Cloned cDNA sequences of the hypoxanthine/guanine phosphoribosyltransferase gene from a mouse neuroblastoma cell line found to have amplified genomic sequences.

Brennand, John; Chinault, A. Craig; Konecki, David; Melton, David W.; Caskey, C. Thomas

doi:10.1073/pnas.79.6.1950

Cited by 111 publications

(45 citation statements)

References 33 publications

(32 reference statements)

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“…The cloned cDNA for mouse HGPRT, pHPT2, was shown by Brennand et al (6) to hybridize with HGPRT sequences in Chinese hamster mRNA and DNA. Thus, it was suitable for use as a radioactively labled probe to examine the HGPRT gene in DNA from our mutants.…”

Section: Resultsmentioning

confidence: 99%

“…agarose gel, and transferred to a nitrocellulose filter. To identify restriction fragments containing HGPRT sequences, DNA on the filter was hybridized to a radiolabeled HGPRT cDNA contained on an HpaII fragment of the recombinant plasmid pHPT2 (6 Fig. 2, as was the case in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The mouse HGPRT cDNA (pHPT2) is a 1,050-base-pair insert at the PstI site of pBR322 which contains the 3' end and about 70% of the mRNA sequence (6 High-molecular-weight DNA was isolated from cultured cells according to Pellicer et al (27) and cleaved with restriction enzymes under conditions recommended by the manufacturer (Bethesda Research Laboratories or New England Biolabs). The fragments were fractionated on agarose gels and transferred to nitrocellulose essentially as described by Southern (35) except that the gel was soaked sequentially in 0.5 N NaOH-1.5 M NaCl for 2 h and 0. labeled pHPT2 HpaII fragment per ml.…”

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Deletion and amplification of the HGPRT locus in Chinese hamster cells.

Fuscoe¹,

Fenwick²,

Ledbetter³

et al. 1983

Mol. Cell. Biol.

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144

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Somatic cell selective techniques and hybridization analyses with a cloned cDNA probe were used to isolate and identify Chinese hamster cell lines in which the X-linked gene for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) has been altered. Two of 19 HGPRT-deficient mutants selected were found to have major DNA deletions affecting the HGPRT locus. Cytogenetic studies revealed that the X chromosome of each deletion mutant had undergone a translocation event, whereas those from the remaining 17 mutants were normal. Phenotypic revertants of the thermosensitive HGPRT mutant RJK526 were isolated, and amplification of the mutant allele was shown to be the predominant mechanism of reversion. Comparisons of restriction enzyme fragments of DNA from deletion versus amplification strains identified two regions of the Chinese hamster genome that contained homology to the cDNA probe. One was shown to be much larger than the 1,600-nucleotide mRNA for HGPRT and to be comprised of linked fragments that contained the functional HGPRT gene. The second was neither transcribed nor tightly linked to the functional gene. These initial studies of HGPRT alterations at the level of DNA thus identified molecular mechanisms of phenotypic variation.The X-linked locus for hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8) has been the focus of many investigations into the mechanisms of mutation and reversion in cultured mammalian cells (7,13,19). In part this is due to the availability of efficient forward and reverse selective protocols (36). In addition, the functional hemizygosity of the gene in most cells has facilitated the design and interpretation of experiments. Finally, the fact that HGPRT mutations in humans can cause gout (20) or the Lesch-Nyhan syndrome (33) has also drawn attention to this gene.In the absence of selective medium, HGPRT is a nonessential enzyme in cultured cells. Thus, there should be few if any constraints upon the types of mutations present in HGPRT-deficient cells. However, it has been difficult to classify or localize the mutation in any particular cell line because genetic techniques such as analysis by mitotic recombination are not yet applicable to mammalian cells. As reviewed by Caskey and Kruh (7), the best evidence for the presence of mutations in the HGPRT gene has come from demonstrations that particular clones produce t Present address:

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Deletion and amplification of the HGPRT locus in Chinese hamster cells.

Fuscoe¹,

Fenwick²,

Ledbetter³

et al. 1983

Mol. Cell. Biol.

Self Cite

144

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“…Thus the mutant phenylalanine hydroxylase gene must be associated with the 7kb allele in the father which he has also transmltted to an unaffected sibling homozygous for the 7 kb band. Since this sibling is also homozygous for the 4kb HindIll band, the (Old & Higgs, 1983) Collagen Sykes, 1983;Prockop & Kivirikko, 1984) Human growth hormone (Moore et al, 1982) al-Antitrypsin (Kidd et al, 1983) Hypoxanthine phosphoribosyltransferase (Jolly et al, 1982;Brennand et al., 1982) Phenylalanine hydroxylase Factor IX (Choo et al, 1982) Factor VIII Glucose-6-phosphate dehydrogenase (Persico et al, 1981) Ornithine transcarbamoylase (Old et al, 1985) Antithrombin-3 (Bock et al, 1983) Low density lipoprotein receptor (Russell et al, 1983); 3-hydroxy-3-methylglutaryl-CoA reductase (Chin et al, 1982); apolipoproteins (Tolleshaug et al, 1983) Oncogenes (Hamlyn & Sikora, 1983) Immunoglobins Arnold et al, 1983) T-cell receptor (Toyonaga et al, 1984) . This is important for future genetic counselling and will help in the understanding of the factors involved in the non-disjunction resulting in the chromosomal trisomy.…”

Section: Vol 226mentioning

confidence: 99%

An appraisal of the application of recombinant DNA techniques to chromosome defects

Ellis¹,

Davies²

1985

Biochemical Journal

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“…Partial deficiency of HPRT leads to severe gouty arthritis (27). Previously, we reported the identification and characterization of the complete murine, hamster, and human cDNA sequences for HPRT (3,28). These cDNAs have been used as probes to identify and isolate the murine, human, and Chinese hamster hprt genomic sequences (30,36).…”

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confidence: 99%

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

Chang¹,

Wager-Smith²,

Tsao³

et al. 1987

Mol. Cell. Biol.

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Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 x 103 to 5 x 104/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3-to 10-fold by cocultivation of the ecotropic *2 and amphotropic PA-12 helper cells. Higher viral titers (8 x 105 to 7.5 x 106) were obtained when nonproducer NIH 3T3 cells or I2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT-(6-thioguanine resistant [6TGF]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418' could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.

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Cloned cDNA sequences of the hypoxanthine/guanine phosphoribosyltransferase gene from a mouse neuroblastoma cell line found to have amplified genomic sequences.

Cited by 111 publications

References 33 publications

Deletion and amplification of the HGPRT locus in Chinese hamster cells.

Deletion and amplification of the HGPRT locus in Chinese hamster cells.

An appraisal of the application of recombinant DNA techniques to chromosome defects

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

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