Efficient recovery of functionally intact mRNA from agarose gels via transfer to an ion-exchange membrane

Holland, Leneè S.; Wangh, Lawrence J.

doi:10.1093/nar/11.10.3283

Cited by 26 publications

(10 citation statements)

References 30 publications

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“…For production of specific primers or fragments to be used in S1 protection experiments, 10 to 50 ,ug of plasmid DNA was first digested with a restriction enzyme producing the desired end, treated with calf intestinal alkaline phosphatase (Boeringer Mannheim Biochemicals), and labeled at the 5' ends with T4 polynucleotide kinase (Bethesda Research Laboratories) and [y-32P]ATP (ICN Pharmaceuticals Inc.); the DNA was then spermine precipitated (19) to remove the bulk of unincorporated nt's and digested with a second restriction enzyme, and the fragments of interest, labeled at only one end, were isolated by elution from polyacrylamide gels (26). 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%

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: Methodsmentioning

confidence: 99%

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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“…To isolate the mRNAs coding for the Aa, BP, and -y chains of fibrinogen, liver mRNA was fractionated in a methylmercuric hydroxide agarose gel and recovered from the gel by transfer to and elution from a DEAE membrane (13). The high resolution of the RNA achieved during electrophoresis was preserved by slicing the DEAE membrane into 1-mm sections.…”

mentioning

confidence: 99%

“…1 from the gel to a DEAE membrane, the membrane was cut into sections 1 mm in width (perpendicular to the original direction of migration of the RNA), and the RNA was eluted as described previously (13). One-fifth of the RNA recovered from each section was used in an in vitro wheat germ translation reaction of 25 p.I, under our standard conditions (13). Then 10 p.1 from each translation reaction was analyzed in an SDS-polyacrylamide gel.…”

mentioning

confidence: 99%

“…Then 10 p.1 from each translation reaction was analyzed in an SDS-polyacrylamide gel. Electrophoresis and autoradiography were performed as before (13,33). After fixation in 45% methanol-10% acetic acid, gels were treated with En3Hance (New England Nuclear Corp.) as described by the manufacturer.…”

mentioning

confidence: 99%

“…To test this possibility, a cDNA probe was prepared by reverse transcription of the mRNA recovered in fractions 3 and 4. Synthesis of cDNA was performed as described previously (13) except that the 20-,lI reaction contained 3.5 ,uM TTP and 100 ,uCi of [a-32P]-TTP (3,200 Ci/mmol; New England Nuclear Corp.).…”

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confidence: 99%

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Xenopus fibrinogen: characterization of the mRNAs for the three subunits.

Holland¹,

Wangh²

1984

Mol. Cell. Biol.

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We purified and characterized the mRNAs coding for each of the three subunits of Xenopus fibrinogen. Purification was accomplished by electrophoretic separation of liver polyadenylated RNA in a fully denaturing gel, followed by recovery of the RNA from the gel via transfer to an ion-exchange membrane. This procedure yielded fractions which were highly enriched for the mRNAs for each of the fibrinogen chains. The fibrinogen mRNAs were identified by two methods: (i) in vitro translation followed by subunit-specific cleavage with the proteases thrombin and batroxobin; and (ii) cross-hybridization with cDNA clones for individual subunits of rat fibrinogen. The results demonstrate that the Aca and -y chains of frog fibrinogen are each coded by a single mRNA species. The Aca mRNA is ca. 3,100 nucleotides in length, which is nearly twice the minimum size required to code for the Aca precursor polypeptide. The y chain mRNA comprises about 1,600 bases and includes only a small untranslated region. In contrast, the BP subunit is synthesized from two mRNAs, one of which is 2,500 and the other 1,800 nucleotides long. The 2,500-base mRNA includes a large noncoding region, whereas the smaller one is near the minimum required size. The larger BP8 mRNA is ca. fivefold more abundant that the smaller species.Fibrinogen, which is synthesized and secreted by the liver, is found in the plasma of all vertebrates. The intact molecule comprises two sets of three nonidentical subunits designated Aax, BP, and -y. During the process of blood coagulation, the proteolytic enzyme thrombin cleaves the NH2-terminal A and B fibrinopeptides from the Aa and BP chains, thus generating fibrin which forms the structural matrix of a blood clot.We have previously described the polypeptide chain composition of fibrinogen from the frog Xenopus laevis (14,33 inducing the acute-phase response in mammals (16,19), was used in an effort to increase fibrinogen production in frogs. Polyadenylated RNA was purified (13) from the liver offrogs that had received a single injection of pure spirits of turpentine (Parks) into the leg muscle at a dose of 83 ,Il/50 g of body weight 12 to 24 h before sacrifice. This mRNA preparation was slightly enriched for the fibrinogen mRNAs relative to albumin mRNA, based on the in vitro translation assay, and was therefore used for the experiments described here. The sizes of all of the fibrinogen translation products and mRNAs were identical whether the RNA had been isolated from normal or turpentine-treated animals.To isolate the mRNAs coding for the Aa, BP, and -y chains of fibrinogen, liver mRNA was fractionated in a methylmercuric hydroxide agarose gel and recovered from the gel by transfer to and elution from a DEAE membrane (13). The high resolution of the RNA achieved during electrophoresis was preserved by slicing the DEAE membrane into 1-mm sections. The RNA eluted from each membrane slice was translated in vitro, and the products were resolved in a sodium dodecyl sulfate (SDS)-polyacrylamide gel (Fig. 1). All translated...

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Electrophoresis and recovery of active mRNA from composite ultra low/medium gelling temperature agarose gels

Fourcroy

1984

Electrophoresis

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Efficient recovery of functionally intact mRNA from agarose gels via transfer to an ion-exchange membrane

Cited by 26 publications

References 30 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.

Xenopus fibrinogen: characterization of the mRNAs for the three subunits.

Electrophoresis and recovery of active mRNA from composite ultra low/medium gelling temperature agarose gels

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