Generation and characterization of new alleles of quiver (qvr) that encodes an extracellular modulator of the Shaker potassium channel.

Our earlier genetic screen uncovered a paraquat-sensitive leg-shaking mutant quiver1 (qvr1), whose gene product interacts with the Shaker (Sh) K+ channel. We also mapped the qvr locus to EY04063 and noticed altered day-night activity patterns in these mutants. Such circadian behavioral defects were independently reported by another group, who employed the qvr1 allele we supplied them, and attributed the extreme restless phenotype of EY04063 to the qvr gene. However, their report adopted a new noncanonical gene name sleepless (sss) for qvr. In addition to qvr1 and qvrEY, our continuous effort since the early 2000s generated a number of novel recessive qvr alleles, including ethyl methanesulfonate (EMS)-induced mutations qvr2 and qvr3, and P-element excision lines qvrip6 (imprecise jumpout), qvrrv7, and qvrrv9 (revertants) derived from qvrEY. Distinct from the original intron-located qvr1 allele that generates abnormal-sized mRNAs, qvr2, and qvr3 had their lesion sites in exons 6 and 7, respectively, producing nearly normal-sized mRNA products. A set of RNA-editing sites are nearby the lesion sites of qvr3 and qvrEY on exon 7. Except for the revertants, all qvr alleles display a clear ether-induced leg-shaking phenotype just like Sh, and weakened climbing abilities to varying degrees. Unlike Sh, all shaking qvr alleles (except for qvrf01257) displayed a unique activity-dependent enhancement in excitatory junction potentials (EJPs) at larval neuromuscular junctions (NMJs) at very low stimulus frequencies, with qvrEY displaying the largest EJP and more significant NMJ overgrowth than other alleles. Our detailed characterization of a collection of qvr alleles helps to establish links between novel molecular lesions and different behavioral and physiological consequences, revealing how modifications of the qvr gene lead to a wide spectrum of phenotypes, including neuromuscular hyperexcitability, defective motor ability and activity-rest cycles.

Structure, Expression, and Function of a Novel Intercalated Disc Protein, Xin.

Xin was first cloned using differential mRNA display from the developing chicken heart. Chick Xin (cXin) participates in a BMP-Nkx2.5-MEF2C pathway to regulating cardiac morphogenesis. Through subsequent EST database searches and cDNA cloning, two mouse Xin genes, mXinα and mXinß were identified and cloned. The human homologue of mXinα (named Cmya1) was mapped to chromosome 3p21.2-p21.3 by radiation hybrid analysis and recently to 3p22.2 by DNA sequencing, which is near the loci for a dilated cardiomyopathy with conduction defect-2 and arrhythmogenic right ventricular dysplasia-5. The predicted human homologue of mXinß (named Cmya3) was mapped to chromosome 2q24.3 by DNA sequencing. Predicted Xin proteins all contain a novel 16-amino acid repeating unit (Xin repeat), a putative DNA binding domain and nuclear localization signal, as well as a proline-rich region. All three Xin genes from chick and mouse have a similar tissue expression profile, which is restricted to striated muscle. The expression of mXinα in Nkx2.5 or MEF2C knockout mouse embryos was drastically reduced, suggesting that mXinα is a downstream target of the Nkx2.5 and MEF2C transcription factors. On the other hand, the expression of mXin was up-regulated when mice were subjected to pressure overload-induced cardiac hypertrophy. Xin protein co-localizes with N-cadherin and ß-catenin throughout mouse embryogenesis and into adulthood. Furthermore, mXinα appears to interact directly with ß-catenin. The Xin repeats bind to actin filaments and may also organize microfilaments into networks. These results may suggest that Xin acts by integrating adhesion, by organizing actin filament arrangement at the insertion sites, and by regulating Wnt/ß-catenin-and N-cadherin-mediated signaling pathways required for cardiac development and cardiac function.

A novel TCTG(G/C) direct repeat and an A/T-rich HMG2-binding site control the expression of the rat cardiac troponin T gene.

We previously showed that the rat cardiac troponin T proximal promoter (-497bp from the transcriptional start site) was sufficient to confer cardiac-specific expression of a reporter gene in both cultured cardiomyocytes and transgenic mice. This promoter consists of two cis-regulatory modules with high sequence similarity. Nucleotides from -319 to -289 (termed D2) and nucleotides from -249 to -209 (termed F41) contain a TCTG(G/C) direct repeat and an A/T-rich sequence. Competition gel mobility shift assays revealed that the same protein factors were bound to both D2 and F41. Deletion and religation studies of the promoter suggested that D2 acted as an enhancer but could not totally substitute for the F41 function. Mutational analyses demonstrated that the direct repeat was required for the DNA-binding and promoter activity. Moreover, cardiac-specific 42kDa proteins and a ubiquitous high mobility group 2 (HMG2) protein were identified to be responsible for the binding to the TCTG(G/C) direct repeat and the A/T-rich sequence, respectively. Overexpression of HMG2 had differential effects on the promoter in cardiomyocytes versus fibroblasts. Our results provided the first evidence to support that HMG2 together with tissue-specific factors could direct a stimulatory or inhibitory effect on thecardiac troponin T gene expression in heart or non-heart tissue, respectively.