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Results for “DNA Mutational Analysis”

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At least 37 records · Page 2Linked to original sources
PubMed1991

Identification of a new haemophilia BM case produced by a mutation located at the carboxy terminal cleavage site of activation peptide.

We describe a novel point mutation due to C----G transversion at nucleotide 20518 in the exon VI of factor IX gene, resulting in the substitution of glycine (GGG) for arginine (CGG) at position 180 in the polypeptide. This point mutation was found in a patient with a haemophilia BM variant. We designated the altered factor IX produced by this new mutation as factor IXMadrid. This mutation blocks the cleavage site involved in the release of the activation peptide at Arg180-Val181. It also abolishes the Aval site (CTCGGG) in exon VI, which can be directly detected with the enzymatic DNA amplification technique (PCR) and offers the possibility of direct analysis in carrier and prenatal diagnosis in kindreds with this mutation.

Amino Acid Sequence↗
PubMed1992

Reverse transcriptase of human immunodeficiency virus type 1: functionality of subunits of the heterodimer in DNA synthesis.

From an in vitro analysis of the DNA-synthesizing abilities of certain specifically mutated forms of the heterodimeric reverse transcriptase of human immunodeficiency virus type 1, we can conclude that in a heterodimer, the functionality of p66 is necessary while the functionality of the p51 subunit is not needed. Conversely, p51 is not able to catalyze DNA synthesis when associated with p66, and yet when the p66 protein is absent, p51 can function. These conclusions applied to DNA synthesis on heteropolymeric RNA and DNA templates.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Spontaneous and ultraviolet-induced mutations on a single-stranded shuttle vector transfected into monkey cells.

The shuttle vector plasmid PCF3A, carrying the supF target gene, can be transfected into monkey COS7 cells as single-stranded or double-stranded DNA. Single strand-derived plasmid progeny exhibited a 10-fold higher spontaneous mutation frequency than double strand-derived progeny. The location of spontaneous mutations obtained after transfection of the single-stranded vector shared similarities with that for double-stranded vectors. However, the nature of base changes was very different. Single-stranded PCF3A DNA was used to study ultraviolet-induced mutagenesis. An earlier report (Madzak and Sarasin, J. Mol. Biol., 218 (1991) 667-673) showed that single-stranded DNA exhibited a lower survival and a higher mutation frequency than double-stranded DNA after ultraviolet irradiation. In the present report, sequence analysis of mutant plasmids is presented. The use of a single-stranded vector allowed us to show the targeting of mutations at putative lesion sites and to determine the exact nature of the base implicated in each mutation. Frameshift mutations were more frequent after transfection of control or irradiated plasmid as single-stranded DNA than as double-stranded DNA. Multiple mutations, observed at a high frequency in the spontaneous and ultraviolet-induced mutation spectra following single-stranded DNA transfection, could be due to an error-prone polymerisation step acting on a single-stranded template.

Animals↗
PubMed1992

[Leber's optic neuropathy: new diagnostic prospects].

The diagnosis of Leber's hereditary optic neuropathy, essentially based on a family history of a similar illness, becomes difficult in sporadic cases. In this disease, mitochondrial DNA mutations have been recently discovered and detected by digestion with restriction enzyme after amplification by the Polymerase Chain Reaction. We present two patients in whom these molecular biological techniques confirmed the diagnosis of Leber's hereditary optic neuropathy in the absence of a convincing family history. These new molecular biological methods, providing a precious diagnostic test, would certainly play a greater part in genetic counselling and would probably be a prerequisite for the development of new forms of therapy.

Adult↗
PubMed1992

Somatic mutation of the APC gene in gastric cancer: frequent mutations in very well differentiated adenocarcinoma and signet-ring cell carcinoma.

We searched for somatic mutations of the adenomatous polyposis coli (APC) gene in DNA samples isolated from 57 sporadic gastric cancers, by means of a ribonuclease (RNase) protection analysis coupled with DNA amplification by the polymerase chain reaction (PCR). Examining 30% of the APC coding region, including a region where somatic mutations in colorectal tumors are known to be clustered, we detected somatic mutations in 12 tumors; seven in 17 very well differentiated adenocarcinomas, two in 19 well or moderately differentiated adenocarcinomas, and three in ten signet-ring cell carcinomas. So far, no somatic mutations have been identified in 11 poorly differentiated adenocarcinomas. Eight of the 17 somatic mutations found in 12 tumors caused truncation of the gene product due to a nonsense mutation and a 1-, 2- or 5-bp deletion; nine others were point mutations that altered amino acids. Our results suggest that inactivation of APC plays a role in development of some gastric cancers, particularly very well differentiated adenocarcinomas and signet-ring cell carcinomas.

Adenocarcinoma↗
PubMed1992

Crude isolation of DNA from unselected human pancreatic tissue and amplification by the polymerase chain reaction of Ki-ras oncogene to detect point mutations in pancreatic cancer.

Human pancreatic tissue obtained during surgery was divided into two samples, one stored in formalin for histology and the other frozen directly for DNA studies. A small bit of the frozen tissue was excised without prior differentiation of malignant from non-malignant tissue and crude DNA prepared. The DNA was used to amplify the Ki-ras oncogenes with the polymerase chain reaction (PCR) using primers for codons 12, 13 and 61. The PCR products were hybridized with oligonucleotides to detect mutations in these codons. No mutations were seen in patients histologically free from pancreatic cancer, having cancer of the papilla of Vater or endocrine pancreatic cancer. In the 9 cases of pancreatic cancer, point mutations were detected in codon 12 in 7 cases and in codon 13 in one patient. Thus, the PCR technique can be used to detect point mutations in Ki-ras oncogenes even in crude DNA without selection of malignant from non-malignant tissue.

Codon↗
PubMedDate not supplied

Detection of single-base mutations in a mixed population of cells: a comparison of SSCP and direct sequencing.

Single-base-pair changes in several genes have been shown to be important in carcinogenesis. We have compared the sensitivity of two commonly used techniques for detection of single-base-pair changes. Defined populations of cells were prepared by mixing wild-type Chinese hamster ovary cells with cells carrying a known mutation in the CTP synthetase gene. RNA was extracted and analyzed for the mutation by single-strand conformational polymorphism and direct sequence analysis of polymerase chain reaction products. We found that both techniques were able to detect the mutation when it was present in 25% of the cells, but that direct sequencing was slightly more sensitive and able to detect the mutation when it was present in only 20% of the population.

Animals↗
PubMed1991

New mutant gene (transthyretin Arg 58) in cases with hereditary polyneuropathy detected by non-isotope method of single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) was analyzed to detect a mutation in the transthyretin (TTR) gene from the mother and son showing polyneuropathy with carpal tunnel syndrome. DNA segments containing TTR coding sequence were amplified by polymerase chain reaction, heat denatured and electrophoresed on a neutral polyacrylamide gel. The single-stranded DNA fragments in the gel were transferred to a nylon membrane and hybridized with biotinylated TTR cDNA probe, followed with chemiluminescent DNA detection. The mobility shift was found in the fragments of exon 3 from the patients' DNA. Sequencing analyses of the exon 3 confirmed a T----G base change, resulting in a Leu 58----Arg substitution. TTR Arg 58 is the first mutant TTR gene that has been detected by SSCP analysis. The rapid and sensitive detection of new mutations at various sites on the TTR gene is hereafter possible by the present method in the facilities for non-radioactive experiments.

Adult↗
PubMed1992

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene.

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene: ts17, ts24, ts69 (WR strain) and ts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed with ts17, and incorporation of 3H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5 orf by marker rescue, and the single nucleotide changes responsible for the ts phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (< 2 kb), intragenic DNA fragments at 39.5 degrees C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.

Alleles↗
PubMed1991

Transient expression of deletion mutants of the herpes simplex virus thymidine kinase-encoding gene in mouse fibroblast cells.

Previous studies have shown that at least three polypeptides of 43, 39 and 38 kDa are translated from separate AUG codons of the thymidine kinase (TK) encoding mRNA of herpes simplex virus type 1. In addition, small tk-specific transcripts initiated within the tk coding region were observed. However, functional activity of these three proteins and their role in establishing of the TK+ cell phenotype is not yet clear. In order to locate the 5' boundary of the gene encoding functionally active TK, we constructed a set of deletion mutants with truncated 5' ends and examined their ability to provide a TK+ phenotype after microinjection into nuclei of LTK- cells. The results demonstrate that nucleotide sequences upstream from the second ATG codon can be removed without affecting the TK+ phenotype. Deletion of the second start codon and its downstream region inactivates the TK function. Those deletion mutants which contain only the third ATG codon are TK-. Thus, the 38-kDa polypeptide that initiates at the third start codon is not endowed with the TK+ activity. Constructs containing deletions up to nt +210 and lacking all 5'-end canonical and aberrant transcription control regions, as well as first start codon, can provide the TK+ function.

Animals↗
PubMed1992

Identification of splicing mutations of the last nucleotides of exons, a nonsense mutation, and a missense mutation of the XPAC gene as causes of group A xeroderma pigmentosum.

Four mutations of the XPAC gene were identified as molecular bases of different UV-sensitive subgroups of xeroderma pigmentosum (XP) group A. One was a G to C transversion at the last nucleotide of exon 4 in GM1630/GM2062, a little less hypersensitive subgroup than the most sensitive XP2OS/XP12RO. The second mutation was a G to A transition at the last nucleotide of exon 3 in GM2033/GM2090, an intermediate subgroup. Both mutations caused almost complete inactivation of the canonical 5' splice donor site and aberrant RNA splicing. The third mutation was a nucleotide transition altering the Arg-211 codon (CGA) to a nonsense codon (TGA) in another allele of GM2062. The fourth mutation was a nucleotide transversion altering the His-244 codon (CAT) to an Arg codon (CGT) in XP8LO, an intermediate subgroup. Our results strongly suggest that the clinical heterogeneity in XP-A is due to different mutations in the XPAC gene.

Adolescent↗
PubMed1992

Oxygen sensitivity of an Escherichia coli mutant.

Genetic evidence indicates that Oxys-6, an oxygen-sensitive mutant of Escherichia coli AB1157, is defective in the region of the hemB locus. Oxys-6 is capable of growth under aerobic conditions only if cultures are initiated at low-inoculum levels. Aerobic liquid cultures are limited to a cell density of 10(7) cells per ml by the accumulation of a metabolically produced, low-molecular-weight, heat-stable material in complex organic media. Both Oxys-6 and AB1157 cells produce the material, but only aerobic cultures of the mutant are inhibited by it. The material is produced by both intact cells and cell extracts in complex media. This reaction also occurs when the amino acid L-lysine is substituted for complex media.

Cloning, Molecular↗
PubMed1992

Genetic alterations in the 61st codon of the H-ras oncogene isolated from archival sections of hepatic hyperplasias, adenomas and carcinomas in control groups of B6C3F1 mouse bioassay studies conducted from 1979 to 1986.

In order to better understand the molecular events in murine hepatocarcinogenesis, the frequency and types of mutations in the murine H-ras proto-oncogene isolated from 184 independent, spontaneously occurring hepatic lesions were determined. Hepatocellular foci, hyperplasias, adenomas and carcinomas were obtained from archival samples of control male (134 samples) and female (50 samples) B6C3F1 mice used in oncogenicity studies that were conducted at Lilly Research Laboratories from 1979 to 1986. The 61st codon region of the H-ras oncogene from these sections was amplified using the polymerase chain reaction. Mutation frequencies were determined by restriction fragment length polymorphism analysis. The types of mutations were characterized by allele-specific oligonucleotide hybridization and confirmed by DNA sequencing. Forty-two per cent of the carcinomas, 44% of the adenomas, 42% of the hyperplasias and 29% of the foci contained mutations at the 61 codon. The mutation spectra for the carcinomas, adenomas and hyperplasias consisted of mostly CAA-AAA transversions, followed by CAA-CGA transitions, followed by CAA-CTA transversions. These results demonstrate that: (i) the frequency of spontaneous mutations in the H-ras 61st codon is equivalent in murine hyperplasias, adenomas and carcinomas, and (ii) sex was not a determining factor in either the mutation frequency or mutation spectrum for the spontaneous lesions. If these lesions represent successive stages in the carcinogenic process, then these results suggest that mutations in the 61st codon of H-ras are early events in spontaneous murine hepatocarcinogenesis.

Adenoma↗
PubMed1992

No genetic differences between affected and unaffected members of a German family with Leber's hereditary optic neuropathy (LHON) with respect to ten mtDNA point mutations associated with LHON.

In order to investigate possible synergistic influences of different mtDNA mutations on penetrance and severity of Leber's hereditary optic neuropathy (LHON), a large German LHON pedigree is characterized with respect to 10 different mutations associated with LHON. All members of the family carry three different mtDNA mutations (at nucleotide 4,216, 11,778 and 13,708) in a homoplasmic form, regardless of whether or not they are clinically affected. Testing for another 7 mutations reveals negative results in all family members. Hence, the variable disease expression of the family members cannot be explained by varying combinations of LHON-associated mtDNA mutations.

Base Sequence↗
PubMed1992

Rapid identification of mutations in the glucocerebrosidase gene of Gaucher disease patients by analysis of single-strand conformation polymorphisms.

To detect mutations in the glucocerebrosidase gene in Gaucher disease patients, we used the recently described technique of single-strand conformation polymorphism (SSCP) analysis in combination with selective amplification. We analyzed exon 8, 9, 10 and 11 of the glucocerebrosidase gene; these exons were sequentially amplified using the selectively amplified products as templates. We found variant SSCP patterns corresponding to the presence or absence of the 6433C mutation, which was detected by NciI digestion analysis, in exon 10. Furthermore, we detected four variant SSCP patterns in exon 8, 10 and 11. Sequencing analysis consistently revealed four single-base substitutions in the corresponding exons, three novel missense mutations (5409A, 6375G and 6682T) and one silent polymorphism (6594A). These mutations were found only in one patient; therefore, these findings have confirmed the marked genetic heterogeneity of Gaucher disease. SSCP analysis in combination with selective amplification is a rapid and sensitive procedure for the screening of the mutations in the glucocerebrosidase gene of patients with Gaucher disease.

Base Sequence↗
PubMed1992

Mutational analysis of a variant of ARS1 from Saccharomyces cerevisiae.

A naturally occurring single base-pair G to A transition, creating a 10/11 near-match close to the essential 11 base-pair core consensus of ARS1, was used to investigate the importance of near-match sequences. The 10/11 near-match can not substitute for the core consensus since an ARS- phenotype is observed when the core consensus is deleted. However, deletion mutations revealed that this near-match together with a short palindromic sequence, also situated in the B-flanking region, comprise a single element crucial for optimal ARS function. The palindrome has the potential of forming a stem-loop structure. Rather precise observations concerning the borders of the B-region were achieved. The four base pairs separating the near-match from the core consensus perform a spacing function where the identity of the bases are unimportant. However, this spacing is highly important since deletion of these four base pairs leads to an ARS- phenotype.

Base Sequence↗