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PubMed1978

Structural comparison of glycophorins and immunochemical analysis of genetic variants.

Differences in amino acid sequence of erythrocyte membrane glycophorin A are correlated with M or N blood group activity. A second sialoglycoprotein, glycophorin B, has an amino acid sequence identical to that of glycophorin AN in the first 23 positions and carries N activity only, suggesting that different structural genes code for the glycoproteins carrying these antigens. Certain genetically variant cells lack glycophorin A, as determined by immunochemical methods, and serological MN activity. Other variants lack MN activity, but contain normal amounts of glycophorin A in the membrane.

Amino Acid Sequence↗

PubMed1985

Isolation and sequence of a human cytochrome P-450 cDNA clone.

A previously reported cDNA clone [pP450(1)] coding for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.), was used as a specific hybridization probe to screen a human liver cDNA library. Restriction mapping showed that two of the colonies isolated contained plasmids coding for overlapping regions of the same cDNA sequence. The clone [pHP450(1)] having the longer cDNA insert (1.25 kilobase pairs) was sequenced. The homology between the rat and human cDNAs is 62% in their coding regions but is only random (24%) in the 3'-noncoding nucleotides. The amino acid sequence deduced from the human cDNA is 50% identical to that of P-450e(U.C.). The homology increases to 72% if conservative changes in amino acid residues are permitted. The hydropathy profile of the polypeptide encoded by pHP450(1) is almost identical to that of P-450e(U.C.). Regions known to be highly conserved in cytochrome P-450 isozymes isolated from rat, rabbit, and mouse were found to be conserved in the amino acid sequence derived from pHP450(1). Analysis by Southern blotting indicated that the human cytochrome P-450 encoded by pHP450(1) is part of a multigene family.

Amino Acid Sequence↗

PubMed1994

Novel variants of the 5S rRNA genes in Eruca sativa.

The 5S ribosomal RNA (rRNA) genes of Eruca sativa were cloned and characterized. They are organized into clusters of tandemly repeated units. Each repeat unit consists of a 119-bp coding region followed by a noncoding spacer region that separates it from the coding region of the next repeat unit. Our study reports novel gene variants of the 5S rRNA genes in plants. Two families of the 5S rDNA, the 0.5-kb size family and the 1-kb size family, coexist in the E. sativa genome. The 0.5-kb size family consists of the 5S rRNA genes (S4) that have coding regions similar to those of other reported plant 5S rDNA sequences, whereas the 1-kb size family consists of the 5S rRNA gene variants (S1) that exist as 1-kb BamHI tandem repeats. S1 is made up of two variant units (V1 and V2) of 5S rDNA where the BamHI site between the two units is mutated. Sequence heterogeneity among S4, V1, and V2 units exists throughout the sequence and is not limited to the noncoding spacer region only. The coding regions of V1 and V2 show approximately 20% dissimilarity to the coding regions of S4 and other reported plant 5S rDNA sequences. Such a large variation in the coding regions of the 5S rDNA units within the same plant species has been observed for the first time. Restriction site variation is observed between the two size classes of 5S rDNA in E. sativa.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1991

Dissecting the activating mutations in v-erbB of avian erythroblastosis virus strain R.

The v-erbB oncogene isolated from the R (or ES4) strain of avian erythroblastosis virus is capable of inducing erythroleukemia and fibrosarcomas. This oncogene differs from the proto-oncogene c-erbB, the avian homolog of the epidermal growth factor receptor, by its lack of an intact ligand-binding domain as well as additional alterations in its cytoplasmic coding sequences. By contrast, the insertionally activated c-erbB, a variant oncogene, which encodes a product that also lacks the ligand-binding domain but is otherwise unaltered in its cytoplasmic coding sequences, is capable of inducing leukemia but cannot induce sarcomas. In this report, we show that the critical changes for activating the sarcomagenic potential displayed by v-erbB R are two point mutations within the tyrosine kinase domain and an internal deletion of 21 amino acids in the carboxyl-terminal regulatory domain. The removal of the carboxyl-terminal autophosphorylation sites is not obligatory. These activating mutations (Arg-263 to His, Ile-384 to Ser, and the deletion of residues 494 to 514), when introduced singly into the insertionally activated c-erbB, all dramatically increase fibroblast-transforming potential. Arg-263 resides near the highly conserved HRD motif of the kinase domain, and its mutation to His increases the autophosphorylation activity. The other two mutations do not alter the intrinsic kinase activity and presumably affect other aspects of the receptor involved in growth signaling. Therefore, the high transforming potential of v-erbB R is a consequence of synergism among multiple activating mutations.

Alpharetrovirus↗

PubMed2003

High-throughput genotyping by DHPLC of the dihydropyrimidine dehydrogenase gene implicated in (fluoro)pyrimidine catabolism.

Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme in the degradation of pyrimidines and pyrimidine base analogs including the anticancer drugs 5-fluorouracil (5-FU) and Xeloda. A decreased DPD enzyme activity has been described in cancer patients experiencing severe and life-threatening toxicity after 5-FU treatment and distinct sequence variants in the DPD gene (DPYD) have been associated with impaired enzyme function. The most prominent mutation in the DPD deficient patient group, a mutation in the splicing donor consensus sequence of intron 14, IVS14+1g>a, resulting in a truncated protein, has been observed in the Caucasian population at frequencies as high as 0.91%-0.94%. This underlines the need for a test system for DPYD mutations in patients undergoing chemotherapy with 5-FU or with Xeloda. To set up a fast and sensitive method to identify variant DPYD alleles, we analyzed 50 healthy individuals by denaturing high performance liquid chromatography (DHPLC). A primer set spanning the whole coding region and the exon-intron boundaries of DPYD was used. In addition, a cDNA-based assay was developed to rapidly identify the 165 base pair deletion in the corresponding RNA of IVS14+1g>a mutation carriers. The optimal mutation detection was elaborated for each of the PCR fragments. DHPLC analysis detected 5 different genetic alterations occurring in the coding region of the gene, as well as 10 intronic sequence variants, respectively. In conclusion, high-throughput screening for DPYD variants by DHPLC may be a reliable tool in the investigation of the molecular basis of DPD deficiency. Furthermore, it will help to identify patients at risk for toxic side effects upon chemotherapy using 5-FU and will facilitate individual treatment of patients.

Adult↗

PubMed1991

Alternative splicing of rat tropoelastin mRNA is tissue-specific and developmentally regulated.

Sequence analysis of cDNA clones coding for rat tropoelastin previously has identified two variants that potentially corresponded to alternatively spliced tropoelastin mRNAs (Pierce et al., 1990). We have now used S1 nuclease protection analysis of total RNA from aorta, skin and lungs of 10-day and 6-week old rats to localize all sites of alternative splicing in the tropoelastin mRNA and to examine tissue-specific and developmental regulation of the use of these sites. This analysis revealed multiple sites of alternative splicing involving rat tropoelastin coding sequences corresponding to exons 12 through 15 of the bovine tropoelastin gene and a single site of alternative splicing at sequences corresponding to exon 33. Messenger RNAs from all three tissues at both developmental stages were alternatively spliced at the same sites; there was no evidence for the use of an alternative splice site unique to a particular tissue or developmental stage. However, both tissue-specific and developmentally regulated differences were apparent in the proportion of rat tropoelastin mRNA alternatively spliced at exon 33. Tropoelastin mRNA from the aorta and lungs of neonatal rats was alternatively spliced at exon 33 ten time more frequently than tropoelastin mRNA from skin. Between 10 days and 6 weeks of development, the use of this site of alternative splicing decreased by twenty-fold in RNA from skin, ten-fold in RNA from lungs and two-fold in RNA from aorta. In contrast, alternative splicing at exons 12 through 15 occurred in a small percentage of the mRNA and use of these sites exhibited minimal tissue-specific differences or developmental regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

PubMed1999

Four amino acid changes are associated with the Aldh3a1 locus polymorphism in mice which may be responsible for corneal sensitivity to ultraviolet light.

We studied the phenotype and the nucleotide sequence for the cDNAs of the Aldh3a1a and Aldh3a1c allelic forms of the dioxin-inducible cytosolic aldehyde dehydrogenase (ALDH3A1) present in inbred mouse strains. This gene is constitutively expressed in cornea, stomach, skin, urinary bladder and lungs. The Aldh3a1a allele is found in most inbred mouse strains and codes for a 'high-activity' corneal enzyme compared to the 'low-activity' encoded by the Aldh3a1c allele in SWR/J strain. The 'low-activity' variant is associated with extensive corneal clouding after a single exposure to ultraviolet light. The ALDH3A1 phenotype was examined in tissues from inbred mouse strains carrying the Aldh3a1a allele including, SJL/J, C57BL/6 J/Ibg, DBA/2 J/Ibg, C3H/Ibg and the Aldh3a1c allele (SWR/J). Only trace levels of ALDH3A1 activity were found in all SWR/J tissues. All other strains had significant levels of ALDH3A1 activity in eye, stomach, skin, less in urinary bladder and lungs and only trace amounts in liver. However, no differences were found in corneal and stomach ALDH3A1 mRNA levels between the 'low-' and 'high-activity' variants. A 1556-bp ALDH3A1 cDNA fragment, containing the entire coding region plus 5' and 3' untranslated regions, was amplified by reverse transcriptase-polymerase chain reaction from SWR/J and DBA/2 J/Ibg mouse strains. Sequence analysis revealed 13 nucleotide changes in the Aldh3a1c allele. Four of these changes result in G88R, I154N, H305R and I352V substitutions, whereas nine changes are silent. The I154N disrupts a potential alpha helix, which belongs to the Rossmann fold. Replacement of Arg with the more ionizable His at position 305 of a beta strand might directly affect catalytic activity of the enzyme. It is likely that structural changes associated with these amino acid changes are responsible for the loss of ALDH3A1 enzymatic activity in SWR/J mice.

Aldehyde Dehydrogenase↗

PubMed1992

DNA mutations associated with the human butyrylcholinesterase J-variant.

The J-variant of human serum butyrylcholinesterase (BChE) causes both an approximately two-thirds reduction of circulating enzyme molecules and a corresponding decrease in the level of BChE activity present in serum. Since the level of serum BChE activity and the duration of succinylcholine apnea are inversely correlated, this marked decrease in activity makes individuals with the J-variant more susceptible than usual subjects to prolonged apnea from succinylcholine. We reinvestigated the same family in which Garry et al. identified the J-variant phenotype. The atypical, fluoride, and K-variant mutations were also identified in members of the 47-person pedigree. DNA amplification by PCR, followed by direct sequencing of the amplified DNA, led to the finding that the J-variant phenotype of human serum BChE was associated with two DNA point mutations in the coding region. One of these was the mutation previously identified with the K-variant phenotype (GCA----ACA; Ala539----Thr). The other was an adenine-to-thymine transversion at nucleotide 1490, which changed amino acid 497 from glutamic acid to valine (GAA----GTA; Glu497----Val). This latter point mutation was named the J-variant mutation (formal name BCHE*497V). The J-variant mutation has not been identified without the K-variant mutation. The J-variant mutation created an RsaI-enzyme RFLP. Two additional point mutations, located in the noncoding regions of the gene, were also found to be linked with the J-variant and K-variant point mutations on the same allele. These noncoding polymorphic mutations had previously been found linked to the atypical and K-variant point mutations. A summary table shows dibucaine, fluoride, and Hoffmann-La Roche compound Ro 2-0683 inhibition numbers for 119 samples whose DNA has been sequenced. Eighteen BChE genotypes are represented.

Amino Acid Sequence↗

PubMed1977

Stem cell (immunoblastic) lymphoma. A variant of B lymphocytic lymphoma.

From a histologic review of cases coded as Hodgkin's disease and reticulum cell sarcoma, 12 cases were selected as examples of stem cell lymphoma in which the preponderant cell has characteristics of the B immunoblast. Clinically, these lesions affect the elderly (average age 57.3 years), disseminate early, and, with a few exceptions, progress rapidly to a fatal termination. Morphologically, the neoplastic stem cells, which have pyroninophilic cytoplasm, form diffuse infiltrates with an admixture of acidophilic cells. As a regional variation, the pattern in these lesions overlaps histologically with that seen in angioimmunoblastic lymphadenopathy. The overlap in patterns is presumptive evidence that the angioimmunoblastic pattern at times may be a precursory expression of the stem cell lymphoma. On the basis of morphologic features, these tumors are interpreted as a variant of B cell lymphomas.

Adult↗

PubMed2005

Distribution of saa gene variants in verocytotoxigenic Escherichia coli isolated from cattle and food.

The pathogenesis of verocytotoxigenic Escherichia coli (VTEC) infection in humans is multifactorial, given that verocytotoxins are the principal virulence factor. Most strains causing serious diseases possess the eae gene that encodes the adhesin intimin, but its presence is not essential for virulence as some cases are caused by eae-negative strains. An autoagglutinating adhesin designated Saa was found in some eae-negative strains. This protein varies in size as a consequence of variation in the number of copies of a 37-aa repeat unit in the C-terminal region. Based on these findings, we designed PCR primers to amplify the region coding for these differences to detect saa gene variants present in VTEC strains isolated in Argentina from cattle and meat. The gene saa was detected in 36 (31.6%) eae-negative strains and 5 variants were found. Strains isolated from cattle possessed 4 saa variants, whereas 2 variants were present in isolates from meat. Saa variant 1 predominated (18 strains) and was distributed in strains isolated both from cattle and from meat. Our study revealed the existence of two novel saa variants, termed 4 and 5, which have a higher number of 111-bp repeats than saa genes previously studied.

Adhesins, Bacterial↗

PubMed1985

Properties of wild-type strains of enterotoxigenic Escherichia coli which produce colonization factor antigen II, and belong to serogroups other than O6.

Enterotoxigenic strains of Escherichia coli, which belonged to serogroups other than O6 and produced colonization factor antigen II, usually produced only coli surface antigen 3 (CS3) and gave weak mannose-resistant haemagglutination of bovine erythrocytes. A non-autotransferring plasmid, NTP165, from a strain of E. coli O168. H16 coded for heat-stable enterotoxin, heat-labile enterotoxin and CS antigens. The CS antigens expressed after acquisition of plasmid NTP165 depended on the recipient strain: a biotype A strain of serotype O6. H16 expressed CS1 and CS3; a biotype C strain of serotype O6. H16 expressed CS2 and CS3; strain K12 and strain E19446 of serotype O139. H28 expressed only CS3. An exceptional wild-type strain, E24377, of serotype O139. H28 produced CS1 and CS3 when isolated; a variant of E24377 which had lost the plasmid coding for CS antigens produced both CS1 and CS3 after the introduction of NTP165.

Antigens, Bacterial↗

PubMed2006

Identification of a novel C5L2 variant (S323I) in a French Canadian family with familial combined hyperlipemia.

OBJECTIVE: A functional acylation stimulating protein (ASP) receptor, C5L2, has been recently identified in ASP-responsive cells. Impaired ASP-mediated triglyceride synthesis has previously been described in a subset of hyperapolipoprotein B/familial combined hyperlipidemia subjects. METHODS AND RESULTS: DNA sequencing of C5L2 coding region in 61 unrelated probands identified a heterozygous variant (G968-->T) in 1 subject, resulting in Ser323-->Ile substitution in the carboxyl terminal region. This variant was not detected in 2176 additional chromosomes by restriction fragment length polymorphism or fluorescence polarization genotyping. Eight family members of the proband were identified with one altered (+/-)C5L2 allele. Nine other family members had the wild-type (+/+)C5L2 sequence. The abnormal allele was associated with increased plasma triglyceride, plasma cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein B and ASP. Of 23 subjects tested in cell-based ASP bioactivity assays, those with C5L2(+/-) variant (n = 2) had a 50% reduction in ASP-stimulated triglyceride synthesis, glucose transport and marked reduction in maximal binding (B(max)). By contrast, a C5L2(+/+) family member responded normally, as did hyperapolipoprotein B normal ASP subjects compared with C5L2(+/+) controls (n = 6). CONCLUSIONS: The S323I variant may alter C5L2 function and might be one molecular basis contributing to familial combined hyperlipidemia.

Adult↗

PubMed1989

Characterization of coronavirus JHM variants isolated from Wistar Furth rats with a viral-induced demyelinating disease.

Murine hepatitis virus (MHV) can cause neurological disease when inoculated intracerebrally (ic) into mice and rats. Specifically the JHM strain of MHV (MHV-JHM) generally causes an acute encephalitis when inoculated ic into 2-day-old Wistar Furth rats. In contrast, JHM generally produces a chronic demyelinating disease with resulting posterior paralysis when inoculated ic into 10-day-old Wistar Furth rats. In addition, while JHM readily produces a productive infection in a mouse fibroblast cell line (L-2), it does not form syncytia or replicate well in a tissue cell line of glial origin (G26-24). We have isolated and characterized three MHV-JHM viral variants from the central nervous system of two Wistar Furth rats with a MHV-JHM-induced demyelinating disease. The pattern of viral-specific mRNA for all three of these variants differed from what was observed for the wild-type parental MHV-JHM that had been passaged only in tissue culture. One of these variants, ATllf cord virus, which induced a chronic demyelinating disease in 2- or 10-day-old intracerebrally inoculated Wistar Furth rats, had a deletion in the coding region of the peplomer glycoprotein mRNA. In addition, this variant formed massive syncytia and replicated well in G26-24 cells. We have not detected this deletion in the other two JHM variants, ATllf brain virus and ATlle brain virus. ATllf brain virus and ATlle brain virus primarily produced an acute encephalitis when reinoculated into 2- or 10-day-old Wistar Furth rats. In addition, these two variants did not form syncytia and had a reduced ability to replicate in G26-24 cells.

Animals↗

PubMed1997

Association between human cancer and two polymorphisms occurring together in the p21Waf1/Cip1 cyclin-dependent kinase inhibitor gene.

BACKGROUND: The cyclin-dependent kinase inhibitor gene p21Waf1/Cip1 plays a role in signaling cellular growth arrest. In response to DNA damage, p21 is induced by the p53 gene, thereby playing a direct role in mediating p53-induced G1 arrest. Alterations in this gene may adversely affect regulation of cellular proliferation and increase susceptibility for cancer. Two polymorphisms have previously been characterized in the p21 gene: a C-->A transversion at codon 31 (ser-->arg) and a C-->T transition 20 nucleotides downstream from the 3' end of exon 3. METHODS: The codon 31 polymorphism in exon 2 of the p21 gene was identified by restriction digestion (Alw26I) of products amplified by polymerase chain reaction (PCR). The polymorphism downstream from exon 3 of the p21 gene was identified by single strand conformation polymorphism (SSCP) analysis of PCR amplified products and was confirmed by PstI enzyme restriction digestion. DNA variant alleles were confirmed by direct DNA sequencing. The entire coding region and the promoter region (p53 binding domain) of the p21 gene were screened for mutations by SSCP analysis or DNA sequencing. RESULTS: The two polymorphisms were found in 18 of 96 tumor samples lacking p53 alterations (18.8%). Nine of 54 prostate adenocarcinoma samples (16.7%) contained both p21 variants, whereas 9 of 42 squamous cell carcinomas of the head and neck (21.4%) displayed both polymorphisms. Of the 110 controls examined, 10 (9.1%) had both alterations. Both p21 polymorphisms occurred together in all samples examined and there was no indication of mutation in the coding region of the p21 gene or in the p53 binding domain of the promoter region. CONCLUSIONS: These data suggest that p21 gene variants may play a role in increased susceptibility for the development of some types of cancer. In the current study, the authors demonstrated that the occurrence of these two polymorphisms is increased in prostate adenocarcinoma and squamous cell carcinoma of the head and neck. The polymorphic sites may be directly responsible for this apparent increased susceptibility or they may be linked to regulatory region alterations.

Adenocarcinoma↗

PubMed1996

An N-terminal domain shared by Fas/Apo-1 (CD95) soluble variants prevents cell death in vitro.

Fas/Apo-1 molecule, also designated as CD95, is a member of the TNF receptor family. Fas cross-linking by its natural ligand or by agonistic mAbs results in rapid induction of apoptosis in susceptible cells. in addition to the Fas full-length mRNA, human activated PBMC and tumor cell lines express several mRNA Fas variants that derive from alternative splicing of the primary transcript. All five variants identified, two of which are newly described here, code for soluble proteins that, with the exception of FasTMDel, are truncated in the extracytoplasmic region and possess short C-terminal amino acid sequences corresponding to a different reading frame. We have identified Abs that recognize all splicing variants and established a sandwich ELISA by which the soluble Fas molecules could be detected in culture supernatants of transfected cell lines and in PBMC following T cell activation. Next, we have studied in detail the functional role of these variants by apoptosis inhibition studies. We found that all soluble proteins block the apoptosis induced by either an agonistic Ab or, more importantly, by the natural Fas ligand in Fas-positive sensitive cell lines. interestingly, this functional property can be assigned to the first 49 amino acids of the mature protein that is the only region shared by the five soluble Fas molecules.

Adult↗

PubMed1997

Small genetic effects in complex diseases: a review of regulatory sequence variants in dyslipoproteinemia and atherosclerosis.

OBJECTIVE: Most reported mutations that affect lipoprotein metabolism are found within the coding sequences of genes. Recently, a few mutations that occur within promoter sequences have been detected. These promoter sequence variants are the topic of the present review. METHODS: Some of these variants are fairly common genomic variants in the promoter regions for candidate genes in lipoprotein metabolism, such as APOA1, APOC3, LPA, and LPL. It is possible that such regulatory sequence variants can result in chronic, modestly altered levels of expression of qualitatively normal gene products. This might have a cumulative effect on quantitative phenotypes, such as plasma lipoprotein concentrations, over the long term. Such an effect might not be detected by existing clinical, biochemical, and/or physiological assays. RESULTS: At present, the most consistent evidence from several lines of experiments indicates that genomic variation in the APOC3 promoter creates slightly elevated plasma triglyceride concentrations within the physiologic range. This altered expression appears to predispose to hypertriglyceridemia in the presence of secondary factors. Genetic variants that produce small effects on promoter function might thus be one component of the predisposition to complex diseases. CONCLUSION: The aggregate of many small effects may create or contribute to a background of susceptibility that, under appropriate conditions, leads to development of frank dyslipidemia and atherosclerosis.

Apolipoprotein A-I↗

PubMed2006

MSX1 mutations contribute to nonsyndromic cleft lip in a Thai population.

Previous studies observed that MSX1 mutations could contribute to nonsyndromic cleft lip with or without cleft palate (CL/P) in some populations. Of the proposed pathogenic mutations, the P147Q variant was predominant in Vietnamese and present in Filipino populations. We investigated whether MSX1 mutations also contribute to nonsyndromic CL/P in the Thai population. Specifically, we performed mutation analysis covering all the coding regions of the MSX1 gene for 100 Thai patients with nonsyndromic CL/P. A total of eight variant sites were identified. Six were in coding regions, including four nonsynonymous changes, 101C > G (A34G), 440C > A (P147Q), 799G > T (G267C), and 832C > T (P278S). The G267C and P278S variants were predicted to be "probably damaging" by PolyPhen, changed themselves as potential exonic splicing enhancers for serine/arginine-rich proteins, and were not present in 162 control individuals of Thai ethnic background. Unlike all of the previously reported potential missense mutations in MSX1, these two novel potential mutations were found in exon 2 on the C-terminal side of the homeodomain protein. Moreover, in contrast to previous reports, we found the P147Q variant in 8 out of 100 Thai controls and an association between the variant and CL/P in our population could not be detected, suggesting that it is not pathogenic. Our data support that MSX1 mutations are found in 2% of cases of CL/P and should be considered for genetic counseling implications, but suggest that the P147Q variant is not pathogenic.

Alleles↗

PubMed1999

Abnormal, error-prone bypass of photoproducts by xeroderma pigmentosum variant cell extracts results in extreme strand bias for the kinds of mutations induced by UV light.

Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a greatly increased susceptibility to sunlight-induced skin cancer. Cells from the majority of patients are defective in nucleotide excision repair. However, cells from one set of patients, XP variants, exhibit normal repair but are abnormally slow in replicating DNA containing UV photoproducts. The frequency of UV radiation-induced mutations in the XP variant cells is significantly higher than that in normal human cells. Furthermore, the kinds of UV-induced mutations differ very significantly from normal. Instead of transitions, mainly C-->T, 30% of the base substitutions consist of C-->A transversions, all arising from photoproducts located in one strand. Mutations involving cytosine in the other strand are almost all C-->T transitions. Forty-five percent of the substitutions involve thymine, and the majority are transversions. To test the hypothesis that the UV hypermutability and the abnormal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells with those from HeLa cells and a fibroblast cell strain, MSU-1.2, for the ability to replicate a UV-irradiated form I M13 phage. The M13 template contains a simian virus 40 origin of replication located directly to the left or to the right of the target gene, lacZalpha, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to approximately 37% of the control value required only 1 photoproduct per template for XP variant cell extracts, but approximately 2.2 photoproducts for HeLa or MSU-1.2 cell extracts. The frequency of mutants induced was four times higher with XP variant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP variant cell extracts, the proportion of C-->A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the template for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, this value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, and virtually all were T-->A transversions. Sequence analysis of the coding region of the catalytic subunit of DNA polymerase delta in XP variant cell lines revealed two polymorphisms, but these do not account for the reduced bypass fidelity. Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cells, bypass of photoproducts involving cytosine in the template for the leading strand differs significantly from that of photoproducts in the lagging strand.

Cell Extracts↗