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Inserts within the mRNA sequence on the 15 S globin mRNA precursor from chicken
Abstract:
[3H]Uridine pulse-labelled nuclear RNA of chicken immature red blood cells contains two RNA species with globin coding sequences sedimenting at 9 S and 15 S, the latter representing most probably the precursor to 9 S globin mRNA. Whether the globin mRNA sequence on the 15 S precursor is interrupted by interspersed sequences was investigated by hybridization of labelled nuclear 15 S RNA with an excess of unlabelled globin complementary DNA. After degradation of nonhybridized RNA by RNAase A the enzyme was removed by proteinase K and a subsequent phenol extraction. The complementary DNA hybrids were melted and the complementary DNA degraded by DNAase I. For the separation of complementary DNA protected RNA polyacrylamide gel electrophoresis in formamide was used. Three RNA fragments with approximately 320, 200 and 120 nucleotides were found in the case of the 15 S RNA, whereas only one 650-700 nucleotide fragment was detected when the same procedure was carried out with nuclear 9 S RNA. This means, that the chicken 15 S globin mRNA precursor contains at least two inserts within the mRNA sequence, which originate presumably by transcription of a gene with intervening sequences.
Insights
Chicken red blood cells contain precursor globin messenger RNA (mRNA) with interspersed sequences. These intervening sequences are found in the 15S precursor RNA, indicating a gene structure with introns.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Chicken immature red blood cells possess nuclear RNA with globin sequences.
- Two RNA species, 9S and 15S, contain globin coding sequences, with 15S likely being the precursor to 9S globin mRNA.
Purpose of the Study:
- To investigate if the globin mRNA sequence within the 15S precursor RNA is interrupted by interspersed sequences.
- To determine the structure of the globin mRNA precursor in chicken red blood cells.
Main Methods:
- Hybridization of radiolabeled nuclear 15S RNA with excess unlabeled globin complementary DNA (cDNA).
- Degradation of non-hybridized RNA using RNase A, followed by proteinase K treatment and phenol extraction.
- Melting of cDNA-RNA hybrids and degradation of cDNA using DNase I.
- Separation of cDNA-protected RNA fragments via polyacrylamide gel electrophoresis in formamide.
Main Results:
- Analysis of 15S RNA revealed three RNA fragments (approx. 320, 200, and 120 nucleotides) after processing.
- Analysis of 9S RNA yielded a single fragment (650-700 nucleotides).
- The presence of multiple fragments from 15S RNA indicates interruptions within the globin mRNA sequence.
Conclusions:
- The 15S globin mRNA precursor in chicken red blood cells contains at least two inserts within the mRNA sequence.
- These inserts suggest that the globin gene in chickens is transcribed from a gene with intervening sequences (introns).
- This finding provides evidence for the existence of split genes in eukaryotes.