MI1IQ is a complex of calmodulin and an epitopetagged 85-kDa fragment representing the amino-terminal catalytic motor domain and the first of 6 calmodulinbinding IQ domains of the mammalian myosin I gene, rat myr-1 (130-kDa myosin I or MI 130 Class I myosins in mammals are mechanochemical molecules with an amino-terminal motor domain containing an ATP and actin-binding region, a neck region with one or more socalled IQ domains to which calmodulin binds, and a carboxylterminal tail region (1). One member of the class I myosins, MYR-1, is ubiquitously expressed in mammalian cells. myr-1 contains up to 6 IQ domains; alternate splice forms containing 4, 5, or 6 IQ domains exist (2). The 130-kDa myosin I isolated from rat liver is a myr-1 gene product (3, 4). Although quantitation of calmodulin has indicated that this preparation contains 6 mol of calmodulin/130-kDa myosin I heavy chain (4), isoforms corresponding to the 5 IQ and 4 IQ variants are also expressed in liver (2) and may be present.The 130-kDa myosin I translocates actin filaments slowly and in a Ca 2ϩ -sensitive manner (5). At 10 M free Ca 2ϩ and above, motility is inhibited. This decrease in motility can be reversed by the addition of exogenous calmodulin, indicating that a calcium-induced dissociation of calmodulin might be responsible for the decrease in motility. Laser trap analyses have indicated that the 130-kDa myosin I translocates actin in a two-step process (6). The authors proposed that the two mechanical steps are coupled to P i and ADP release, respectively. Transient kinetic analyses have indicated that the mechanical step coupled to ADP release is unlikely to contribute to force generation or to motility, but could be a system for providing a strain-sensitive ADP release mechanism. This, together with the slow ATP-induced dissociation of actin-myosin I, suggests that this myosin is best suited for maintenance of tension (7).We have coexpressed in baculovirus calmodulin and a fragment representing the first 728 amino acids of MYR-1, which codes for the amino-terminal motor domain and 1 IQ domain; we refer to this complex as MI 1IQ 1 (8). MI 1IQ translocates actin filaments in vitro. Unlike the parent molecule, the rate of actin translocation is not affected by the Ca 2ϩ concentration over the range of pCa 4 -7 and the rates of movement of MI 130 and MI 1IQare comparable. The availability of a homogeneous preparation from the baculovirus expression system has allowed us to explore in detail the kinetics of this construct representing the motor domain and the first IQ domain. Our results indicate that the truncated myosin I possesses kinetic properties indistinguishable from the parent molecule. Thus, the 5 deleted IQ domains and their associated calmodulins play no role in defining the unloaded properties of the myosin I head. Furthermore, our results indicate that, when bound to actin, myosin

The muscle and species-specific differences in enzymatic activity between Placopecten and Argopecten striated and catch muscle myosins are attributable to the myosin heavy chain. To identify sequences that may modulate these differences, we cloned and sequenced the cDNA encoding the myosin heavy chains of Placopecten striated and catch muscle. Deduced protein sequences indicate two similar isoforms in catch and striated myosins (97% identical); variations arise by differential RNA splicing of five alternative exons from a single myosin heavy chain gene. The first encodes the phosphate-binding loop; the second, part of the ATP binding site; the third, part of the actin binding site; the fourth, the hinge in the rod; and the fifth, a tailpiece found only in the catch muscle myosin heavy chain. Both Placopecten myosin heavy chains are 96% identical to Argopecten myosin heavy chaina isoforms. Because subfragment-1 ATPase activities reflect the differences observed in the parent myosins, the motor domain is responsible for the variations in ATPase activities. In addition, data show that differences are due to Vmax and not actin affinity. The sequences of all four myosin heavy chain motor domains diverge only in the flexible surface loop near the nucleotide binding pocket. Thus, the different ATPase activities of four molluscan muscle myosins are likely due to myosin heavy chain sequence variations within the flexible surface loop that forms part of the ATP binding pocket of the motor domain.

Traditional carrier screening assays are designed to look for only the most common mutations within a gene owing to cost considerations. Although this can yield high detection rates in specific populations for specific genes (such as cystic fibrosis in Caucasians), they are suboptimal for other ethnicities or for patients of mixed or unknown ethnic background. Next-generation DNA sequencing provides an opportunity to provide carrier screening using more comprehensive mutation panels that are limited primarily by information about the clinical impact of detected sequence changes. We describe a next-generation DNA sequencing-based assay capable of reliably screening patient samples in a timely and comprehensive manner. The analytic accuracy in a research setting has been documented. Here, we describe the additional studies performed to ensure the accuracy (analytic validity) and robustness of our assay for use in clinical practice and provide data from our experience offering this testing. Our clinical experience using this approach to screen 11,691 in vitro fertilization patients has identified 449 mutant alleles: 447 in carriers and 2 in an affected individual. In total, we found 87 distinct mutations in 14 different genes. Approximately one quarter of the mutations found are not included in traditional, limited, mutation panels, including 16 known mutations unique to our panel, and novel truncating mutations in several genes.

MYRThe myr-1 gene, which includes a motor domain followed by 6 calmodulin-binding, so-called IQ domains, and a carboxylterminal tail region (2), codes for rat liver 130-kDa myosin I (3, 4) or MI 130 , 1 as it is also known (5). Quantitation of the amount of calmodulin associated with the purified 130-kDa myosin I heavy chain has indicated that the heavy chain copurifies with 6 molecules of calmodulin (4), although isoforms corresponding to the 5 IQ and 4 IQ variants are also expressed in liver (2).myr-1 is most closely related in sequence to brush border myosin I (BBMI), which in microvilli cross-links the core bundle of actin filaments to the membrane (6, 7). Although expression of BBMI is essentially confined to intestine (8, 9), myr-1 is widely expressed (2). In NRK cells, MYR-1 is associated with the plasma membrane and in cell protrusions such as lamellipodia and membrane ruffles (10). Some 130-kDa myosin I is found in association with several subcellular fractions from rat liver, but the majority of this isoform in liver cells is most likely cytoskeleton-associated (11). These localization studies, together with recent kinetic analyses indicating among other things that the ATP-induced dissociation of actin-MI 130 is slow, are consistent with a role for MYR-1 in maintenance of tension of the cytoskeleton (5).The purified native 130-kDa myosin I translocates actin filaments in a Ca 2ϩ -regulated manner. Motility is highest at pCa 7-8, and then decreases with increasing Ca 2ϩ concentration. At free Ca 2ϩ concentrations above 0.1 M, motility can be restored by the addition of exogenous calmodulin (12). One interpretation supported by in vitro actin binding assays is that calmodulin dissociates in the presence of Ca 2ϩ and that addition of excess calmodulin favors reassociation and therefore reestablishment of motility.To assist in the molecular characterization of the 130-kDa myosin I, we have expressed in insect cells using the baculovirus expression system, a truncated myosin I heavy chain of 85 kDa representing the amino-terminal 728 amino acids of MYR-1, which includes the motor domain and the first of the 6 IQ domains that comprise the so-called neck region. This truncated myosin I heavy chain was co-expressed with calmodulin. The purified truncated myosin I heavy chain and its associated calmodulin are referred to here as MI 1IQ . Biochemical analyses of this truncated myosin I have permitted evaluation of properties conferred on the parent 130-kDa myosin I molecule by the motor domain and the association of calmodulin to the first of 6 IQ domains. MI 1IQ exhibits steady state ATPase activities resembling the parent molecule. Our results also indicate that binding of calmodulin to the first IQ domain in MYR-1 is insensitive to Ca 2ϩ . Furthermore, we show that the truncated myosin I translocates actin filaments in vitro at a rate resembling that of the parent molecule, although unlike the parent molecule, the truncated form shows no sensitivity to Ca 2ϩ in the range of 0.1-100 M. These results are discu...

Diabetic cardiomyopathy is characterized by LV systolic and diastolic dysfunction, the latter correlating with decreased exhaled NO. The NO pathway is intact, suggesting impaired availability of NO as contributor to cardiomyopathy.

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ATPase activities of molluscan adductor muscle myosins show both muscle and species specific differences: ATPase activity of catch muscle myosin is lower than that of phasic muscle myosin; a 4-5-fold difference exists between the activities of phasic striated muscle myosins from the bay scallop (Argopecten irradians) and sea scallop (Placopecten magellanicus). To characterize the light chains of these myosins we determined the cDNA sequences of the essential light chains and the regulatory light chains from Placopecten striated and catch muscle. The nucleotide sequences of the essential light chains from Placopecten striated and catch muscle myosins are identical and show 94% identity and 98% homology to the Argopecten essential light chain indicating that the tissue and species specific differences in ATPase activities are not due to the essential light chain. We identified three regulatory light chain isoforms, one from striated and two from catch muscle. Sequence differences were restricted to nucleotides encoding some of the N-terminal 52 amino acids. The three recombinant Placopecten regulatory light chain isoforms and the Argopecten regulatory light chain were incorporated into hybrid myosins that contained the essential light chain and heavy chain from Placopecten striated, Placopecten catch, or Argopecten striated muscle. Measurement of the ATPase activities of these hybrids indicates clearly that it is the myosin heavy chain and not the regulatory light chains that are responsible for the muscle and species specific differences in enzymatic activities. Analysis of genomic DNA indicated that these regulatory light chain isoforms are products of a single regulatory light chain gene that is alternatively spliced in the 5' region only.

Loss of function variants in the PCDH15 gene can cause Usher syndrome type 1F, an autosomal recessive disease associated with profound congenital hearing loss, vestibular dysfunction, and retinitis pigmentosa. The Ashkenazi Jewish population has an increased incidence of Usher syndrome type 1F (founder variant p.Arg245X accounts for 75% of alleles), yet the variant spectrum in a panethnic population remains undetermined. We sequenced the coding region and intron-exon borders of PCDH15 using next-generation DNA sequencing technology in approximately 14,000 patients from fertility clinics. More than 600 unique PCDH15 variants (single nucleotide changes and small indels) were identified, including previously described pathogenic variants p.Arg3X, p.Arg245X (five patients), p.Arg643X, p.Arg929X, and p.Arg1106X. Novel truncating variants were also found, including one in the N-terminal extracellular domain (p.Leu877X), but all other novel truncating variants clustered in the exon 33 encoded C-terminal cytoplasmic domain (52 patients, 14 variants). One variant was observed predominantly in African Americans (carrier frequency of 2.3%). The high incidence of truncating exon 33 variants indicates that they are unlikely to cause Usher syndrome type 1F even though many remove a large portion of the gene. They may be tolerated because PCDH15 has several alternate cytoplasmic domain exons and differentially spliced isoforms may function redundantly. Effects of some PCDH15 truncating variants were addressed by deep sequencing of a panethnic population.