A human dihydrofolate reductase pseudogene and its relationship to the multiple forms of specific messenger RNA

Masters, Jeffrey N.; Yang, Ji-Young; Cellini, Alessandra; Attardi, Giuseppe

doi:10.1016/s0022-2836(83)80032-1

Cited by 54 publications

(23 citation statements)

References 38 publications

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“…6C). Furthermore, the species which were present in all three cell lines were increased in amount in VA2-B and 6A3 cells, relative to HeLa cells, in approximately the same proportion as the 3.8-, 1.0-, and 0.8-kb DHFR mRNAs (25,27).…”

Section: Resultsmentioning

confidence: 86%

“…The first primer extension reactions were carried out with 6A3 polysomal poly(A)+ RNA as a template and the 52-nt EcoRI-Hinfl fragment (Fig. 1A) derived from pHD84 (25,27) as a primer ( Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

“…When the first restriction digest contained many fragments, the fragment of interest was isolated from the rest by elution from an agarose (36) or polyacrylamide (26) [6] containing 0.1 mM EDTA) and transferred to a Zeta Probe filter (Bio-Rad Laboratories) by electrophoresis in 1/3 x borate buffer as described previously (18). After baking the filter for 3 h at 80°C, the hybridization was carried out essentially as previously described (25).…”

Section: Methodsmentioning

confidence: 99%

“…These multiple mRNA species differ mainly in the length of the 3'-untranslated tail (10,25,32), which can be up to severalfold longer than the reading frame, as is the case for the major human DHFR mRNA (3.8-kilobase [kb] mRNA). Furthermore, there is good evidence indicating that the 3'-end tails of the multiple mRNAs are colinear (25,32). These observations would be compatible with the idea that the same transcript could produce different forms of mRNA by processing and polyadenylation at alternative sites.…”

mentioning

confidence: 99%

“…Thus, four major polyadenylated [poly(A)+] species of DHFR mRNA in mouse cells (32), three species in Chinese hamster cells (22), and three main species in human cells (27) have been identified. These multiple mRNA species differ mainly in the length of the 3'-untranslated tail (10,25,32), which can be up to severalfold longer than the reading frame, as is the case for the major human DHFR mRNA (3.8-kilobase [kb] mRNA). Furthermore, there is good evidence indicating that the 3'-end tails of the multiple mRNAs are colinear (25,32).…”

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confidence: 99%

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Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site.

Masters¹,

Attardi²

1985

Mol. Cell. Biol.

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The 5' ends of dihydrofolate reductase (DHFR)-specific transcripts have been mapped in the 5'-flanking region of the amplified DEHFR gene of the human methotrexate-resistant cell line 6A3 by primer extension and Si protection experiments. The main 5' end, at position -71 relative to the first nucleotide of the DHFR reading frame, corresponds to the recently identified main transcription initiation site for the DHFR gene and pertains to transcripts representing approximately 99% of the DHFR-specific polysomal polyadenylic acid-containing RNA, and including the previously described DHFR mRNAs with sizes of 3.8, 1.0, and 0.8 kilobases. At least six other minor 5' ends have been mapped to nucleotide positions -449 to -480 upstream of the DHFR gene and pertain to approximately 1% of the DHFR-specific polysomal polyadenylic acid-containing RNA. These upstream initiating transcripts appear to include five major discrete species with sizes of 4.3, 3.8, 3.1, 2.1, and 1.0 kilobases and four minor ones with sizes of 7.3, 5.0, 1.4, and 0.8 kilobases. These species, with the exception of those of 3.1-and 2.1-kilobase sizes, also have been found in VA2-B cells, the parental line of 6A3, and in HeLa cells. The upstream initiating transcripts present in all three cell lines are increased in amount in 6A3 cells as compared with the other cell lines, in about the same proportion as the three identified DHFR mRNAs.The analysis of the mode of expression of the dihydrofolate reductase (DHFR) gene in mammalian cells, which has been greatly facilitated by the availability of cell lines with amplified genes, has revealed an unsuspected complexity both in these and the parental cell lines, in the form of the existence of multiple species of DHFR mRNAs. Thus, four major polyadenylated [poly(A)+] species of DHFR mRNA in mouse cells (32), three species in Chinese hamster cells (22), and three main species in human cells (27) have been identified. These multiple mRNA species differ mainly in the length of the 3'-untranslated tail (10,25,32), which can be up to severalfold longer than the reading frame, as is the case for the major human DHFR mRNA (3.8-kilobase [kb] mRNA). Furthermore, there is good evidence indicating that the 3'-end tails of the multiple mRNAs are colinear (25,32). These observations would be compatible with the idea that the same transcript could produce different forms of mRNA by processing and polyadenylation at alternative sites.In the present work, an extensive mapping study of the 5' ends of DHFR-specific transcripts in human cell lines with an amplified or normal DHFR gene complement has unexpectedly revealed the presence, besides a main transcription initiation point at position -71 relative to the first nucleotide (nt) of the DHFR reading frame (11), of at least six additional minor sites several hundred nt's upstream of the DHFR gene, which correspond to 5' ends of DHFR-specific transcripts. These upstream initiating transcripts are found both in polysomal RNA, where they include several discrete species in the...

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Section: Resultsmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site.

Masters¹,

Attardi²

1985

Mol. Cell. Biol.

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Amplification of the thymidylate synthase gene in anN 10-propargyl-5,8-dideazafolic-acid-resistant human leukemia, MOLT-3 cell line developed in pteroylglutamic acid, but not in leucovorin

Miyachi

Takemura

Kobayashi

et al. 1996

J Cancer Res Clin Oncol

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The types of folates used during the development of resistance to methotrexate have been suggested to play an important role in the mechanisms of established resistance. In this study, effects of reduced and oxidized folates on the development of resistance to a thymidylate synthase (TS) inhibitor, N10-propargyl-5, 8-dideazafolic acid (CB3717), were examined in the human leukemia cell line MOLT-3. MOLT-3 cells were made resistant to CB3717 by soft-agar cloning in RPMI-1640 medium with either pteroylglutamic acid (PGA) or a more physiological folate (10 nM leucovorin). A 40-fold CB3717-resistant subline developed in PGA (MOLT-3/CB3717(40)-PGA) showed amplification of the TS gene with a concomitant increased level in the gene expression. A 200-fold CB3717-resistant subline (MOLT-3/CB3717(200)-PGA), which was derived from MOLT-3/CB3717(40)-PGA, showed further enhancement of amplification of the TS gene. In contrast, even a 200-fold CB3717-resistant subline developed in leucovorin (MOLT-3/CB3717(200)-LV) showed neither amplification nor overexpression of the TS gene. Both MOLT-3/CB3717(200)-PGA and MOLT-3/CB3717(200)-LV cells showed decreased membrane transport of PGA as well as methotrexate. These results suggest that the types of folates used during the development of CB3717 resistance may play a role in resistance, and that impaired transport of PGA, in CB3717-resistant MOLT-3 cells developed in PGA, might have accelerated amplification of the TS gene.

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The role of folates in the development of methotrexate resistance in human leukemia cell line K562

Miyachi

Takemura

Andō

et al. 1992

J Cancer Res Clin Oncol

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The effect of reduced and oxidized folates on the development of methotrexate (MTX) resistance has been examined in human leukemia cell line K562 (K562/S). K562/S cells were made resistant to MTX by soft-agar cloning either in RPMI-1640 medium (K562/MTX-PGA) or in folic-acid-free RPMI-1640 medium containing 10 nM leucovorin (K562/MTX-LV). The optimal concentrations of leucovorin for the growth of K562/S, K562/MTX-PGA and K562/MTX-LV cells were 1 nM, 5 nM and 10 nM respectively. K562/MTX-PGA cells were 24-fold resistant to MTX as noted by impaired MTX transport. In contrast, K562/MTX-LV cells were 26-fold resistant to MTX as noted by gene amplification of dihydrofolate reductase. Furthermore cross-resistance to cytosine arabinoside was only demonstrated in K562/MTX-PGA, while the K562/MTX-LV cells showed no significant cross-resistance to cytosine arabinoside. These results suggest that the type and level of folates used during the development of MTX resistance may play a role in the mechanism for MTX resistance. Leukemia cells that are grown in leucovorin might serve as a model for acquired MTX resistance in vivo.

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A human dihydrofolate reductase pseudogene and its relationship to the multiple forms of specific messenger RNA

Cited by 54 publications

References 38 publications

Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site.

Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site.

Amplification of the thymidylate synthase gene in anN 10-propargyl-5,8-dideazafolic-acid-resistant human leukemia, MOLT-3 cell line developed in pteroylglutamic acid, but not in leucovorin

The role of folates in the development of methotrexate resistance in human leukemia cell line K562

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