In the yeast Saccharomyces cerevisiae the 5′ and 3′ splice junctions and the internal branch acceptor site (TACTAAC box) are highly conserved intron elements. Analyses of mutants have demonstrated the importance of each of these elements in the splicing process. In the present report we show by three different analytical approaches (splicing‐dependent beta‐galactosidase expression, in vitro splicing assays and in vivo RNA analyses) that at least two of these elements (the TACTAAC and 3′ splice signals) also have to fulfill certain spacing requirements to allow efficient splicing to occur. In particular, the spacing of the 3′ splice site from the 2′‐5′ branch site is a critical factor in determining the efficiency for completion of the final reactions of splicing, intron release and exon‐exon joining. Whereas insertions within this region have little or no effect on the first reactions in splicing (the 5′ cleavage and 2′‐5′ branch formation), they dramatically affect the efficiency of the final reactions. In contrast, a 15‐base deletion between these two sites has no detectable effect on splicing efficiency. We also show that the 5′ cleavage and branch formation can take place, albeit inefficiently, in transcripts in which all of the yeast sequences downstream of the branch site have been replaced by Escherichia coli sequences. We conclude from these studies that, in yeast, the 5′ and 3′ splice sites are recognized independently from one another, but always in conjunction with the TACTAAC signal.
We have investigated the topology of the human jJ2-adrenerglc receptor expressed inEscherichia col, using the genetic method described by B wth and coworkers. We found that fusin with alkali phhatse beyond a certain point on the human 2-adrenerglc receptor sequence were assembled into the bacterial membrane with the same topology as the human P2-adrenergic receptor in the mammalan membrane.
We constructed a translational fusion between the Saccharomyces cerevisiae actin gene and the Escherichia coli (-galactosidase structural gene such that expression of 0-galactosidase activity required accurate splicing of the actin intron. Using this chimeric gene, we generated a series of internal deletions which removed the TACTAAC box or, in addition, TACTAAC-like sequences within the intron. Analysis of the fusion transcripts produced in these deletions allowed us to conclude that the TACTAAC-like sequence TACTAAG can substitute, albeit inefficiently, for the authentic TACTAAC box in the splicing process. These results indicate that the yeast splicing machinery can utilize a cryptic TACTAAC box, but there are requirements for primary sequence and proper position.A key step in the splicing process is the definition of the precise boundaries of the intron by the splicing machinery. In the yeast Saccharomyces cerevisiae, a subset of nuclear genes encoding mRNAs contain intervening sequences (IVS) (34). Previous work has identified two regions within the intron which are required for splicing. The sequence at the 5' intron-exon junction, although subject to substantial variation about a consensus sequence in metazoans, in yeasts is an essentially 100%o conserved hexanucleotide (11). This apparent lack of variance in yeast splicing signals allowed the identification of an additional conserved sequence within the intron, the so-called TACTAAC box, a heptanucleotide found in all yeast introns 4 to 53 nucleotides upstream of the AG at the 3' splice junction. The importance of these conserved sequences in the splicing process was demonstrated by the observation that deletion of either the 5' consensus sequence (4) or the TACTAAC box (11,12,26) abolishes splicing and leads to the accumulation of fulllength precursor (11,12,26).While consensus sequences at the 5' (GTAAGT) and 3' [(PY)nAG] intron-exon junctions had been recognized in metazoan introns (1, 21), there has been no evidence to suggest an analog of the TACTAAC box. This apparent difference has recently been verified with the development of extracts which carry out the splicing reaction in vitro. A detailed analysis of the transcripts in both mammalian and yeast extracts (22,24), and more recently the characterization of intermediates in the splicing process in vivo (3,28,37), has shown that splicing of pre-mRNA in both systems occurs via a common intermediate. This intermediate has been termed a lariat, because the 5' end of the intron is joined by a 2'-5' phosphodiester bond to a site within the intron. The location of this 2'-5' linkage, or branch (36), is within the TACTAAC box in yeasts (3). In mammalian cells, branch points are determined primarily by their location with respect to the 3' splice site regardless of the sequence surrounding the branch point, and an obvious consensus sequence is less readily identifiable (9,24,27,30,31).The novel nature of these intermediates argues that splicing must proceed by fundamentally similar mechanisms in * Corresp...
The biological role of simple repetitive DNA consisting of short, tandemly arrayed sequences, is still enigmatic. Previously we have described how simple quadruplet repeats (sqr) isolated from a sex-specific snake satellite {Elaphe radiata) are also "sex-specifically" arranged in mouse and man (Epplen et al, '81, '82a). In the mouse, these sqr are interspersed on all chromosomes, but a substantial part is concentrated in the pericentromeric region of the Y-chromosome (Epplen et al., '82b). In one of the subcloned snake satellite DNA components (pErs5), the sqr are adjacent to a single-copy sequence that in turn identifies a male-specific putative mRNA in the mouse (Epplen et al., '82b). The isolated stretch of sqr themselves cross-hybridizes with at least two species of poly (A)+ RNA from mice occurring in both sexes (Epplen et al., '82b, '83). By examining one of the non-sex-specific transcripts, we demonstrate here substantial open reading frames in the corresponding 2.5-kilobase &b)-long mouse cDNA clone, including some 130 copies of sqr. The results are discussed in the context of mechanisms of primary sex-determination in vertebrates, in particular the gonadal induction mechanism in heterogametic sex determination.
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