The beta-tropomyosin gene in chicken contains two mutually exclusive exons (exons 6A and 6B) which are used by the splicing apparatus in myogenic cells, respectively, before (myoblast stage) and after (myotube stage) differentiation. The myoblast splicing pattern is shown to depend on multiple sequence elements that are located in the upstream intron and in the exon 6B and that exert a negative control over exon 6B splicing. This regulation of splicing is due, at least in part, to a secondary structure of the primary transcript, which limits in vivo the accessibility of exon 6B in myoblasts.

The POLI gene, encoding DNA polymerase a (pola) in Saccharomyces cerevisiae, is transiently transcribed during the cel cycle at the G1/S phase boundary. Here we show that yeast pola is present at every stage of the ceUl cycle, and its level only slightly increases following the peak of POLI transcription. POLI mRNA synthesis driven by a GAL] promoter can be completely abolished without affecting the growth rate of logarithmically growing yeast cultures for several cell divisions, although the amount of the pola polypeptide drops below the physiological level. Moreover, a-factor-arrested cells can enter S phase and divide synchronously even if POLI transcription is abolished. These results indicate that the level of yeast pola is not rate limiting and de novo synthesis of the enzyme is not required for entrance into S phase.Eukaryotic DNA polymerase a (pola), together with the tightly bound DNA primase, plays an essential role in lagging strand synthesis and initiation of DNA replication at an origin (1-3). The gene encoding pola in Saccharomyces cerevisiae (POLI) is transiently transcribed during the cell cycle at the G1/S phase boundary (4) concomitantly with several DNA synthesis genes (for a review, see refs. 5 and 6). A conserved promoter sequence, the Mlu I cell cycle box (MCB), mediates this transcriptional control and is present twice in the POLI gene (7-9). One component of the transcription factor(s) that binds to the MCB is the SWI6 gene product, whose function is relevant for cell cycle-dependent transcription of yeast DNA synthesis genes (10-12). However, SWI6 deletion is not lethal but leads to deregulated constitutive transcription of these genes (10, 11). Moreover, it has been shown that the level of proteins required for Saccharomyces cerevisiae DNA replication (replication factor A) or for entrance into S phase (CDC46 gene product) does not show any large fluctuation during the cell cycle (13,14), although the transcription of the corresponding genes is clearly periodic (14,15). A nearly constant amount of essential replication proteins has been observed also in actively cycling cells from other eukaryotes, including mammalian pola, RF-A, proliferating cell nuclear antigen (PCNA), DNA ligase (13,(16)(17)(18)(19), and Schizosaccharomyces pombe pola, PCNA, and DNA ligase (20)(21)(22). However, the transcription of the corresponding genes in these organisms does not appear to be cell cycle regulated. These findings leave uncertain as to whether the transcriptional activation of DNA replication genes observed in Saccharomyces cerevisiae is required for the onset of DNA replication in S phase.Our goal in the present work was to establish whether the amount of pola in Saccharomyces cerevisiae is rate limitingThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. and periodic transcription of the POLI gene is necessary for entrance into S pha...

To assess the role of eukaryotic DNA primase in vivo, we have produced conditional and lethal point mutations by random in vitro mutagenesis of the PRII and PRI2 genes, which encode the small and large subunits of yeast DNA primase. We replaced the wild-type copies of PRII and PRI2 with two pril and two pri2 conditional alleles. When shifted to the restrictive temperature, these strains showed altered DNA synthesis and reduced ability to synthesize high molecular weight DNA products, thus providing in vivo evidence for the essential role of DNA primase in eukaryotic DNA replication. Furthermore, mapping of the mutations at the nucleotide level has shown that the two pril and twopri2 conditional alleles and onepri2 lethal allele have suffered single base-pair substitutions causing a change in amino acid residues conserved in the corresponding mouse polypeptide.The essential function of DNA polymerase a and the tightly associated DNA primase during replication of eukaryotic chromosomes is supported by several lines of biochemical and genetic evidence (1, 2). The catalytic properties of eukaryotic DNA primases, either free or in association with DNA polymerase, are almost identical in yeast and in more complex eukaryotic cells (1). The enzyme catalyzes the synthesis of discrete-length oligoribonucleotides when RNA synthesis is coupled with chain elongation by DNA polymerase, but the mechanism determining the switch from primer synthesis to DNA chain elongation is not yet understood. In Saccharomyces cerevisiae, DNA primase activity is associated with two polypeptides of 58 and 48 kDa (p58 and p48, respectively) (3). Studies with anti-p48 and anti-p58 antibodies and an affinity labeling procedure have shown that both polypeptides are needed for DNA primase activity (4-6). Cloning and characterization of the single essential genes POLI (7,8), PRII (4, 9), and PRI2 (5), which encode, respectively, DNA polymerase a and the two primase subunits p48 and p58 of the S. cerevisiae complex, have made it possible to address several questions about the role of the complex subunits, their interactions, and modulation of their expression (10)(11)(12)(13)(14)(15). Characterization of some poll temperature-sensitive mutants has provided information about the functional significance of amino acid sequences that are conserved among DNA polymerases (13-15); these studies have supported previous structural and functional work in vitro. Moreover, comparison of the sequences of cloned genes has revealed a high degree of conservation between the amino acid sequences of yeast and mouse primase subunits (refs. 5,9,and 16; B. Tseng, personal communication).Although the evidence for the role of DNA primase in replication of the eukaryotic genome is based on in vitro studies, the isolation of conditional lethal mutants allows the assessment ofthe primase function in vivo in different aspects of DNA metabolism. Since pril and pri2 mutants were previously not available, we have pursued the goal of producing such mutants by random in vit...

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