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Biomedical subjects

J E Cleaver

Publications and source records attributed to J E Cleaver.

At least 55 records · Page 3Linked to original sources
PubMed1995

Xeroderma pigmentosum variant cells are resistant to immortalization.

Xeroderma pigmentosum (XP) is a human repair-deficient disorder that is caused by mutations in any of eight genes (A-G, V). The genes for complementation groups A-G have been cloned fully or in part, but the gene for the XP variant (XPV) has yet to be cloned. The lack of progress with XPV is in large part due to the rarity of stably transformed cell lines. We have attempted to immortalize fibroblasts from several XPV patients to obtain cell lines with which to characterize this disease and clone the appropriate gene. We have found, as have other investigators, that this XP group is very difficult to immortalize. We used a variety of approaches, including transfection with pSV ori- (a plasmid containing the simian virus (SV) 40 large T antigen) followed by spontaneous transformation, which provided stable immortal lines from Cockayne syndrome A and B, but not from XPV; transfection with pSV ori- and exposure to 3 Gy of X-rays; transfection with pSV ori-, exposure to 2 Gy of X-rays, and treatment with 1 mM ethyl methanesulfonate; transfection with human papilloma virus-16; and infection with SV40. Even though we used as many as 2 x 10(8) cells in some experiments, we were able to immortalize only one of our lines, XP30RO. Because the biochemical defect in XPV cell lines involves the capacity to replicate damaged DNA templates, perhaps the XPV gene product could be a replication factor that interacts with SV40 T antigen, and whose absence from XPV cell lines presents difficulties for the immortalization process to proceed.

Antigens, Polyomavirus Transforming↗
PubMed1995

Induction and repair of (6-4) photoproducts in normal human and xeroderma pigmentosum variant cells during the cell cycle.

The reduced rate of (6-4) photoproduct repair observed in some cell lines may represent a more severe repair deficiency in some cohort of the cell cycle, such as S-phase. Radioimmunoassay was used to determine the kinetics of (6-4) photoproduct repair in normal human fibroblasts and xeroderma pigmentosum variant cells fractionated into different phases of the cell cycle by counterflow centrifugal elutriation. Ultraviolet fluence response curves indicated that the same amount of (6-4) photoproduct damage was induced at all phases of the cell cycle. The extent of (6-4) photoproduct repair in asynchronous XP variant cells was significantly reduced compared to normal human cells. However, the rate and extent of (6-4) photoproduct repair was constant throughout the cell cycle in both normal and XP variant cells. Hence, the UV hypersensitive and hypermutable phenotypes observed in XP variant cells are not attributable to cell cycle-dependent deficiencies in excision repair nor the yield of photodamage through the cell cycle.

Cell Cycle↗
PubMed1995

Sister chromatid exchanges in cells defective in mismatch, post-replication and excision repair.

Three processes associated with DNA damage and genomic instability have been defined experimentally as operating during or soon after DNA replication: mismatch repair, post-replication repair and sister chromatid exchange. All these processes appear to operate on damage and/or errors in newly replicated DNA. Both mismatch repair and post-replication repair involve resynthesis of up to 1 kb of newly synthesized DNA: mismatch repair operates on single-base or slippage errors; post-replication repair operates on persistent gaps in newly synthesized DNA caused by damage on parental strands. Using colon cancer cells with different mismatch repair capacity, together with normal cells and excision-repair-defective and post-replication-repair-defective xeroderma pigmentosum (XP) cells, we analysed possible interactions between these processes. No evidence for overlap of mismatch repair with excision or post-replication repair was found. However, post-replication-repair-defective XP variant cells that were SV40 transformed showed higher UV-induced sister chromatid exchange frequencies than did untransformed cells. This suggests that sister chromatid exchanges in the XP variant are closely involved with UV-induced replication errors that are enhanced by transformation.

Caffeine↗
PubMed1995

Nucleotide excision repair: variations associated with cancer development and speciation.

Nucleotide excision repair requires the action of multiple interacting proteins that locate damage in DNA, remove it as a short oligonucleotide and synthesize a replacement patch. Mutations in genes coding for these proteins give rise to a wide range of diseases involving skin carcinogenesis, neuronal decline and developmental disorders of bone and central nervous system. Complex clinical symptoms of more than one clinical disorder may occur because of mutations that influence protein-protein interactions. Significant differences in repair occur between individuals and species for which the molecular basis and phenotypic consequences have yet to be explained.

Animals↗
PubMed1994

Genetic testing.

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Genetic Diseases, Inborn↗
PubMed1994

Prenatal diagnosis of xeroderma pigmentosum and Cockayne syndrome.

In a study of fetal cells from a series of 12 pregnancies in ten families at risk for the ultraviolet light-sensitive, DNA repair-deficient diseases xeroderma pigmentosum (XP) and Cockayne syndrome (CS), we detected one XP and two CS homozygote fetuses. The diagnoses were confirmed by analysis of fetal skin fibroblasts or second amniotic samples after termination of the pregnancies. The measurement of ultraviolet light sensitivity and DNA repair depended on properties common to the seven excision repair-deficient XP complementation groups (A-G) and the two CS complementation groups (A, B). No XP variant families were included in the study, because the variant requires different testing techniques. Reliable and rapid diagnosis proved possible in all but one of the 12 pregnancies, supporting the use of these methods until the spectrum of mutations in the various XP and CS genes of the U.S. population is fully characterized and a DNA sequence-based diagnostic procedure becomes available.

Amniocentesis↗
PubMed1994

Mutation and expression of the XPA gene in revertants and hybrids of a xeroderma pigmentosum cell line.

A series of ultraviolet (UV)-resistant cell lines have been generated from a UV-sensitive XP group A cell line homozygous for a stop codon (TGA) in the chromosome 9 XPA gene. Three lines generated by chemical mutagenesis acquired the ability to excise (6-4) photoproducts but not cyclobutane dimers from the whole genome; two lines generated by a fusion procedure with hamster cells acquired the ability to excise both (6-4) photoproducts and cyclobutane dimers from the whole genome. A central region of the hamster XPA gene was cloned and sequenced. With the use of species-specific primers in the polymerase chain reaction, we found that the hybrid cell lines do not contain a hamster XPA gene. Sequence analysis showed that all of the UV-resistant cell lines contain reversions of the human stop codon, resulting in missense mutations (glycine or leucine for arginine) or wild-type sequences. The concentration of XPA protein in revertant cell lines was about one-half that in normal cells, which would be expected from heterozygous cells; there was no evidence that the mutant proteins were less stable than the wild-type proteins. These results are consistent with the idea that the XPA protein initiates repair by binding to damaged sites with various affinities, depending on the photoproduct and the transcriptional state of the region. A concentration of XPA protein near 50% is needed before repair can proceed into nontranscribed regions of the genome. The revertant cell lines represent a class of missense mutations in the XPA gene that may have altered specificity and that can be used to understand some of the regulatory differences in repair of photoproducts in various regions of the genome.

Amino Acid Sequence↗
PubMed1994

Trichothiodystrophy: clinical spectrum, central nervous system imaging, and biochemical characterization of two siblings.

Trichothiodystrophy (TTD), an autosomal recessive disorder characterized by sulfur-deficient brittle hair, identifies a group of genetic disorders with an altered synthesis of high-sulfur matrix proteins and a defect in excision repair of ultraviolet damage in fibroblasts of most TTD patients. In contrast to patients with xeroderma pigmentosum (XP), TTD patients do not have an increased frequency of skin cancers. TTD patients may be grouped into four categories: 1) those without photosensitivity and without a defect in excision repair of UV damage; 2) those without photosensitivity and with an excision-repair defect in the same gene as in XP-D (complementation group D); 3) those with photosensitivity and with the XP-D repair defect; 4) those with photosensitivity and with a repair defect distinct from that in XP-D. We present a brother and sister in the third category of TTD. Clinically, the patients have brittle hair, short stature, ichthyosis, photosensitivity, nail and dental dysplasias, cataracts, mental retardation, and pyramidal tract abnormalities. Diagnosis was made by hair mount, which shows the characteristic banding pattern with polarizing microscopy, and by hair amino acid analysis, which demonstrated decreased high-sulfur matrix proteins. Fibroblasts cultured from skin biopsies had a marked DNA excision repair defect similar to the repair defect seen in XP-D. We have documented a unique dysmyelinating disorder on magnetic resonance imaging of the brain that might explain their mental retardation, marked hyperactivity, and neurologic deficits. Following the discovery that the human excision repair cross complementing rodent ultraviolet group 2 (ERCC2) gene is able to correct the ultraviolet sensitivity of XP-D cell strains, the ERCC2 cDNA from previous TTD patients was sequenced and shows frameshifts, deletions and point mutations in the ERCC2 gene. Molecular analysis of our patients is in progress. Molecular analysis of the defects in ERCC2 in clinically distinct patients with XP,XP/Cockayne's syndrome, and TTD may provide insight into the molecular mechanisms of these genetically related but clinically distinct disorders.

Brain↗
PubMed1994

Excision of cyclobutane dimers in genomic and episomal DNA in human cells.

Direct determination has been made of cyclobutyl pyrimidine dimer induction and excision repair in an episomal SV40 DNA population in vivo. Maintaining SV40-transformed human (GM637) cells in confluent culture results in amplification of a mutant SV40 episome to high copy number. T4 endonuclease V was used to quantify the induction and repair of cyclobutane dimers in the SV40 episome and genomic DNA of the same cells. Differences in both parameters were observed; cyclobutane dimers were induced at 1.5-2-fold greater frequency in episomal DNA and excised at a reduced rate compared to genomic DNA in the host cells.

Cell Line, Transformed↗
PubMed1993

Mechanisms involved in rejoining DNA double-strand breaks induced by ionizing radiation and restriction enzymes.

DNA double-strand breaks are considered to be the most deleterious lesion induced by ionizing radiation. However, the mechanism of rejoining of these lesions has not been extensively studied at the molecular level. We have used a shuttle vector, pHAZE, to analyze the mechanism of rejoining of DNA double-strand breaks in human cells. The advantage of this vector system is that, unlike many previously described shuttle vectors, it has a large target gene for the detection of deletions and it is maintained as a freely replicating episome with chromatin conformation in the nucleus of human cells. In this study we compare data obtained on the spectrum of mutations induced in pHAZE by ionizing radiation (alpha-particles) and restriction enzymes (PvuII, ClaI, and PvuI). Unlike ionizing radiation, restriction enzymes induce double-strand breaks in DNA with known end structures at defined locations and therefore provide a model system for analyzing cellular responses to DNA double-strand breaks. Exposure of human cells containing the vector to alpha-particle irradiation produced both point mutations and large deletions in pHAZE. When the junction regions of the deletions were sequenced it was found that 65% were rejoined with up to 6 bp of homology at the junction region. Analysis of restriction-enzyme-induced mutations suggests that double-strand break ends are modified to facilitate rejoining and that the type of modification is characteristic for different end structures. Double-strand breaks with cohesive ends appear to have fewer modifications introduced at the break points before rejoining than breaks with blunt ends. When considered in relation to the data obtained with ionizing radiation this suggests that the presence of cohesive sequences either at, or in proximity to, the ends enhances rejoining of DNA double-strand breaks.

Base Sequence↗
PubMed1993

A single-site mutation in the XPAC gene alters photoproduct recognition.

The XPAC (xeroderma pigmentosum group A complementing) gene, which is located on chromosome 9, carries a variety of point mutations in XP group A patients. We investigated the role of the XPAC gene product in excision repair by generating revertants of an XP group A cell line (XP12RO) that have increased resistance to ultraviolet light. One of these cell lines, XP129, can repair (6-4) pyrimidine-pyrimidone photoproducts normally but has reduced repair of cyclobutane dimers, as in XP12RO. Sequence analysis of cDNA from the XPAC gene indicated that XP12RO contains a termination codon at amino acid position 207, resulting in a reduced amount of mRNA and no detectable protein. In the revertant XP129 line, this termination codon has been mutated further and now encodes glycine in one allele instead of the wild-type arginine. The mRNA level detected by allele-specific polymerase chain reaction amplification was greater for the reverted sequence than for the chain-terminating sequence. These observations indicated that a point mutation resulting in a mis-sense mutation in the XPAC gene and altered expression of the XPAC protein can alter the substrate specificity of the excision repair system, and imply that the XPAC gene product plays an important role in photoproduct recognition.

Amino Acid Sequence↗