagen promoter and suggest a model whereby the factors which bind to these sequences cooperate in the formation of a transcription initiation complex.

Animals↗
PubMed1992

Specific point mutations that activate v-abl are not found in Philadelphia-negative chronic myeloid leukaemia, Philadelphia-negative acute lymphoblastic leukaemia or blast transformation of chronic myeloid leukaemia.

The involvement of the BCRlABL fusion gene in patients with Philadelphia (Ph) chromosome positive chronic myeloid leukaemia (CML) and acute lymphoblastic leukaemia (ALL) is well characterised, but the molecular events underlying the cases of Ph-negative CML and ALL that lack BCR gene involvement and those that cause transformation of Ph-positive CML are unknown. The murine ABL gene can be activated by genetic events that do not involve the BCR gene, including the introduction of two specific point mutations in exons VII and XI respectively, as found in the homologous sequence of the v-abl oncogene. We therefore sought evidence for analogous point mutations in the ABL gene in patients with Ph-negative, BCR-negative CML (n = 25), Ph-negative ALL (n = 18) and in Ph-positive CML in transformation (n = 28). We used restriction fragment length polymorphism and single strand conformational polymorphism techniques to analyse DNA amplified fragments of selected ABL coding regions from leukaemia cells. We identified only normal wild-type DNA sequences. The absence of these transforming point mutations does not exclude the possibility that the ABL gene in such patients could be activated by other means.

Adult↗
PubMed1988

An apparent deletion of an oligonucleotide detected by RNA fingerprint in the nondiabetogenic B variant of encephalomyocarditis virus is caused by a point mutation.

The diabetogenic D variant of encephalomyocarditis virus (EMC-D) was previously shown to be different from the nondiabetogenic B variant of encephalomyocarditis virus (EMC-B) by a single spot in an oligonucleotide fingerprint after RNase T1 digestion of their genomic RNAs. An oligoribonucleotide was missing from EMC-B but was present in EMC-D. The oligoribonucleotide specific to EMC-D was isolated from a two-dimensional polyacrylamide gel and sequenced as 5'-ACAAUCUCACUUUUCCAACAACAG-3'. Molecular hybridizations of EMC-D and EMC-B genomic RNAs with a DNA primer complementary to the EMC-D-specific oligoribonucleotide revealed that the absence of a corresponding spot in EMC-B was due to a point mutation rather than a deletion. By sequencing a cloned cDNA of EMC-B corresponding to the EMC-D-specific oligoribonucleotide, the point mutation was identified as a G for EMC-B and an A for EMC-D transversion at base 9 of the oligonucleotide. Comparative sequence analysis of eight randomly picked RNA segments around the EMC-D-specific oligoribonucleotide revealed that there were no base changes between EMC-D and EMC-B. It is concluded that the diabetogenic EMC-D viral genome differs from the nondiabetogenic EMC-B viral genome by at least a point mutation.

Animals↗
PubMed1984

A novel combination of K-ras and myc amplification accompanied by point mutational activation of K-ras in a human lung cancer.

Amplifications of two oncogenes, c-K-ras-2 and c-myc, were found in a human lung giant cell carcinoma (LGCC) Lu-65, which is maintained in nude mice. The extent of c-K-ras-2 and myc amplifications were estimated to be 10- and 8-fold, respectively, by means of the Southern hybridization procedure. In addition, NIH3T3 cells were transformed by transfection of Lu-65 DNA and the transforming gene was identified as c-K-ras-2. c-K-ras-2 genes were cloned from a gene library of Lu-65 and a single point mutation causing a substitution of cysteine for glycine in codon 12 was found by DNA sequencing. It was concluded that the amplification of the c-myc and c-K-ras-2 genes are accompanied by point mutational activation of c-K-ras-2 in the human LGCC Lu-65. This is the first report of multiple gene amplification accompanied by a point mutation of oncogenes in human cancer cells, providing further support for the idea that co-operation of at least two activated cellular oncogenes is required for carcinogenesis.

Amino Acid Sequence↗
PubMed1991

Targeted introduction of a diphtheria toxin-resistant point mutation into the chromosomal EF-2 locus by in vivo homologous recombination.

We have introduced a specific point mutation into the gene for chromosomal elongation factor 2 (EF-2) in Chinese hamster ovary cells (CHO-K1) by in vivo homologous recombination. To obtain a selectable construct for gene-targeting, we modified a diphtheria toxin-resistant mutant EF-2 gene (Gly717 to Arg) by deleting its promoter and first exon so that homologous recombinants could be distinguished from randomly integrated transformants, and also by inserting a second positive selection marker, the neomycin-resistance gene, into the 3'-flanking region to increase the selective accuracy. More than 80% of the clones surviving after selection for resistance to both the toxin and neomycin were the desired homologous recombinants in which the wild-type, toxin-sensitive EF-2 gene was replaced by the modified gene giving resistance to both the toxin and neomycin. This result shows that the specific point mutation was co-introduced with a second selective marker into an endogenous chromosomal gene and that the modified gene was expressed.

Animals↗
PubMed1996

The multiple endocrine neoplasia type 2B point mutation alters long-term regulation and enhances the transforming capacity of the epidermal growth factor receptor.

The RET proto-oncogene encodes a member of the receptor tyrosine kinase family. Multiple endocrine neoplasia type 2B (MEN 2B) is caused by the mutation of a conserved methionine to a threonine in the catalytic domain of the RET kinase. When the MEN 2B point mutation was introduced into the epidermal growth factor (EGF) receptor (M857T EGFR), the intrinsic tyrosine kinase activity of the mutant receptor was similar to that of wild-type EGF receptor and remained ligand-dependent. However, the mutant receptor showed an enhanced transforming capacity compared to the wild-type receptor as judged by its ability to mediate the growth of NIH 3T3 cells in soft agar. Using the oriented peptide library approach to examine substrate specificity, the M857T mutation was found to be associated with a decrease in the selectivity of the receptor for Phe and an increase in the selectivity for acidic residues at the P + 1 position as compared to wild-type EGF receptor. Short-term responses to EGF were similar in cells expressing wild-type and M857T EGF receptors. However, significant differences in receptor down-regulation were observed between the two receptors. These data demonstrate that the MEN 2B point mutation alters the substrate specificity of receptor tyrosine kinases and suggest that the enhanced oncogenesis associated with the MEN 2B mutation may be due in part to alterations in receptor regulation.

3T3 Cells↗
PubMed2003

Five novel and four recurrent point mutations in the antithrombin gene causing venous thrombosis.

We analyzed the antithrombin (AT) gene in 9 unrelated Japanese patients with thrombotic disease. All 7 exons, the splice junctions, and the 5'-flanking region of the AT gene were amplified by polymerase chain reaction and sequenced directly. Nine different point mutations, all in the heterozygous state, were identified. Five novel (M-32T, M89K, L146H, Q159X, and L409P) and 2 previously reported (R132X and R359X) point mutations were identified in patients with type 1 deficiency. Two different missense mutations, R393C and R393H, located in the protease reactive site were detected in patients with type 2 deficiency. No other sequence abnormalities in the AT gene were detected by direct sequencing. None of the mutations was present in 100 alleles from 50 unrelated Japanese control subjects Although type 1 deficiency was diagnosed in patient 7 on the basis of approximately 50% AT antigen and activity levels, the data indicated that the novel L409P mutation is a type 2 pleiotropic effects (PE) deficiency because its location in the C-terminal portion of the reactive site is similar to the locations of reported PE type mutations, and it is highly conserved among other serpins.

Adolescent↗
PubMed2006

Multiple cryptic splice sites can be activated by IDS point mutations generating misspliced transcripts.

Mutations in the gene encoding the enzyme iduronate-2-sulfatase (IDS) were reported as the cause of the X-linked recessive lysosomal disease, mucopolysaccharidosis II (MPS II). Amongst the different mutations, it emerges that nearly 10% are nucleotide substitutions causing splicing mutations. We now report the molecular characterisation of three MPS II patients with multiple aberrant transcripts due to three different point mutations. The c.418+1G>C that occurred in the invariant splice-site motif, produced only aberrantly spliced transcripts. Whilst the mutations affecting variant motifs (c.419G>T) or coding regions (c.245C>T) led to aberrantly spliced transcripts in addition to correctly spliced transcripts with the respective predicted missense mutation, p.G140V or p.A82V. A combination of experimental tests and computational approaches were used to understand the molecular basis underlying the altered transcription patterns. In addition, by using real-time reverse transcriptase polymerase chain reaction, the reduction of mRNA amount in two patients observed was likely due to nonsense-mediated mRNA decay pathway. Overall, our results further emphasised the importance of cloning and sequencing independent transcripts to reveal less abundant, aberrant products, which often could not be detected by direct sequencing. Moreover, the different splicing patterns observed in the three patients as a consequence of point mutations show how sensitive the balance is between constitutive and cryptic splice sites in the IDS gene. The generation of such diverse transcripts, together with their level of expression, could contribute to the profound phenotypic variability reported in MPS II.

Adolescent↗