130-kDa smooth muscle myosin light chain kinase is transcribed from a CArG-dependent, internal promoter within the mouse mylk gene

The 130-kDa smooth muscle myosin light chain kinase (smMLCK) is a Ca2+/CaM-regulated enzyme that plays a pivotal role in the initiation of smooth muscle contraction and regulation of cellular migration and division. Despite the critical importance of smMLCK in these processes, little is known about the mechanisms regulating its expression. In this study, we have identified the proximal promoter of smMLCK within an intron of the mouse mylk gene. The mylk gene encodes at least two isoforms of MLCK (130 and 220 kDa) and telokin. Luciferase reporter gene assays demonstrated that a 282-bp fragment (-167 to +115) of the smMLCK promoter was sufficient for maximum activity in A10 smooth muscle cells and 10T1/2 fibroblasts. Deletion of the 16 bp between -167 and -151, which included a CArG box, resulted in a nearly complete loss of promoter activity. Gel mobility shift assays and chromatin immunoprecipitation assays demonstrated that serum response factor (SRF) binds to this CArG box both in vitro and in vivo. SRF knockdown by short hairpin RNA decreased endogenous smMLCK expression in A10 cells. Although the SRF coactivator myocardin induced smMLCK expression in 10T1/2 cells, myocardin activated the promoter only two- to fourfold in reporter gene assays. Addition of either intron 1 or 6 kb of the 5' upstream sequence did not lead to any further activation of the promoter by myocardin. The proximal smMLCK promoter also contains a consensus GATA-binding site that bound GATA-6. GATA-6 binding to this site decreased endogenous smMLCK expression, inhibited promoter activity in smooth muscle cells, and blocked the ability of myocardin to induce smMLCK expression. Altogether, these data suggest that SRF and SRF-associated factors play a key role in regulating the expression of smMLCK.     

MCM3AP is transcribed from a promoter within an intron of the overlapping gene for GANP

MCM3 acetylase (MCM3AP) and germinal-centre associated nuclear protein (GANP) are transcribed from the same locus and are therefore confused in databases because the MCM3 acetylase DNA sequence is contained entirely within the much larger GANP sequence and the entire MCM3AP sequence is identical to the carboxy terminus of GANP. Thus, the MCM3AP and GANP genes are read in the same reading frame and MCM3AP is an N-terminally truncated region of GANP. However, we show here that MCM3AP and GANP are different proteins, occupying different locations in the cell and transcribed from different promoters. Intriguingly, a promoter for MCM3AP lies within an intron of GANP. This report is an interesting example in nature of two separate gene products from the same locus that perform two entirely different functions in the cell. Therefore, to avoid further confusion, they should now be referred to as separate but overlapping genes.     

7SL RNA from Schizosaccharomyces pombe is encoded by a single copy essential gene

We have identified an abundant ribonucleoprotein particle from Schizosaccharomyces pombe with properties related to those of the vertebrate signal recognition particle (SRP), including cytoplasmic localization, association with microsomes and ribosomes at low, but not high, salt concentrations and high resistance to micrococcal nuclease. The 256-nucleotide RNA component carries a 5'-triphosphate group and shows close secondary structure, and limited primary sequence homology to vertebrate 7SL RNA. 7SL-like RNAs were also detected in a number of other fungi. The single copy gene (SRP7) encoding S.pombe 7SL was disrupted by insertion of a transposon carrying the selective marker LEU2, and the disrupted gene was used to replace one chromosomal SRP7 gene in a diploid strain. Haploid srp7[unk] strains fail to germinate.     

The lethal toxin from Australian funnel-web spiders is encoded by an intronless gene

Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome.     

cis-elements involved in alternative splicing in the rat beta-tropomyosin gene: the 3'-splice site of the skeletal muscle exon 7 is the major site of blockage in nonmuscle cells.

We have been using the rat beta-tropomyosin (beta-TM) gene as a model system to study the mechanism of alternative splicing. The beta-TM gene spans 10 kb with 11 exons and encodes two distinct isoforms, namely skeletal muscle beta-TM and fibroblast TM-1. Exons 1-5, 8, and 9 are common to all mRNAs expressed from this gene. Exons 6 and 11 are used in fibroblasts, as well as in smooth muscle cells, whereas exons 7 and 10 are used exclusively in skeletal muscle cells. Our previous studies localized the critical elements for regulated alternative splicing to sequences within exon 7 and the adjacent upstream intron. We also demonstrated that these sequences function, in part, to regulate splice-site selection in vivo by interacting with cellular factors that block the use of the skeletal muscle exon in nonmuscle cells (1). Here we have further characterized the critical cis-acting elements involved in alternative splice site selection. Our data demonstrate that exon 7 and its flanking intron sequences are sufficient to regulate the suppression of exon 7 in nonmuscle cells when flanked by heterologous exons derived from adenovirus. We have also shown by both in vivo and in vitro assays that the blockage of exon 7 in nonmuscle cells is primarily at its 3'-splice site. A model is presented for regulated alternative splicing in both skeletal muscle and nonmuscle cells.     

A second ferritin L subunit is encoded by an intronless gene in the mouse

Multiple homologous sequences for the ferritin L subunit are present in mammalian genomes, but so far, only one expressed gene has been described. Here we report the isolation of a cDNA from a mouse bone marrow library, corresponding to an isoform of the mouse ferritin L subunit. This new subunit, that we named Lg, differs from the L subunit of ten amino acids. Specific amplification of mouse genomic DNA using the polymerase chain reaction (PCR) confirmed the presence of this Lg sequence in the mouse genome but also suggested that it must be encoded by an intronless gene. Using a series of different Lg-specific oligonucleotides as probes, we subsequently isolated a genomic clone containing an uninterrupted sequence, identical to the Lg cDNA. This Lg gene lacks introns and does not contain the 28 base pairs (bp) conserved motif usually present at the 5 end of most ferritin mRNAs, which confers translational regulation by iron. When transiently transfected into K562 cells, this Lg genomic clone is actively transcribed, suggesting that, although it possesses the characteristics of a processed pseudogene, it is likely to correspond to the gene encoding this new ferritin subunit.     

The small MbtH-like protein encoded by an internal gene of the balhimycin biosynthetic gene cluster is not required for glycopeptide production

The balhimycin biosynthetic gene cluster of the glycopeptide producer Amycolatopsis balhimycina includes a gene (orf1) with unknown function. orf1 shows high similarity to the mbtH gene from Mycobacterium tuberculosis. In almost all nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters, we could identify a small mbtH-like gene whose function in peptide biosynthesis is not known. The mbtH-like gene is always colocalized with the NRPS genes; however, it does not have a specific position in the gene cluster. In all glycopeptide biosynthetic gene clusters the orf1-like gene is always located downstream of the gene encoding the last module of the NRPS. We inactivated the orf1 gene in A. balhimycina by generating a deletion mutant. The balhimycin production is not affected in the orf1-deletion mutant and is indistinguishable from that of the wild type. For the first time, we show that the inactivation of an mbtH-like gene does not impair the biosynthesis of a nonribosomal peptide.