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1.
P. Hösli C. H. M. M. de Bruyn F. J. J. M. Oerlemans M. Verjaal R. E. Nobrega 《Human genetics》1977,37(2):195-200
Summary A previously developed simple ultramicromethod has been used for the rapid prenatal diagnosis of hypoxanthine-guanine phosphoribosyl transferase (HG-PRT) deficiency. The method is based on the incubation of small numbers of visually selected, lyophilized fibroblasts (in the present study five cells per incubation) with radioactive substrate in an end volume of 0.3 l. Fibroblasts derived from the amniotic fluid of a 15-week male fetus in a heterozygote for the X-linked Lesch-Nyhan syndrome showed a severe degree of HG-PRT deficiency. In total 50 fibroblasts were used. The diagnosis was confirmed upon termination of the pregnancy by the demonstration of HG-PRT deficiency in fetal erythrocytes and cultured skin fibroblasts.HG-PRT: hypoxanthine-guanine phosphoribosyl transferase 相似文献
2.
Subjects heterozygous for the Lesch-Nyhan syndrome with a deficiency of the X-linked gene for the enzyme hypoxanthine-guanine phosphoribosyl transferase (PRT) would be expected to have two populations of erythrocytes in roughly equal proportions—one type with the normal enzyme and the other type exhibiting the mutant form of the enzyme. In contrast to this prediction, previous studies utilizing an X-linked gene for another enzyme as a marker for the PRT locus have suggested that erythrocytes from heterozygotes consist largely of cells with the normal form of the enzyme. We have recently described a mutant form of hypoxanthine-guanine phosphoribosyl tranferase with altered kinetic properties which allow it to be measured in artificial mixtures with the normal enzyme. The mutant enzyme could not be detected in erythrocyte lysates from a proven heterozygote for both the normal and this mutant form of the enzyme. This provides additional evidence that either inactivation of the X-chromosome in erythropoietic tissue from the heterozygote for PRT deficiency is not random or that random X-chromosome inactivation is followed by selection against erythrocyte precursors with the mutant enzyme.This study was supported in part by USPHS Research Grant No. AM14362, USPHS Training Grant No. AM05620, and a grant (RR-30) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. 相似文献
3.
C.H.M.M. de Bruyn T.L. Oei P. Hösli 《Biochemical and biophysical research communications》1976,68(2):483-488
An ultra-microchemical method using radioactive substrates has been developed for enzyme activity measurements at the single cell level. In order to demonstrate the possibilities of this radiochemical microassay, activity measurements of hypoxanthine-guanine phosphoribosyl transferase (HG-PRT) and of adenine phosphoribosyl transferase (A-PRT) in isolated human fibroblasts are described. There was a linear relationship between the number of cells incubated and the enzyme activities found. It was observed that the HG-PRT activity in single, skin derived, fibroblasts did not differ from that in amniotic fluid derived fibroblasts, thus providing a new, quantitative assay for rapid prenatal diagnosis in the Lesch-Nyhan syndrome. 相似文献
4.
Mutant clones of human diploid fibroblasts deficient in the enzyme, hypoxanthine-guanine phosphoribosyl transferase (HGPRT) were selected by their ability to grow in medium containing the cytotoxic purine analogue, 6-thioguaninine (6TG). The optimal conditions for mutant selectiom were 6TG concentrations between 1 and 5 μg ml?1 and cell plating densities ~ 103 cells cm?2.Nine spontaneous and four radiation-induced 6TG-resistant mutants had <2% of the parental strain HGPRT activity and were unable to grow in medium containing azaserine. These mutants were phenotypically stable during > 25 population doublings in non-selective medium and five mutants that were examined showed no gross change from the normal human karyotype.Evidence is presented to show that 6TG is a better selective agent than 8-azaguanine (8AG) for HGPRT-deficient mutants of human diploid fibroblasts. 相似文献
5.
Spermatocytes in the late prophase of first meiotic division isolated from sterile males retain higher activities for three X-linked enzyme5, phosphoglycerate kinase (PGK)-1, glucose-6-phosphate dehydrogenase (G6PD), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) than those of fertile males. The sterilty of the male is presumed to be owing to the rearrangement of X-chromosome material and the possibility of abnormal meiotic X-chromosome inactivation is discussed. 相似文献
6.
The resistance of Chinese hamster epithelial liver cells (CHEL) and Chinese hamster fibroblasts (V79) towards toxic purine analogues has been determined. The liver cells are more sensitive than fibroblasts to 6-thioguanine (6-TG), 8-azaguanine (8-AZ) and 2,6-diaminopurine (DAP). The hypoxanthine-guanine (HGPRT) and adenine phosphoribosyl transferase (APRT) activities of extracts of CHEL cells were lower than those of corresponding extracts of V79. The level of 5'-nucleotidase was about 5-fold higher in the epithelial cells. It appears that HGPRT and APRT activities of extracts of liver epithelial cells are masked or reduced by 5'-nucleotidase activity and other inhibitors. The significance of these findings is discussed. 相似文献
7.
M.P. Uitendaal T.L. Oei C.H.M.M. de Bruyn P. Hösli 《Biochemical and biophysical research communications》1976,71(2):574-578
A new method making use of a radiochemical enzyme assay at the single cell level is presented to investigate metabolic cooperation, a widely studied form of cellular communication. In this case metabolic cooperation between normal human fibroblasts and fibroblasts derived from a patient deficient for the enzyme hypoxanthine-guanine phosphoribosyl transferase has been studied.A mixture of an equal number of both cell types was cultured in close physical contact and after trypsinisation, replating and culturing the cells for several hours in a high dilution, quantitative enzyme measurements with individual cells isolated from the mixture were carried out. From the distribution curve of the enzyme activities of the individual cells the conclusion could be drawn that a macromolecule, either the enzyme itself or DNA or mRNA, coding for that enzyme, is transferred from normal to mutant cells. 相似文献
8.
We have used [3H]guanine incorporation as a rapid and sensitive assay of xanthine-guanine phosphoribosyl transferase (GPT) activity in SV40 transformed human fibroblasts. The SV40 early promoter is more efficient than the Rous sarcoma virus long terminal repeat for transient expression of the gpt gene. The assay works well in a derivative of AT5BIVA which lacks hypoxanthine-guanine phosphoribosyl transferase (hprt-) and we show here how the assay has been adapted to work in the hprt+ AT5BIVA parent. 相似文献
9.
X-linked mutant alleles associated with prenatal male lethality are difficult to analyze because only heterozygous females
are readily available for study. Genomic analysis of the mutant allele is facilitated by the construction of somatic cell
hybrids because this enables the segregation of the X Chromosomes (Chrs) that carry the mutant and wild-type alleles. We describe
here a method that ensures that the X Chr carrying the mutant allele is retained in somatic cell hybrids in an active selectable
state. This is achieved by mating heterozygous females to males that carry a mutation at the hypoxanthine phosphoribosyl transferase
(Hprt) locus. The resultant F1 females are compound heterozygotes, and when cells from these females are fused to HPRT− Chinese hamster cells and subjected
to selection in HAT medium, the only survivors are those hybrid cells that retain an active X Chr carrying the mutant allele
together with the wild-type Hprt allele. We use hybrids constructed by this method to demonstrate that there are no gross deletions or genomic rearrangements
present in three mottled alleles associated with prenatal male lethality.
Received: 8 January 1996 / Accepted: 29 February 1996 相似文献
10.
M.J. Howell 《International journal for parasitology》1981,11(3):235-242
Using polyethylene glycol, hybrid cells were formed between rat fibroblasts lacking the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT, B.C. 2.4.2.8) and cells of the liver fluke Fasciola hepatica. The hybrid cells survived in a medium containing hypoxanthine, aminopterin and thymidine (HAT) indicating that the enzyme deficiency of the parental rat cells had been corrected. Isoelectric focusing in agarose gels showed that the HGPRT activity in the hybrids was of F. hepatica rather than rat origin. F. hepatica chromosomes could not be identified with certainty in hybrids; and fluke antigens, other than HGPRT, could not be detected in them or in culture medium in which they had grown. 相似文献
11.
Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension. 总被引:7,自引:2,他引:5
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A method for fusion of protoplasts bearing amplified plasmids and human diploid fibroblasts or other cell types in suspension is described. Transient expression of plasmid-encoded proteins occurs in up to 50% of the human cells, as demonstrated for simian virus 40 T antigen by immunofluorescence and the Escherichia coli xanthine-guanine phosphoribosyl transferase by autoradiography. In contrast, frequencies of stable transformants were similar to those obtained by the CaPO4 coprecipitation technique. However, experiments with both methods involving the recombinant pRSVneo (in which the Rous sarcoma virus long terminal repeat regulates expression of the antibiotic-inactivating aminoglycoside phosphotransferase) revealed a much higher frequency of colonies in G418 selective medium with constructions in which the early region of simian virus 40 DNA was present as well. We propose a role for the simian virus 40 T antigen in enhancing stable transformation in this system. 相似文献
12.
The effect of phorbol myristate acetate, phorbol dibutyrate, ethanol, dimethylsulfoxide, phenol, and seven metabolites of phenol on metabolic cooperation were assessed as a function of mutant cell recovery from populations of cocultivated hypoxanthine-guanine phosphoribosyl transferase-deficient mutant (HGPRT–) and wild-type (HGPRT+) Chinese hamster V79 lung fibroblasts. Phorbol myristate acetate and phorbol diputyrate, two established tumor promoters, were potent inhibitors of metabolic cooperation. Ethanol and dimethylsulfoxide, solvents commonly used to prepare chemicals for testing, weakly inhibited metabolic cooperation. Phenol and phenylglucuronide had no effect on metabolic cooperation. Four oxidative metabolites (1,4-benzoquinone, catechol, hydroxyquinol and quinol) inhibited metabolic cooperation. Phenylsulfate weakly inhibited metabolic cooperation. Conversely, 2-methoxyphenol, a methylated derivative of catechol, appeared to enhance metabolic cooperation. These results generallyAbbreviations CAS
Chemical Abstracts Service
- DMSO
dimethylsulfoxide
- ETOH
ethanol
- HGPRT
hypoxanthine-guanine phosphoribosyl transferase
- HGPRT+
HGPRT-competent
- HGPRT–
HGPRT-te]deficient
- MC
metabolic cooperation
- MC+
metabolic cooperation-competent
- MC–
metabolic cooperation-deficient
- MEM
minimum essential medium
- PDBu
phorbol dibutyrate
- PMA
phorbol myristate acetate
- 6TG
6-thioguanine
- 6TGr
6-thioguanine-resistant
- 6TGs
6-thioguanine-sensitive
- V79/MC assay
Chinese hamster V79 lung fibroblast assay for metabolic cooperation 相似文献
13.
Correction of certain mutant phenotypes by intimate contact with normal cells, i.e. ‘metabolic cooperation’, is an easily studied form of cell communication. Metabolic cooperation between normal cells and mutant cells deficient in hypoxanthine-guanine or adenine phosphoribosyl transferase (HGPRTase and APRTase respectively) appears to be the result of transfer of the enzyme product, nucleotide or nucleotide derivative, from normal to mutant cells. This process shows selectivity in that mutant derivatives of mouse L cells are unable to function as recipients of HGPRTase or APRTase products, while hamster and human fibroblasts with these enzyme deficiencies, exhibit correction of the mutant phenotype, when in contact with normal donor cells. There is also selectivity with respect to substances transferred, since other mutant phenotypes, i.e. G-6 PD deficiency, are not corrected by contact with normal cells. Species specificities do not appear to influence metabolic cooperation, therefore heterospecific cell mixtures provide an opportunity to cytologically distinguish cells and study individual cell interactions. 相似文献
14.
Chinese hamster ovary cells in the exponential phase of growth were harvested and separated by the method of centrifugal elutriation into subpopulations enriched with up to 95% G1 phase, 70% S phase and 65% G2 + M phase cells. Cell cycle distributions were routinely monitored by flow cytometry. Following elutriation, aliquots of cells from each of the enriched cell fractions were incubated in the presence or absence of 4 mM of 2-[(aminopropyl)amino] ethanethiol (WR-1065) for 30 min at 37 degrees C. The cells were then irradiated with 60Co gamma-rays or fission-spectrum neutrons from the JANUS research reactor. Both cell killing and mutagenesis were determined. Regardless of the radiation quality used, cells enriched in G1 phase were the most sensitive to radiation-induced mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase locus. The relative magnitude of protection exerted by WR-1065 differed for each of the elutriator separated cell populations. The greatest magnitude of protection, however, was observed for G1-enriched populations, regardless of the radiation quality used or the biological end-point tested. 相似文献
15.
The genotoxicity of lucidin,a natural component of Rubia tinctorum L., and lucidinethylether,a component of ethanolic Rubia extracts 总被引:1,自引:0,他引:1
Johannes Westendorf Barbara Poginsky Hildegard Marquard Gunnar Groth Hans Marquardt 《Cell biology and toxicology》1988,4(2):225-239
The genotoxic activity of lucidin (1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone), a natural component of Rubia tinctorum L., was tested in a battery of short-term tests. The compound was mutagenic in five Salmonella typhimurium strains without metabolic activation, but the mutagenicity was increased after addition of rat liver S9 mix. In V79 cells, lucidin was mutagenic at the hypoxanthine-guanine phosphoribosyl transferase gene locus and active at inducing DNA single-strand breaks and DNA protein cross-links as assayed by the alkaline elution method. Lucidin also induced DNA repair synthesis in primary rat hepatocytes and transformed C3HI M2-mouse fibroblasts in culture. We also investigated lucidinethylether, which is formed from lucidin by extraction of madder roots with boiling ethanol. This compound was also mutagenic in Salmonella, but only after addition of rat liver S9 mix. Lucidinethylether was weakly mutagenic to V79 cells which were cocultivated with rat hepatocytes. The compound did not induce DNA repair synthesis in hepatocytes from untreated rats, but positive results were obtained when hepatocytes from rats pretreated with phenobarbital were used. We conclude that lucidin and its derivatives are genotoxic.Abbreviations DMBA
7,12-dimethylbenz(a)anthracene
- HA
hydroxyanthraquinones
- LUE
lucidinethylether
- PRH
primary rat hepatocytes
- UDS
unscheduled DNA synthesis 相似文献
16.
Amelia Martínez-Ramón Santiago Grisolía 《Biochemical and biophysical research communications》1980,96(3):1011-1016
While it has been known for many years that tissues from Lesch-Nyhan patients are deficient in hypoxanthine-guanine phosphoribosyl transferase, it has been demonstrated more recently that erythrocyte lysates show an increase in the levels of enzymes which participate in “de novo” synthesis of pyrimidines. Also, we were able to demonstrate, using autoradiography, an increased rate of incorporation of aspartate into the nucleus of intact cultured fibroblasts from a Lesch-Nyhan patient. We have now confirmed this finding. In this paper we show increased incorporation of aspartate into the nucleus of cells from three other patients. Interestingly, the incorporation of orotate is decreased in the fibroblasts from the four patients studied. The incorporation of leucine is the same in Lesch-Nyhan fibroblasts as in normal fibroblasts. The significance of these findings is briefly discussed. 相似文献
17.
Rapid assay for detection of Escherichia coli xanthine-guanine phosphoribosyltransferase activity in transduced cells. 总被引:15,自引:6,他引:9
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Cultured mammalian cells transduced with the Escherichia coli gene, Ecogpt, synthesize the bacterial enzyme xanthine-guanine phosphoribosyl transferase (XGPT) (1). This paper describes a method for measuring XGPT activity in crude cell extracts by following the conversion of 14C-xanthine (X) to 14C-xanthine monophosphate (XMP) and 14C-xanthosine (XR) by thin layer chromatography. The method is rapid, easy to use, sensitive and linear over a wide range of XGPT activity and has been useful for detecting XGPT in cells that were transiently transfected or stably transformed with Ecogpt. During our studies, we have found that a human cell line (XP20S) converts xanthine to XMP. This activity is probably catalyzed by a variant hypoxanthine-guanine phosphoribosyltransferase (HGPT) since the low activity is readily inhibited by hypoxanthine. A low level of conversion of X to XMP may explain why some cell lines are not killed in a medium containing mycophenolic acid and X. 相似文献
18.
An electrophoretic method for detecting hypoxanthine-guanine phosphoribosyl transferase variants 总被引:6,自引:0,他引:6
Vazken M. Der Kaloustian Raymond Byrne William J. Young Barton Childs 《Biochemical genetics》1969,3(3):299-302
An electrophoretic method that detects hypoxanthine-guanine phosphoribosyl transferase variants is described.Aided by National Institutes of Health Grants HD 00486, HD 00004, GM 14155, and AM 10813. 相似文献
19.
Hybridization of mutant cell lines deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C.: 2.4.2.8) from a variety of established rodent sources with HGPRT plus human cells yielded progeny cells which grew in selective medium containing hypoxanthine, aminopterin and thymidine (HAT). The same result was obtained when the human cell used was an HGPRT minus transformed line derived from a patient with the Lesch-Nyhan syndrome. Electrophoretic analysis indicated that all HAT-resistant progeny clones contained an active HGPRT enzyme which was indistinguishable from the wild type enzyme of the corresponding normal rodent cells. In contrast, no HAT-resistant cells have been obtained when the same HGPRT minus rodent cells were subjected to fusion processes in the absence of human cells or when they fused with similarly derived HGPRT minus mutant cells of other rodents. Reversion in expression of the rodent gene for HGPRT was detected in clones which retained one or more human chromosomes and in clones which contained no detectable human chromosomal material. The observed re-expression of rodent HGPRT in HAT-resistant clones suggests that HGPRT plus as well as HGPRT minus human cells contributed a factor which determined the expression of respective rodent structural genes for HGPRT. In contrast, HGPRT minus rodent cells were unable to induce the synthesis or normal HGPRT in the cells derived from the patient with the Lesch-Nyhan syndrome. 相似文献
20.
N S Magnuson L E Perryman D P Suttle J L Robinson P H Mason K M Marta 《Comparative biochemistry and physiology. B, Comparative biochemistry》1985,81(3):781-786
In an attempt to determine the metabolic defect causing severe combined immunodeficiency (SCID) in horses in which altered purine metabolism has been observed, various parameters of purine and pyrimidine metabolism were evaluated. The activities of nine purine enzymes (adenosine kinase, purine nucleoside phosphorylase, deoxyadenosine kinase, deoxycytidine kinase, 5'-nucleotidase, AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase were measured in fibroblasts. All activities determined for SCID horses were normal. Uptake of 10 microM adenosine or 2'-deoxyadenosine (a growth inhibitory concentration for SCID fibroblasts) by SCID fibroblasts was identical to that found for normal fibroblasts in the presence of both 1 and 50 microM phosphate. The Km determined for the transport of both adenosine and 2'-deoxyadenosine was 35 microM. In the presence of p-nitrobenzylthioguanosine (a nucleoside transport inhibitor), 2'-deoxyadenosine uptake was inhibited to the same extent in all fibroblast lines tested. To determine if the last step in pyrimidine biosynthesis might be altered in SCID fibroblasts, UMP synthase activities were evaluated but found to be normal (0.5 nmol UMP formed/min/mg protein). 相似文献