FRET analysis of actin-myosin interaction in contracting rat aortic smooth muscle.

We examined the interaction of smooth muscle myosin with alpha-actin and beta-actin isoforms during the contraction of A7r5 smooth muscle cells and rat aortic smooth muscle. The techniques of confocal microscopy and fluorescence resonance energy transfer (FRET) analysis were utilized in examining A7r5 cells and rat aortic rings contracted with phorbol 12,13-dibutyrate. Visual evaluation of confocal images of A7r5 smooth muscle cells contracted by phorbol 12,13-dibutyrate indicated significant disassociation of myosin from alpha-actin but not beta-actin. Whole-cell FRET analysis confirmed these observations (alpha-actin-myosin -67%, beta-actin-myosin -2%). Time course studies further showed that alpha-actin-myosin complex increased significantly (40%) within 1.5 min after the addition of phorbol 12,13-dibutyrate and then declined as contraction progressed. FRET analysis of rat aortic rings at different intervals of contraction indicated significant increases in alpha-actin-myosin at the initiation (79%) and plateau (67%) in force development, but not during the intermediate period of slowly developing tension (-4%). By comparison, beta-actin-myosin complex was unchanged except during slow force development, in which the association was significantly decreased (-30%). Similar to that of alpha-actin-myosin, Alexa 488 - phalloidin staining fluorescence indicated increased tissue F-actin content at the initiation (21%) and plateau (62%) in force. FRET images indicated the development of thickened cables and patches of alpha-actin-myosin in tissue throughout the interval of contraction. The results provide direct evidence of dynamic remodeling of the contractile protein during vascular smooth muscle contraction and suggest that FRET analysis may be a powerful tool for assessment of tissue protein-protein associations.

Detection of histidine decarboxylase in rat aorta and cultured rat aortic smooth muscle cells.

OBJECTIVE: Having previously demonstrated release of histamine from mast-cell-deficient rat aorta, the objective of this study was to determine and localize histamine synthesis capability in the aorta by detecting histidine decarboxylase (HDC), the enzyme that catalyzes histamine formation. MATERIALS AND METHODS: Experiments were conducted with nested reverse transcription-polymerase chain reaction (nRT-PCR) to detect HDC mRNA and with immunofluorescence and western blot analysis to detect HDC protein in rat aorta, cultured rat aortic smooth muscle (RASMC) and endothelial cells (RAEC). RESULTS: Gel electrophoresis of nRT-PCR products indicated HDC mRNA in liver, aorta and RASMC but not in RAEC or kidney. Sequence analysis confirmed that the band observed in RASMC was the target HDC amplicon. Immunofluorescence indicated the presence of HDC protein in RASMC and not in RAEC. Western Blot analysis revealed HDC protein (55 kDa) in liver, aorta, RASMC but not in RAEC or kidney. CONCLUSIONS: The results of this study are the first to demonstrate the presence of HDC mRNA and protein in rat aorta and more specifically in RASMC, indicative of their capability to synthesize histamine.

Characterization of the region containing the jcpk PKD gene on mouse Chromosome 10.

The jcpk gene on mouse Chromosome 10 causes a severe, early onset form of polycystic kidney disease (PKD) when inherited in an autosomal recessive manner. In order to positionally clone this gene, high resolution genetic and radiation hybrid maps were generated along with a detailed physical map of the approximately 500-kb region containing the jcpk gene. Additionally, sixty-nine kidney-specific ESTs were evaluated as candidates for jcpk and subsequently localized throughout the mouse genome by radiation hybrid mapping analysis. Previous studies indicating non-complementation of the jcpk mutation and 67Gso, a new PKD translocation mutant had suggested that 67Gso represents a new allele of jcpk. Fluorescence in situ hybridization (FISH) analysis using key bacterial artificial chromosome clones from the jcpk critical region, refined the 67Gso breakpoint and provided support for the allelism of jcpk and 67Gso.

High-resolution genetic and physical mapping of modifier-of-deafwaddler (mdfw) and Waltzer (Cdh23v).

Modifier-of-deafwaddler (mdfw) and waltzer (Cdh23v) are loci on mouse chromosome 10 encoding factors that are essential for the function of auditory hair cells. The BALB/cByJ-specific mdfw allele encodes a necessary and sufficient modifier that induces progressive early onset hearing loss in CBy-dfw2J heterozygotes. Recessive mutations in the waltzer locus result in circling behavior and congenital deafness. In this report we present a high-resolution integrated genetic and physical map of mdfw and Cdh23(v). Our genetic analyses localize mdfw between markers D10Mit60 and 148M13T7 within a 1.01-cM region. The Cdh23v critical interval is fully contained within the mdfw region and localizes between markers 146O23T7 and 148M13T7 within a 0.35-cM interval that is represented in an approximately 500-kb BAC contig. Our data suggest that mdfw and Cdh23v are allelic.

Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D.

Mouse chromosome 10 harbors several loci associated with hearing loss, including waltzer (v), modifier-of deaf waddler (mdfw) and Age-related hearing loss (Ahl). The human region that is orthologous to the mouse 'waltzer' region is located at 10q21-q22 and contains the human deafness loci DFNB12 and USH1D). Numerous mutations at the waltzer locus have been documented causing erratic circling and hearing loss. Here we report the identification of a new gene mutated in v. The 10.5-kb Cdh23 cDNA encodes a very large, single-pass transmembrane protein, that we have called otocadherin. It has an extracellular domain that contains 27 repeats; these show significant homology to the cadherin ectodomain. In v(6J), a GT transversion creates a premature stop codon. In v(Alb), a CT exchange generates an ectopic donor splice site, effecting deletion of 119 nucleotides of exonic sequence. In v(2J), a GA transition abolishes the donor splice site, leading to aberrant splice forms. All three alleles are predicted to cause loss of function. We demonstrate Cdh23 expression in the neurosensory epithelium and show that during early hair-cell differentiation, stereocilia organization is disrupted in v(2J) homozygotes. Our data indicate that otocadherin is a critical component of hair bundle formation. Mutations in human CDH23 cause Usher syndrome type 1D and thus, establish waltzer as the mouse model for USH1D.

Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D.

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital sensorineural hearing loss, vestibular dysfunction and visual impairment due to early onset retinitis pigmentosa (RP). So far, six loci (USH1A-USH1F) have been mapped, but only two USH1 genes have been identified: MYO7A for USH1B and the gene encoding harmonin for USH1C. We identified a Cuban pedigree linked to the locus for Usher syndrome type 1D (MIM 601067) within the q2 region of chromosome 10). Affected individuals present with congenital deafness and a highly variable degree of retinal degeneration. Using a positional candidate approach, we identified a new member of the cadherin gene superfamily, CDH23. It encodes a protein of 3,354 amino acids with a single transmembrane domain and 27 cadherin repeats. In the Cuban family, we detected two different mutations: a severe course of the retinal disease was observed in individuals homozygous for what is probably a truncating splice-site mutation (c.4488G-->C), whereas mild RP is present in individuals carrying the homozygous missense mutation R1746Q. A variable expression of the retinal phenotype was seen in patients with a combination of both mutations. In addition, we identified two mutations, Delta M1281 and IVS51+5G-->A, in a German USH1 patient. Our data show that different mutations in CDH23 result in USH1D with a variable retinal phenotype. In an accompanying paper, it is shown that mutations in the mouse ortholog cause disorganization of inner ear stereocilia and deafness in the waltzer mouse.

Scraggly, a new hair loss mutation on mouse chromosome 19.

By use of chlorambucil, we have generated a mouse mutation called scraggly (sgl) that exhibits skin and hair defects. Homozygous mutant mice exhibit hair loss, skin defects, and abnormalities in sebaceous lipid composition. We have constructed a high-resolution genetic map of mouse Chromosome (Chr) 19 that links this mutation to the anonymous DNA marker D19Umi1. An additional cross, (BALB/c x CAST/Ei) F(1) x BALB/c, was used to map markers around this mutation as well as to map the potential candidate genes, Fgf8 and Cyp17. Allelism tests between sgl and asebia (ab), another hair loss mutation on mouse Chr 19, showed that these genes were separate and distinct.

A high-resolution genetic map around waltzer on mouse chromosome 10 and identification of a new allele of waltzer.

A new autosomal recessive mouse mutation characterized by deafness and circling behavior was recovered during mutagenesis experiments with chlorambucil (CHL). On the basis of allelism tests and linkage analyses, this mutation appears to represent a new allele of waltzer (v) that maps to mouse Chromosome (Chr) 10. We have designated this new allele, Albany waltzer (vAlb). A high-resolution map of the region around v was constructed from data from two intersubspecific backcrosses involving Mus musculus castaneus. The analysis of 648 backcross mice has allowed vAlb to be localized 1.1 +/- 0.4 cM distal to D10Mit60 and 0.2 +/- 0.2 cM proximal to a cluster of four markers, D10Mit172, D10Mit112, D10Mit48, and D10Mit196. An independent backcross was used to confirm the map order and distances in the vAlb backcross. The two linkage maps were consistent, indicating that the lesion in vAlb, which is presumed to be a deletion based on the known action of CHL, is small and has not significantly altered the map at this level of detection. Additionally, three genes (Ros1, Grik2, and Zfa) were eliminated as possible candidates for vAlb, and several SSLP markers were separated genetically.

Evidence that two phenotypically distinct mouse PKD mutations, bpk and jcpk, are allelic.

Numerous mouse models of polycystic kidney disease (PKD) have been described. All of these diseases are transmitted as single recessive traits and in most, the phenotypic severity is influenced by the genetic background. However, based on their genetic map positions, none of these loci appears to be allelic and none are candidate modifier loci for any other mouse PKD mutation. Previously, we have described the mouse bpk mutation, a model that closely resembles human autosomal recessive polycystic kidney disease. We now report that the bpk mutation maps to a 1.6 CM interval on mouse Chromosome 10, and that the renal cystic disease severity in our intersubspecific intercross progeny is influenced by the genetic background. Interestingly, bpk co-localizes with jcpk, a phenotypically-distinct PKD mutation, and complementation testing indicates that the bpk and jcpk mutations are allelic. These data imply that distinct PKD phenotypes can result from different mutations within a single gene. In addition, based on its map position, the bpk locus is a candidate genetic modifier for jck, a third phenotypically-distinct PKD mutation.

Fine genetic map of mouse chromosome 10 around the polycystic kidney disease gene, jcpk, and ankyrin 3.

A chlorambucil (CHL)-induced mutation of the jcpk (juvenile congenital polycystic kidney disease) gene causes a severe early onset polycystic kidney disease. In an intercross involving Mus musculus castaneus, jcpk was precisely mapped 0.2 cM distal to D10Mit115 and 0.8 cM proximal to D10Mit173. In addition, five genes, Cdc2a, Col6a1, Col6a2, Bcr, and Ank3 were mapped in both this jcpk intercross and a (BALB/c x CAST/Ei)F1 x BALB/c backcross. All five genes were eliminated as possible candidates for jcpk based on the mapping data. The jcpk intercross allowed the orientation of the Ank3 gene relative to the centromere to be determined. D10Mit115, D10Mit173, D10Mit199, and D10Mit200 were separated genetically in this cross. The order and genetic distances of all markers and gene loci mapped in the jcpk intercross were consistent with those derived from the BALB/c backcross, indicating that the CHL-induced lesion has not generated any gross chromosomal abnormalities detectable in these studies.

Recovery of probes linked to the jcpk locus on mouse chromosome 10 by the use of an improved representational difference analysis technique.

Representational difference analysis (RDA) is a subtractive hybridization technique by which the differences between two complex genomes can be isolated. An improved version of this technique was used to isolate DNA segments that map to a narrow genetic region adjacent to the jcpk locus on Chromosome 10 of the mouse. A mutation at this locus acts recessively and causes an early onset polycystic kidney disease. Genomic subtractions involving DNA from C57BL/6 (B6) and its partially congenic partner, B6-jcpk/jcpk, produced 39 restriction fragments (difference products), 25 of which were unique and represented differences in BglII sites between these two strains. Although none identified the jcpk locus itself, 7 of these were mapped to an interval between 3.4 and 6.5 cM distal to the jcpk locus. Five of these 7 difference products were developed by subtracting B6-jcpk/jcpk from B6 DNA, but only 1 of the 5 was isolated using the original RDA technique. The other 4 were obtained by an improved technique that included size selection of difference products after the third round of subtractive hybridization and amplification. The remaining 2 of the mapped products resulted from the reciprocal subtraction experiment using the improvements. Thus, by this improved technique and two-way subtraction, we were able to add seven new markers to a relatively small genetic region on Chromosome 10.

New mouse model for polycystic kidney disease with both recessive and dominant gene effects.

In the course of studying the genetics of chlorambucil mutagenesis, we have uncovered a new model for autosomal polycystic kidney disease (PKD). In the homozygous condition, the gene, jcpk, causes a very severe disease characterized by cysts in all segments of the nephron. Death usually occurs before 10 days of age. Extrarenal involvement was also noted; enlarged bile ducts, pancreatic ducts, and gall bladder often accompanied the PKD. In addition, approximately 25% of the aged +/jcpk heterozygotes show evidence of glomerulocystic disease. This gene maps to Chromosome 10 between two DNA markers, D10Mit20 and D10Mit42. Because this gene causes extrarenal abnormalities and because it has a heterozygote effect, it may be an informative animal model for the commonly occurring human adult dominant PKD.

Multiple sites of crossing over within the Eb recombinational hotspot in the mouse.

The Eb gene of the mouse major histocompatibility complex (MHC) contains a well-documented hotspot of recombination. Twelve cases of intra-Eb recombination derived from the b, d, k and s alleles of the Eb gene were sequenced to more precisely position the sites of meiotic recombination. This analysis was based on positioning recombination breakpoints between nucleotide polymorphisms found in the sequences of parental haplotypes. All twelve cases of recombination mapped within the second intron of the Eb gene. Six of these recombinants, involving the k and s haplotypes, mapped to two adjoining DNA segments of 394 and 955 base pairs (bp) in the 3' half of the intron. In an additional two cases derived by crossing over between the d and s alleles, breakpoints were positioned to adjoining segments of 28 and 433 bp, also in the 3' half of the intron. Finally, four b versus k recombinants were mapped to non-contiguous segments of DNA covering 2.9 kb and 1005 bp of the intron. An analysis of the map positions of crossover breakpoints defined in this study suggests that the second intron of the Eb gene contains a recombinational hotspot of approximately 800-1000 bp which contains at least two closely linked recombinationally active sites or segments. Further examination of the sequence data also suggests that the postulated location for the recombinational hotspot corresponds almost precisely to an 812 bp sequence that shows nucleotide sequence similarity to the MT family of middle repetitive DNA.