A Novel Myosin-like Protein (Myocilin) Expressed in the Connecting Cilium of the Photoreceptor: Molecular Cloning, Tissue Expression, and Chromosomal Mapping

We have isolated a human cDNA clone encoding a novel acidic protein of MW 55,000 that we designated “myocilin” since it has homology to myosin and is localized preferentially in the ciliary rootlet and basal body of the connecting cilium of photoreceptor cells. The deduced amino acid sequence of human myocilin showed significant homologies with nonmuscle myosin ofDictyostelium discoideumin the N-terminal region and also with olfactomedin of bullfrog in the C-terminal region. Myocilin contained a leucine zipper-like motif similar to that seen in kinectin and other cytoskeletal proteins. These findings suggest that myocilin is a novel cytoskeletal protein involved in the morphogenesis of ciliated neuroepithelium such as photoreceptor cells. The myocilin gene (MYOC) was mapped to human chromosome 1q23–q24 by fluorescencein situhybridization.     

Cloning and Characterization of a Gene (RFN22) Encoding a Novel Brain Expressed Ring Finger Protein (BERP) That Maps to Human Chromosome 11p15.5

We have recently identified a novel RING finger protein expressed in the rat brain, which associates with myosin V and α-actinin-4. Here we have cloned and characterized the orthologous human BERP cDNA and gene (HGMW-approved symbol RNF22). The human BERP protein is encoded by 11 exons ranging in size from 71 to 733 bp, and fluorescence in situ hybridization shows that the BERP gene maps to chromosome 11p15.5, 3′ to the FE65 gene. The human BERP protein is 98% identical to the rat and mouse proteins, and we have identified a highly conserved potential orthologue in Caenorhabditis elegans. BERP belongs to the RING finger–B-box–coiled coil (RBCC) subgroup of RING finger proteins, and a cluster of these RBCC protein genes is present in chromosome 11p15. Chromosome region 11p15 is thought to harbor tumor suppressor genes, and deletions of this region occur frequently in several types of human cancers. These observations indicate that BERP may be a novel tumor suppressor gene.     

Assignment of the βB1 Crystallin Gene (CRYBB1) to Human Chromosome 22 and Mouse Chromosome 5

By using primers complementary to the rat βB1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences.CRYBB1was assigned to the group 5 region in 22q11.2–q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products ofCRYBB1were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other β crystallin genes (βB2(−1), βB3, and βA4) have previously been mapped to the same regions in human and mouse. We demonstrate that the βB1 and βA4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major β crystallins genes that are expressed in the bovine lens.     

Visualization of Melanosome Dynamics within Wild-Type and Dilute Melanocytes Suggests a Paradigm for Myosin V Function In Vivo

Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va⁻) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid (∼1.5 μm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va–dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This “capture” model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an ∼120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, ∼0.14 μm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do not drive obvious spreading of melanosomes over 90 min. We conclude that long-range, bidirectional, microtubule-dependent melanosome movements, coupled with actomyosin Va–dependent capture of melanosomes in the periphery, is the predominant mechanism responsible for the centrifugal transport and peripheral accumulation of melanosomes in mouse melanocytes. This mechanism represents an alternative to straightforward transport models when interpreting other myosin V mutant phenotypes.     

Characterization of Three Novel Human Cadherin Genes (CDH7, CDH19, and CDH20) Clustered on Chromosome 18q22–q23 and with High Homology to Chicken Cadherin-7

Full-length coding sequences of two novel human cadherin cDNAs were obtained by sequence analysis of several EST clones and 5′ and 3′ rapid amplification of cDNA ends (RACE) products. Exons for a third cDNA sequence were identified in a public-domain human genomic sequence, and the coding sequence was completed by 3′ RACE. One of the sequences (CDH7L1, HGMW-approved gene symbol CDH7) is so similar to chicken cadherin-7 gene that we consider it to be the human orthologue. In contrast, the published partial sequence of human cadherin-7 is identical to our second cadherin sequence (CDH7L2), for which we propose CDH19 as the new name. The third sequence (CDH7L3, HGMW-approved gene symbol CDH20) is almost identical to the mouse “cadherin-7” cDNA. According to phylogenetic analysis, this mouse cadherin-7 and its here presented human homologue are most likely the orthologues of Xenopus F-cadherin. These novel human genes, CDH7, CDH19, and CDH20, are localized on chromosome 18q22–q23, distal of both the gene CDH2 (18q11) encoding N-cadherin and the locus of the six desmosomal cadherin genes (18q12). Based on genetic linkage maps, this genomic region is close to the region to which Paget's disease was linked. Interestingly, the expression patterns of these three closely related cadherins are strikingly different.     

FXY2/MID2, a Gene Related to the X-Linked Opitz Syndrome Gene FXY/MID1, Maps to Xq22 and Encodes a FNIII Domain-Containing Protein That Associates with Microtubules

Opitz G/BBB syndrome (OS) is a genetically heterogeneous disorder with an X-linked locus and an autosomal locus linked to 22q11.2. OS affects multiple organ systems with often variable severity even between siblings. The clinical features, which include hypertelorism, cleft lip and palate, defects of cardiac septation, hypospadias, and anorectal anomalies, indicate an underlying disturbance of the developing ventral midline of the embryo. The gene responsible for X-linked OS, FXY/MID1, is located on the short arm of the human X chromosome within Xp22.3 and encodes a protein with both an RBCC (RING finger, B-box, coiled coil) and a B30.2 domain. The Fxy gene in mice is also located on the X chromosome but spans the pseudoautosomal boundary in this species. Here we describe a gene closely related to FXY/MID1, called FXY2, which also maps to the X chromosome within Xq22. The mouse Fxy2 gene is located on the distal part of the mouse X chromosome within a region syntenic to Xq22. Analysis of genes flanking both FXY/MID1 and FXY2 (as well as their counterparts in mouse) suggests that these regions may have arisen as a result of an intrachromosomal duplication on an ancestral X chromosome. We have also identified in both FXY2 and FXY/MID1 proteins a conserved fibronectin type III domain located between the RBCC and B30.2 domains that has implications for understanding protein function. The FXY/MID1 protein has previously been shown to colocalize with microtubules, and here we show that the FXY2 protein similarly associates with microtubules in a manner that is dependent on the carboxy-terminal B30.2 domain.     

Biosynthesis and intracellular movement of the melanosomal membrane glycoprotein gp75, the human b (brown) locus product

A 75-kDa melanosomal glycoprotein (gp75) is the product of a gene that maps to the b (brown) locus, a genetic locus that determines coat color in the mouse. The b locus is conserved (88% identity) between mouse and human. The mouse monoclonal antibody TA99 was used to study the biosynthesis and processing of gp75. gp75 was synthesized as a 55-kDa polypeptide, glycosylated by addition and processing of five or more Asnlinked carbohydrate chains through the cis and trans Golgi, and transported to melanosomes as a mature 75kDa form. Synthesis and processing of gp75 was rapid (T_1/2 < 30 min), and early steps in processing were required for efficient export of gp75 to melanosomes. Fully processed mature gp75 was quite stable (T_1/2 = 22–24 h) in the melanosome. Digestion of high-mannose carbohydrate chains with endo-β-N-acetylglucosaminidase H revealed two alternative processed forms of gp75 that differed in the number or composition of complex-type carbohydrate chains. The rate of synthesis and movement through intracellular membrane compartments was the same for both glycosylated forms. Studies with inhibitors of steps in oligosaccharide processing showed that alternative forms of gp75 were generated during trimming reactions by mannosidase IA/IB and that further maturation resulted in the two mature forms of gp75. We propose that the kinetics of biosynthesis and processing reflect events in the biogenesis and maturation of melanosomes.     

Linkage of the Murine Transforming Growth Factor α Gene with Igk, Ly-2, and Fabp1 on Chromosome 6

TGFα is a mitogenic polypeptide found in the conditioned media of transformed cell lines as well as in various solid tumors. Although its physiological role in normal tissues is uncertain, the autocrine action of TGFα on the EGF receptor is postulated to play a role in tumorigenesis. To explore the possibility that pre-existing mouse mutants might have concordance with the mouse TGFα locus (Tgfa) we sought to establish the chromosomal localization of the murine TGFα gene. Using Southern analysis we have detected NcoI and PvuII RFLP in the TGFα gene of progenitor RI mouse strains. These RFLPs have been used to analyze four different RI sets of DNA and to assign Tgfa to the 35-cM region of chromosome 6. Linkage has been established and the data suggest that the distance between Igk and wa-1 anchor loci may be less than 8 cM and that the gene order for the proximal to mid region of mouse chromosome 6 may be: Ggc-Xmmv27-[Brp-1, Lvp-1, Ms6-4]-[Igk, Ly2, Ly3 Odc-rs5, Rn7s-6, Fabp1]-[Tgfa/wa-1]-IL5-R. Homology of synteny has been further defined between the proximal region of mouse chromosome 6 and with the 2p 13-p11 region of human chromosome 2 encompassing TGFA, IGK, CD8A, and FABP1 .     

DNA Sequence, Chromosomal Localization, and Tissue Expression of the Mouse Proteasome SubunitLmp10(Psmb10) Gene

Proteasomes are nonlysosomal multicatalytic proteases involved in antigen processing. Three of the 10 mammalian proteasome β subunits (LMP2, LMP7, and LMP10) are induced by IFN-γ. Two of these (LMP2 and LMP7) are encoded in the major histocompatibility complex of both human (chromosome 6) and mouse (chromosome 17). However, the human homologue ofLmp10, MECL1,is found on chromosome 16. Here we show that in mice,Lmp10is a single-copy gene localized to chromosome 8, in a region of conserved synteny with human chromosome 16. Sequencing of a 129/SvJ strain genomic clone revealed that the gene has eight exons spanning 2.3 kb. Characterization of a full-length mouse cDNA clone indicates thatLmp10encodes a protein of 273 amino acids with a calculated molecular weight of 29 kDa and an isoelectric point of 6.86. Northern analysis ofLmp2, Lmp7,andLmp10showed expression in heart, liver, thymus, lung, and spleen, but not in brain, kidney, skeletal muscle, or testis.     

A frameshift mutation in the melanophilin gene causes the dilute coat colour in rabbit (Oryctolagus cuniculus) breeds

In rabbit, the dilute locus is determined by a recessive mutated allele (d) that causes the dilution of both eumelanic and pheomelanic pigmentations. In mice, similar phenotypes are determined by mutations in the myosin VA, Rab27a and melanophilin (MLPH) genes. In this study, we investigated the rabbit MLPH gene and showed that a mutation in this gene appears responsible for the dilute coat colour in this species. Checkered Giant F1 families segregating for black and grey (diluted or blue) coat colour were first genotyped for a complex indel in intron 1 of the MLPH gene that was completely associated with the coat colour phenotype (θ = 0.00; LOD = 4.82). Then, we sequenced 6357 bp of the MLPH gene in 18 rabbits of different coat colours, including blue animals. A total of 165 polymorphisms were identified: 137 were in non‐coding regions and 28 were in coding exons. One of them was a frameshift deletion in exon 5. Genotyping the half‐sib families confirmed the complete cosegregation of this mutation with the blue coat colour. The mutation was analysed in 198 rabbits of 23 breeds. All Blue Vienna and all other blue/grey/ash rabbits in other breeds (Californian, Castor Rex, Checkered Giant, English Spot, Fairy Marburg and Fairy Pearly) were homozygous for this deletion. The identification of MLPH as the responsible gene for the dilute locus in rabbit provides a natural animal model for human Griscelli syndrome type 3 and a new mutant to study the role of this gene on pigmentation.     

The Structure and Expression of the Murine Wildtype p53-Induced Phosphatase 1 (Wip1) Gene

The human wildtype p53-induced phosphatase 1 (Wip1; GenBank symbol Ppm1d) gene encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner. We have cloned and sequenced the mouse Wip1 gene and its encoded mRNA. The mouse Wip1 gene is composed of six exons and spans over 36 kb of DNA. The mouse cDNA sequence predicts a 598-amino-acid protein with a molecular mass of roughly 66 kDa. Comparison of human and mouse Wip1 sequences revealed 83% overall identity at the amino acid level. The 5′-flanking region of exon 1 had promoter elements characteristic of a housekeeping gene. The Wip1 coding sequences share conserved functional regions with other PP2Cs from a diverse array of species. Expression of Wip1 mRNA was detected ubiquitously in adult and embryonic tissues, though expression in the testis was much higher than in other tissues. Wip1 has been mapped near the p53 gene on mouse chromosome 11.     

Lop12, a Mutation in Mouse Crygd Causing Lens Opacity Similar to Human Coppock Cataract

A new cataract mutation was discovered in an ongoing program to identify new mouse models of hereditary eye disease. Lens opacity 12 (Lop12) is a semidominant mutation that results in an irregular nuclear lens opacity similar to the human Coppock cataract. Lop12 is associated with a small nonrecombining segment that maps to mouse Chromosome 1 close to the eye lens obsolescence mutation (Cryge^Cat2-Elo), a member of the γ-crystallin gene cluster (Cryg). Using a systemic candidate gene approach to analyze the entire Cryg cluster, a G to A transition was found in exon 3 of Crygd associated with the Lop12 mutation and has been designated Crygd^Lop12. The mutation Crygd^Lop12 leads to the formation of an in-frame stop codon that produces a truncated protein of 156 amino acids. It is predicted that the defective gene product alters protein folding of the γ-crystallin(s) and results in lens opacity.     

Cloning of the murine unconventional myosin gene Myo9b and identification of alternative splicing

We report the cloning of a cDNA for the mouse unconventional myosin Myo9b, the orthologue of the rat myr5 and human MYOIXb genes. A full-length spleen cDNA of 7087 bp encoding a protein of 1961 amino acids was isolated. By RT–PCR, we show that Myo9b is expressed in a wide range of tissues, including heart, brain, muscle and inner ear. In addition, we have identified two alternatively spliced exons. Equivalent exons have not been previously reported for either the human or rat homologues. These exons are located in the Myo9b specific actin-binding site insert of the head domain and in the tail region. A third splice form utilizing an alternative reading frame within the 3′UTR is also described. Several polymorphisms within the coding region were identified; of interest is an in-frame 33 bp imperfect duplication within the tail region that was observed only in the C57Bl/6 strain. Myo9b has been previously mapped to mouse chromosome 8 and is a candidate for the mouse mutations myodystrophy and quinky.