G.G. Brownlee

The nucleotide sequence of somatic 5 S RNA from Xenopus laevis

The 5 S ribosxnal RNA of human, mouse, rabbit and rat cells have the same nucleotide sequence [l-3] , although this differs considerably from that of I:: co/i 141. In order to detertnine the extent of homology among eukaryotes we have studied the 5 S RNA of Xenvplrs luevis, the South African toad. T, and pancreatic ribonuclease digests of uniformly “2P-labelled 5 S RNA were studied and the results allow a sequence to be derived which differs only in eight positions from the mammalian sequence.

Rapid gel sequencing of RNA by primed synthesis with reverse transcriptase

Abstract The known 75-residue sequence adjacent to poly(A) in chicken ovalbumin messenger RNA was used as a model sequence to develop a generally applicable rapid gel sequencing method for RNA based on the “plus and minus” method of Sanger & Coulson (1975). The mRNA was copied in the presence of a specific “primer”, d(pT 10 -G-C), and reverse transcriptase to give 32 P-labelled complementary DNA. After reannealing mRNA, minus incubations (lacking one of the four deoxynucleoside triphosphates) were performed with reverse transcriptase. For plus reactions the Klenow fragment of DNA polymerase I ( Escherichia coli ) was used in the presence of Mn 2+ and a single deoxynucleoside triphosphate. The sequence could be read from residues 20 to 191 (counting from the poly(A)) from the pattern of bands on an autoradiograph of a 12% acrylamide gel. The new region of sequence (76 to 191) was considered to be greater than 95% accurate and probably entirely correct to residue 130 but beyond that, residues 131 to 191, there are a few uncertainties due to limitations of the method. The sequence does not overlap with the carboxyl-terminal region of ovalbumin and furthermore, the 3′ non-coding region is likely to be at least 250 residues long.

Sequence studies of the 5 S DNA of Xenopus laevis

Abstract Nucleotide sequence studies of 32 P-labelled complementary RNA transcripts of the 5 S DNA of Xenopus laevis have shown that the gene region codes for the oocyte-type 5 S RNA. Genes for the somatic-type 5 S RNA, known to have 6 of its 120 bases differing from the oocytes 5 S RNA, do not comprise more than 3% of the genes within this 5 S DNA. Twelve closely related oligomers have been identified in varying yields as sequences from the sense strand of the spacer region of 5 S DNA. One of the most common of these sequences (present in more than 3 copies per repeat) is 5′-A 4 -C-T-C-A 3 -C-T 3 -G-3′; this oligomer itself may have been derived from an ancient duplication. The tandem arrangement of these oligomers has been shown. They comprise at least half and perhaps all of the A + T-rich part of the spacer (which is 60% of each repeat).

Translation of messenger RNA for immunoglobulin light chains in a cell-free system from Krebs II ascites cells

Attempts to isolate the messenger RNA (mRNA) for immunoglobulin light (L) chains have been hampered by the lack of a reliable assay for the mRNA. Recently, however, the synthesis of a mouse light chain in a cell-free system from rabbit reticulocytes was reported [ 11. We have employed an alternative heterologous assay system, derived from Krebs II ascites cells, to identify the mRNA. Previous work has shown that both globin and lens mRNAs are faithfully translated in this system [2, 31. As a source of the mRNA we chose the mouse tumour MOPC 21 engaged in the production of an IgGl (K) immunoglobulin [4] . The light chain of this molecule has been fully sequenced [S, 61. We show here that an RNA fraction from this myeloma directs the synthesis in the ascites system of MOPC 21 light chains.

The 3′ terminal sequence of chicken ovalumin messenger RNA and its comparison with other messenger RNA molecules

The sequence of 75 bases adjacent to the 3′ terminal poly(A) region of chicken ovalbumin messenger RNA was established by copying into 32 P-labelled DNA and sequencing the DNA directly. The sequence immediately adjacent to the poly(A) differs from a mouse MOPC 21 immunoglobulin light chain mRNA; this confirms that there is no obligatory sequence homology here. But, about 20 residues internal to the poly(A), the sequence (5′)A-A-U-A-A-A-(3′) is conserved in the four different mRNAs, rabbit α- and β-globin, immunoglobulin light chain and ovalbumin, although its significance is unknown.

Comparative hybridization studies with an immunoglobulin light chain mRNA fraction and non-immunoglobulin mRNA of mouse

The messenger RNA (mRNA) for the mouse immunoglobulin light chain has been partially purified from several different mouse myelomas [l-S] . A mRNA fraction prepared from the tumour line MOPC 21 has been shown to consist of between 25% and 50% pure L-chain mRNA [6]. Such a mRNA preparation is of potential value, in DNA-RNA hybridization experiments using the DNA excess technique [7,8] for assessing the number of genes for both the variable (V) and the constant(C) region of the immunoglobulin molecule. Such experiments should help to define the extent of antibody diversity encoded as stable information in the genome and the corresponding importance of diversification. We report here that the hybridization pattern of a 13 S mRNA fraction, which contains L-chain mRNA (the LE fraction), consists of components which hybridize with repetitive and with non-repetitive elements in the DNA. A similar biphasic pattern is also shown to occur with a mRNA fraction prepared from mouse fibroblasts (which do not contain immunoglobulin mRNA) and a mRNA fraction from the nonmembrane bound polysomes of myeloma cells. A