Probable cloning artefacts previously interpreted as unusual leader sequences of rodent ornithine decarboxylase mRNAs--a cautionary tale

Messenger RNAs that have structurally unusual 5' leaders attract interest and provoke conjecture. Cloning and sequencing of two rodent ornithine decarboxylase (ODC) cDNAs, those for mouse [Kahana and Nathans, Proc. Natl. Acad. Sci. USA 82 (1985) 1673-1677] and, recently as published in this journal, for rat [Van Kranen et al., Gene 60 (1987) 145-155], have indicated the presence of such features. In both cases, the leader is unusually long and contains multiple AUG start codons preceding that which encodes the N terminus of the protein. In addition, the leader of the rat clone contains a 54-nt perfect inverted repeat. Because ODC expression appears to be regulated translationally, functional implications immediately suggest themselves. Certain unusual features of the mouse cDNA have proven artefactual [Brabant et al., Proc. Natl. Acad. Sci. USA 85 (1988) 2200-2204; Katz and Kahana, J. Biol. Chem. 263 (1988) 7604-7609]. It is likely that the putative leader sequence of rat ODC cDNA also resulted from a cloning artefact.     

Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei.

The process of trans splicing is essential to the maturation of all mRNAs in the Trypanosomatidae, a family of protozoan parasites, and to specific mRNAs in several species of nematode. In Trypanosoma brucei, a 39-nucleotide (nt) leader sequence originating from a small, 139-nt donor RNA (the spliced leader [SL] RNA) is spliced to the 5' end of mRNAs. An intermediate in this trans-splicing process is a Y structure which contains the 3' 100 nt of the SL RNA covalently linked to the pre-mRNA via a 2'-5' phosphodiester bond at the branch point residue. We mapped the branch points in T. brucei alpha- and beta-tubulin pre-mRNAs. The primary branch acceptors for the alpha- and beta-tubulins are 44 and 56 nt upstream of the 3' splice sites, respectively, and are A residues. Minor branch acceptors were detected 42 and 49 nt upstream of the alpha-tubulin splice site and 58 nt upstream of the splice site in beta-tubulin. The regions surrounding these branch points lack homology to the consensus sequences determined for mammalian cells and yeasts; there is also no conservation among the sequences themselves. Thus, the identified sequences suggest that the mechanism of branch point recognition in T. brucei differs from the mechanism of recognition by U2 RNA that has been proposed for other eucaryotes.     

Cloning and nucleotide sequence of the murine hsp84 cDNA and chromosome assignment of related sequences

The nucleotide (nt) sequence of mouse 84-kDa heat shock protein (Hsp) cDNA has been determined using a combination of molecular cloning and oligodeoxynucleotide priming on poly(A) + RNA. The cDNA was 2.5 kb long, not including the poly(A) tail. It contained a 5' leader of about 94 nt that was G + C-rich, and a 243-nt 3'-untranslated region that was A + T-rich in the vicinity of the polyadenylation signal. Gene hsp84 codes for an acidic polypeptide of 724 amino acid (aa) residues. Mouse Hsp84 had 81% and 63% aa homology to Drosophila melanogaster Hsp82 and yeast Hsp90, respectively. The nucleotide sequence had 74% and 59% homology to Drosophila and yeast hsp sequences, respectively, in the coding regions of these genes. This homology did not extend to the 5' - and 3'-untranslated regions. Chromosomal analysis indicated that hsp84-related sequences are on at least three different chromosomes.     

Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro.

Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.     

Structure of the gene for human butyrylcholinesterase. Evidence for a single copy

We have isolated five genomic clones for human butyrylcholinesterase (BChE), using cDNA probes encoding the catalytic subunit of the hydrophilic tetramer [McTiernan et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6682-6686]. The BChE gene is at least 73 kb long and contains four exons. Exon 1 contains untranslated sequences and two potential translation initiation sites at codons -69 and -47. Exon 2 (1525 bp) contains 83% of the coding sequence for the mature protein, including the N-terminal and the active-site serine, and a third possible translation initiation site (likely functional), at codon -28. Exon 3 is 167 nucleotides long. Exon 4 (604 bp) codes for the C-terminus of the protein and the 3' untranslated region where two polyadenylation signals were identified. Intron 1 is 6.5 kb long, and the minimal sizes of introns 2 and 3 are estimated to be 32 kb each. Southern blot analysis of total human genomic DNA is in complete agreement with the gene structure established by restriction endonuclease mapping of the genomic clones: this strongly suggests that the BChE gene is present in a single copy.     

Immunochemical detection of the alpha-subunit of the G-protein, GZ, in membranes and cytosols of mammalian cells

An antiserum (AS 98) was raised against a synthetic peptide deduced from published cDNA sequences of the alpha-subunit of the putative G-protein, GZ (Fong et al. Proc. Natl. Acad. Sci. USA 85, 3066-3070, 1988; Matsuoka et al. Proc. Natl. Acad. Sci. USA 85, 5384-5388, 1988). In membrane and cytosol preparations of many but not all tested mammalian tissues, AS 98 predominantly recognized two proteins of 40 and 43 kDa Mr. Whereas high levels of a 40 kDa GZ alpha-subunit were found in rat liver membranes and in brain cytosol, AS 98 failed to detect the alpha-subunit of GZ in brain membranes.     

Analysis of the human apolipoprotein B gene; complete structure of the B-74 region

In this paper we describe the nucleotide sequence of the B-74 region of human apolipoprotein B-100 mRNA. This region comprises the 3'-proximal three-quarters of the mRNA and contains 10,089 nucleotides (nt), 9786 of which are coding. Combining our data with the published sequence of the 5'-proximal one-quarter (i.e., the B-26 region [Protter et al., Proc. Natl. Acad. Sci. USA 83 (1986) 5678-5682] assigns 14,059 nt to the apoB-100 mRNA. The coding sequence spans 13,548 nt or 4516 amino acids (leader peptide excluded). The B-74 part of the apoB gene is built up of five exons separated by small introns, and is dominated by an unusually large exon of 7.5 kb. The derivation of two (EcoRI and XbaI) restriction fragment length polymorphisms occurring in the coding region is discussed.     

Characterization of mRNA species related to human liver cytochrome P-450 nifedipine oxidase and the regulation of catalytic activity

P-450NF is the major enzyme in human liver involved in the metabolism of the calcium-channel blocker nifedipine. By screening a bacteriophage lambda gt11 expression library, a cDNA clone designated NF 10 with an insert length of 2.8 kilobases (kb) was isolated. This clone was sequenced and found to be highly similar in its overlapping section with sequences of two other cDNA clones previously isolated from the same expression library, NF 25 (Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S., and Guengerich, F. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8064-8068) and HLp (Molowa, D. T., Schuetz, E. G., Wrighton, S. A., Watkins, P. B., Kremers, P., Mendez-Picon, G., Parker, G. A., and Guzelian, P. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5311-5315). However, clone NF 10 had an extra 814 or 813 bases of 3'-noncoding sequence relative to NF 25 or HLp, respectively, and this additional sequence contained a second consensus polyadenylation signal. Specific oligonucleotides were synthesized to differentiate between these three clones at the mRNA level. Oligonucleotides specific to the protein coding region of each clone were found to hybridize to mRNAs of 2.2 and 3.0 kb in size at a ratio of approximately 10:1. The major species of hybridizable mRNA was specific to clone NF 25, and levels of this mRNA could be correlated with levels of immunochemically detectable P-450NF and nifedipine oxidase activity in individual human liver samples. In addition, an oligonucleotide specific to the 3'-noncoding region of clone NF 10 hybridized only with the 3.0-kb mRNA. We conclude that alternative use of the second polyadenylation signal present in clone NF 10 results in production of the 3.0-kb mRNA species and that a pretranslational control mechanism is primarily involved in the regulation of nifedipine oxidase activity.     

Isolation of cDNA clones of human argininosuccinate lyase and corrected amino acid sequence

In the present study, we isolated clones of human argininosuccinate lyase (ASL) cDNA from a liver cDNA library using a clone of rat ASL cDNA and analyzed human ASL cDNA nucleotide sequence. The results reveal that the sequence of human ASL cDNA published by O'Brien et al. in 1986 [Proc. Natl. Acad. Sci USA 83, 7211-7215] had one-base deletions at three independent positions in the coding regions near the COOH-terminus, which caused frame-shift variations in the amino acid sequence. Amino acid sequencing of peptides prepared from purified human liver ASL showed our predicted amino acid sequence to be correct.