Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A shuttle vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in cultured skin cells from a patient with the skin-cancer-prone, DNA repair-deficient disease xeroderma pigmentosum and in repair-proficient cells. After replication in the human cells, progeny plasmids were purified. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of Escherichia coli carrying a suppressible amber mutation in the beta-galactosidase gene. Plasmid survival in the xeroderma pigmentosum cells was less than that of pZ189 harvested from repair-proficient human cells. The point-mutation frequency in the 150-base-pair tRNA marker gene increased up to 100-fold with ultraviolet dose. Sequence analysis of 150 mutant plasmids revealed that mutations were infrequent at potential thymine-thymine dimer sites. Ninety-three percent of the mutant plasmids from the xeroderma pigmentosum cells showed G X C----A X T transitions, compared to 73% in the normal cells (P less than 0.002). There were significantly fewer transversions (P less than 0.002) (especially G X C----T X A) and multiple base substitutions (P less than 0.00001) than when pZ189 was passaged in repair-proficient cells. The subset of mutational changes that are common to ultraviolet-treated plasmids propagated in both repair-proficient and xeroderma pigmentosum skin cells may be associated with the development of ultraviolet-induced skin cancer in humans.
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PMID:Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells. 346 53

The effects of cis-diamminedichloroplatinum(II) (cis-DDP) and trans-DDP adducts on mammalian transcription in vivo have been investigated. A plasmid containing the beta-galactosidase (beta-gal) reporter gene was modified with either of the two platinum compounds and transfected into human or hamster cell lines. A 2-3 fold higher level of transcription was observed in both cell lines from plasmids containing trans-DDP adducts as compared to plasmids modified by cis-DDP. This difference in transcriptional activity was not decreased in human and rodent nucleotide excision repair deficient cell lines, indicating that more efficient excision repair of the trans-DDP adducts was not the cause of its lower ability to block transcription in this assay. For this conclusion to be valid, it is assumed that trans-DDP adducts are repaired primarily by the nucleotide excision repair pathway, as is the case with the adducts of cis-DDP. The possibility that trans-DDP adducts are preferentially bypassed by RNA polymerase was examined by monitoring the elongation of beta-gal mRNA on damaged templates in vivo. Nascent beta-gal mRNA transcripts were recovered from excision repair deficient xeroderma pigmentosum A cells transfected with platinated plasmids, and the extent of RNA synthesis was measured by using ribonuclease protection. Fourfold more trans-DDP than cis-DDP adducts were required to inhibit transcription elongation by 63%. RNA polymerase II bypassed cis- and trans-DDP DNA adducts with efficiencies of 0-16% and 60-70%, respectively. These data provide insight into the differential toxicity of the two platinum isomers.
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PMID:DNA adducts of cis-diamminedichloroplatinum(II) and its trans isomer inhibit RNA polymerase II differentially in vivo. 757 87

Xeroderma pigmentosum (XP) is a human hereditary disease characterized by a defect in DNA repair after exposure to ultraviolet light. Among the seven groups of XP, group A (XP-A) patients show the most severe deficiency in excision repair and a wide variety of cutaneous and neurological disorders. We have cloned homologs of the human XPA gene from chicken, Xenopus, and Drosophila, and sequence analysis revealed that these genes are highly conserved throughout evolution. Here, we report characterization of the Drosophila homolog of the human XPA gene (Dxpa). The Dxpa gene product shows DNA repair activities in an in vitro repair system, and Dxpa cDNA has been shown to complement a mutant allele of human XP-A cells by transfection. Polytene chromosome in situ hybridization mapped Dxpa to 3F6-8 on the X chromosome, where no mutant defective in excision repair was reported. Northern blot analysis showed that the gene is continuously expressed in all stages of fly development. Interestingly, the Dxpa protein is strongly expressed in the central nervous system and muscles as revealed by immunohistochemical analysis using anti-Dxpa antibodies, consistent with the results obtained in transgenic flies expressing a Dxpa-beta-galactosidase fusion gene driven by the Dxpa promoter.
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PMID:Expression and functional analyses of the Dxpa gene, the Drosophila homolog of the human excision repair gene XPA. 767 33

The effect of UV photoproducts or benzo[a]pyrene-diol-epoxide-I (BPDE-I) adducts in DNA on the transient expression of a reporter gene was measured in mammalian cells. The plasmid pRSVCAT was UV irradiated or treated with BPDE-I in vitro and co-transfected with undamaged pRSVBGAL into mouse and human fibroblasts. Variations in transfection efficiency among different cell lines were corrected by adjusting the volumes of cell extracts used in the chloramphenicol acetyl transferase (CAT) assays to contain equal beta-galactosidase (BGAL) activity. The expression of the CAT gene was found to decrease exponentially after transfection of pRSVCAT containing increasing numbers of DNA lesions per molecule. The average number of BPDE-I adducts per plasmid molecule was measured by ELISA; the average number of pyrimidine dimers was estimated from the dose kinetics for the disappearance of the supercoiled form of irradiated plasmid DNA treated with Micrococcus luteus UV endonuclease. By expressing the inhibition of CAT activity in terms of the average number of lesions per gene, we were able to compare directly the effects of two different carcinogen lesions on transient transcription. We observed comparable kinetics of inhibition of gene expression by BPDE-I adducts and pyrimidine dimers in DNA. D0 values determined by linear regression analysis of dose-response curves for inhibition of CAT activity were 4.9 BPDE-I adducts or 6.6 pyrimidine dimers per gene in excision-proficient human fibroblasts; the corresponding values in mouse cells were 4.4 BPDE-I adducts or 5.5 pyrimidine dimers. Similar threshold densities of BPDE-I adducts and pyrimidine dimers were observed before inhibition of transcription from pRSVCAT was detected. No threshold was observed in experiments with human fibroblasts deficient in excision repair (xeroderma pigmentosum group A); calculated D0 values were 1.2 pyrimidine dimers of 2.1 BPDE-I adducts. Our results permit direct comparisons of the magnitude of inhibition of gene transcription by distinct DNA lesions, and suggest that BPDE-I adducts and UV-induced cyclobutane pyrimidine dimers in template DNA block transcription with similar efficacy.
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PMID:Inhibition of reporter gene expression in mammalian cells. Effects of distinct carcinogen lesions in DNA. 820 75

A recombinant nonreplicating human adenovirus type 5, Ad5HCMVsp1lacZ, expressing the lacZ gene under control of the human cytomegalovirus immediate early promoter, was used to assess the effect of heat-shock (HS) on DNA repair of a UV-damaged reporter gene. Host cell reactivation (HCR) of beta-galactosidase (beta-gal) activity for UV-irradiated Ad5HCMVsp1lacZ was used as an indicator of DNA repair in the transcribed strand of an active gene. Repair was examined in heat-shock (HS) pretreated and mock-treated normal fibroblasts, normal lung epithelial cells, xeroderma pigmentosum group A, C, D and G fibroblasts (XP-A, XP-C, XP-D and XP-G), Cockayne's syndrome group A fibroblasts (CS-A), SV40-transformed normal fibroblasts (GM637f) and 5 tumour cell lines (SKOV-3, HeLa, HT29, SCC-25 and U20S). HS enhanced reactivation (HSER) of the reporter gene was detected in normal cells, HT29 tumour cells and XP-C fibroblasts. HSER was reduced or absent in all other XP, CS and tumour cell lines tested. HSER in normal and XP-C cell lines, but not CS-A, XP-A, XP-D or XP-G cells, suggests that HS treatment can enhance the repair of UV-damaged DNA through an enhancement of transcription coupled repair (TCR) or a mechanism which involves the TCR pathway. Since this response was absent in the SV40-transformed fibroblast cell line and 4 of 5 tumour cell lines examined, HSER of beta-gal activity for UV-irradiated Ad5HCMVsp1lacZ also requires some cellular function(s) affected by transformation.
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PMID:Heat-shock enhanced reactivation of a UV-damaged reporter gene in human cells involves the transcription coupled DNA repair pathway. 867 26

Xeroderma pigmentosum type G (XPG) is a human genetic disease exhibiting extreme sensitivity to sunlight. XPG patients are defective XPG endonuclease, which is an enzyme essential for DNA repair of the major kinds of solar ultraviolet (UV)-induced DNA damages. Here we describe a novel dynamics of this protein within the cell nucleus after UV irradiation of human cells. Using confocal microscopy, we have localized the immunofluorescent, antigenic signal of XPG protein to foci throughout the cell nucleus. Our biochemical studies also established that XPG protein forms a tight association with nuclear structure(s). In human skin fibroblast cells, the number of XPG foci decreased within 2 h after UV irradiation, whereas total nuclear XPG fluorescence intensity remained constant, suggesting redistribution of XPG from a limited number of nuclear foci to the nucleus overall. Within 8 h after UV, most XPG antigenic signal was found as foci. Using beta-galactosidase-XPG fusion constructs (beta-gal-XPG) transfected into HeLa cells, we have identified a single region of XPG that is evidently responsible both for foci formation and for the UV dynamic response. The fusion protein carrying the C terminus of XPG (amino acids 1146-1185) localized beta-gal specific antigenic signal to foci and to the nucleolus regions. After UV irradiation, antigenic beta-gal translocated reversibly from the subnuclear structures to the whole nucleus with kinetics very similar to the movements of XPG protein. These findings lead us to propose a model in which distribution of XPG protein may regulate the rate of DNA repair within transcriptionally active and inactive compartments of the cell nucleus.
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PMID:Ultraviolet-induced movement of the human DNA repair protein, Xeroderma pigmentosum type G, in the nucleus. 871 Aug 77

Cells from patients with xeroderma pigmentosum (XP) variant are thought to be defective in postreplication repair. This DNA repair pathway is not well defined in human cells and the exact genetic defect of XP variant is unknown. In another cancer-prone hereditary disorder, hereditary nonpolyposis colon cancer, tumors are characterized by a DNA mismatch repair defect with microsatellite instability. Since there are some similarities between postreplication repair and mismatch repair, we investigated microsatellite instability, the hallmark of a DNA mismatch repair defect, in a lymphoblastoid cell line from a patient with XP variant. Two normal lines and one nucleotide excision repair-defective XP group A line were used as controls. In a host cell microsatellite instability assay, the recently developed shuttle vector pZCA29 was transfected into these cells and replicated plasmid recovered after 3 days. The plasmid contains two CA repeat tracts that interrupt the reading frame of the lacZ gene. Reversion to active beta-galactosidase, detectable by a color reaction of bacterial transformants, represents the frequency of frameshift mutations in the CA repeat tracts during replication of the plasmid, and thereby the host cells' microsatellite instability. We did not find any significant differences in the mutation frequencies of the plasmids after passage through either cell line. This indicates that there is no microsatellite instability in the examined XP variant cell line.
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PMID:Assessment of microsatellite instability in a cell line from a patient with xeroderma pigmentosum variant. 955 84

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are human hereditary disorders characterized at the cellular level by an inability to repair certain types of DNA damage. Usually, XP and CS are clinically and genetically distinct. However, in rare cases, CS patients have been shown to have mutations in genes that were previously linked to the development of XP. The linkage between XP and CS has been difficult to study because few permanent cell lines have been established from XP/CS patients. To generate permanent cell lines, primary fibroblast cultures from two patients, displaying characteristics associated with CS and belonging to XP complementation group G, were transformed with anorigin-of-replication-deficient simian virus 40 (SV40). The new cell lines, summation operatorXPCS1LVo- and summation operatorXPCS1ROo-,were characterized phenotypically and genotypically to verify that properties of the primary cells are preserved after transformation. The cell lines exhibited rapid growth in culture and were shown, by immunostaining, to express the SV40 T antigen. The summation operatorXPCS1LVo- and summation operatorXPCS1ROo- cell lines were hypersensitive to UV light and had an impaired ability to reactivate a UV-irradiated reporter gene. Using polymerase chain reaction (PCR) amplification and restriction enzyme cleavage, the summation operatorXPCS1ROo- cells were shown to retain the homozygous T deletion at XPG position 2972. This mutation also characterizes the parental primary cells and was evident in the XPG RNA. Finally, to characterize the XPG DNA repair deficiency in these cell lines, an episomal expression vector containing wild-type XPG cDNA was used to correct UV-induced damage in a beta-galactosidase reporter gene.
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PMID:Complementation of transformed fibroblasts from patients with combined xeroderma pigmentosum-Cockayne syndrome. 971 45

The genetic disorders xeroderma pigmentosum (XP) and Cockayne syndrome (CS) exhibit deficiencies in the repair of UV-induced DNA damage. CS fibroblasts retain proficient nucleotide excision repair (NER) of inactive (or bulk) DNA, but are deficient in the transcription-coupled repair (TCR) of active genes. In contrast, XP complementation group C (XP-C) fibroblasts retain proficient TCR, but are deficient in bulk DNA repair. The remaining NER-deficient XP groups exhibit deficiencies in both repair pathways. Ad5HCMVsp1lacZ is a recombinant adenovirus vector that is unable to replicate in human fibroblasts, but can efficiently infect and express the beta-galactosidase reporter gene in these cells. We have examined the host cell reactivation (HCR) of beta-galactosidase activity for UV-irradiated Ad5HCMVsp1lacZ in non-irradiated and UV-irradiated normal, XP-B, XP-C, XP-D, XP-F, XP-G, CS-A and CS-B fibroblasts. HCR of beta-galactosidase activity for UV-irradiated Ad5HCMVsp1lacZ was reduced in non-irradiated cells from each of the repair-deficient groups examined (including XP-C) relative to that in non-irradiated normal cells. Prior irradiation of cells with low UV fluences resulted in an enhancement of HCR for normal and XP-C strains, but not for the remaining XP and CS strains. HCR of the UV-damaged reporter gene in UV-irradiated XP and CS strains was similar to measurements of TCR reported previously for these cells. These results suggest that UV treatment results in an induced repair of UV-damaged DNA in the transcribed strand of an active gene in XP-C and normal cells through an enhancement of TCR or a mechanism which involves the TCR pathway.
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PMID:UV-enhanced reactivation of a UV-damaged reporter gene suggests transcription-coupled repair is UV-inducible in human cells. 993 45

We have used a non-replicating recombinant adenovirus, Ad5HCMVlacZ, which expresses the beta-galactosidase (beta-gal) reporter gene, to examine the time course of UV-inducible repair of UV-damaged DNA in human fibroblasts. Host cell reactivation (HCR) of beta-gal activity for UV-irradiated Ad5HCMVlacZ was examined in non-irradiated and UV-irradiated nucleotide excision repair (NER) proficient normal human fibroblasts, xeroderma pigmentosum (XP) group C fibroblasts which are defective in the global genomic repair (GGR) pathway of NER and Cockayne syndrome (CS) fibroblasts which are defective in the transcription coupled repair (TCR) pathway of NER. HCR was deficient in untreated XP-C and CS cells indicating that both TCR and GGR are involved in removal of photolesions from the transcribed strand of the reporter gene in unirradiated human cells as reported previously. Prior UV-irradiation of cells with low UV fluences resulted in a transient enhancement of HCR in normal and XP-C fibroblasts that reached a maximum when cells were infected at 25-35 h after UV. In contrast, UV-enhanced HCR was delayed in CS-B cells, reaching levels similar to that in normal cells only when cells were infected between 40 and 60 h after UV exposure. These results are consistent with a UV-induced up-regulation of GGR through a TCR dependent pathway in CS cells.
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PMID:Enhanced host cell reactivation of a UV-damaged reporter gene in pre-UV-treated cells is delayed in Cockayne syndrome cells. 1612 67


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