Presence of a putative 15S precursor to β-globin mRNA but not to α-globin mRNA in Friend cells

Abstract

Dimethyl sulfoxide-induced Friend cells were labeled for periods of 5-60 min. The denatured RNA was fractionated by sucrose gradient centrifugation and the distribution of a-and ,-globin-specific [3HJRNA was determined by hybridization to hybrid plasmids containing mouse a-and lobin DNA, respectively. After 5 min of labeling, a 15S peak of ,-globin-specific (but not a-gobin-specific) [ In this paper we report on the kinetics of labeling as well as on the isolation of radioactive globin-specific 15S RNA and lOS RNA from short-term labeled Friend cells. Analysis of these RNAs by fingerprinting techniques and by hybridization to cloned mouse a-and fl-globin cDNA showed that the 15S peak contained f3-globin-specific RNA but not a-globin-specific RNA, as well as additional sequences not found in mature globin mRNAs. After short labeling periods, a-globin-specific RNA The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.of slightly higher molecular weight than the corresponding mature mRNA was detected.MATERIALS AND METHODS Growth and dimethyl sulfoxide-induction of Friend cells, RNA extraction, labeling procedures, and fingerprinting and sequencing were as reported previously (9). The analytical and preparative applications of hybrid isolation by poly(I)-Sephadex chromatography also have been described (9).Hybrid plasmids consisting of PCRI (12) by using hybridization to filter-bound a-or ,B-globin hybrid DNA as described by Weinberg et al. (20). (Fig. 1 a-c). The large oligonucleotides were characterized by digestion with pancreatic RNase and identified with the oligonucleotides isolated earlier from a mixture of mouse a-and (3-globin mRNA; the numbering used in Fig. 1 is the same as in the previous work (9). The identification of oligonucleotides as being derived from mouse a-or fl-globin mRNA had earlier been made by matching partial nucleotide sequences with the known amino acid sequences; the present work confirms all but one of these assignments. RESULTS

Precursors of distinct size for chicken αA, αD and β globin mRNAs

“…It was maximal between 2-6 X l0 s M r and represented molecules the size of mature globin mRNA as well as the '15 S' globin pre-mRNA. The latter species is the best described globin precursor in the erythroid systems studied [3][4][5][6][7][8][9]. At steady state it accounts for ~2000 molecules/cell.…”

Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing.

“…This has been suggested by kinetic analysis of globin (1,8,14), immunoglobulin (28), and adenovirus mRNA synthesis (20). However, none of these reports could exclude the possibility that introns can also be removed during transcription.…”

Comparison of cloned mouse alpha- and beta-globin genes: conservation of intervening sequence locations and extragenic homology.

“…In addition, the mature form of the a-globin mRNA appears to be synthesized via a small 850-base precursor, approximately 250 bases larger than the cytoplasmic a-globin mRNA (25,26). It is likely that certain aspects of the structure and organization of one a-globin gene will allow us to account for its joint expression with other a-globin genes located on the same chro-…”