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Head Region Mutations Copyright 1998 by the Genetics Society of America Molecular Genetic Dissection of Mouse Unconventional Myosin-VA: Head Region Mutations Jian-Dong Huang,* M. Jamie T. V. Cope,²,1 Valerie Mermall,³ Marjorie C. Strobel,* John Kendrick-Jones,² Liane B. Russell,²² Mark S. Mooseker,³,§,** Neal G. Copeland,* and Nancy A. Jenkins,* *ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, ²MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom, ³Department of Biology, §Department of Pathology, **Department of Cell Biology, Yale University, New Haven, Connecticut 06520, ²²Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Manuscript received September 19, 1997 Accepted for publication December 23, 1997 ABSTRACT The mouse dilute (d) locus encodes unconventional myosin-VA (MyoVA). Mice carrying null alleles of dilute have a lightened coat color and die from a neurological disorder resembling ataxia and opisthotonus within three weeks of birth. Immunological and ultrastructural studies suggest that MyoVA is involved in the transport of melanosomes in melanocytes and smooth endoplasmic reticulum in cerebellar Purkinje cells. In studies described here, we have used an RT-PCR-based sequencing approach to identify the mutations responsible for 17 viable dilute alleles that vary in their effects on coat color and the nervous system. Seven of these mutations mapped to the MyoVA motor domain and are reported here. Crystallo- graphic modeling and mutant expression studies were used to predict how these mutations might affect motor domain function and to attempt to correlate these effects with the mutant phenotype. HE mouse dilute (d) locus encodes unconventional motor protein is used for the long-range transport of Tmyosin-VA (MyoVA). Mice homozygous for null SER from the cell body to the dendritic shaft. This mutations at dilute have a lightened coat color and die hypothesis is consistent with recent studies indicating from a neurological disorder resembling ataxia and that the movement of membranous organelles involves opisthotonus (arching of the head and neck) within both actin- and microtubule-based motors and with cur- three weeks of birth. The pigment defect in dilute mice rent models suggesting that microtubules provide the does not result from abnormal pigment production. tracks for movement over long distances while actin Rather, the lightened coat color results from the irregu- ®laments provide for movement within local regions of lar clumping of melanosomes within the perinuclear the cytoplasm (Atkinson et al. 1992; Langford 1995). regions of the melanocyte and the subsequent uneven It is also consistent with immuno¯uorescence and im- release of the granules into the hair shaft (Russell and munoelectron microscopy studies showing that MyoVA- Russell 1948). This phenotype is consistent with im- associated organelles are present on both microtubules munolocalization experiments suggesting that MyoVA and actin ®laments (Evans et al. 1997). functions in melanosome transport and/or melano- During the past century hundreds of forward muta- Provance Wu some tethering ( et al. 1996; et al. 1997). tions to dilute have been identi®ed. Most of these alleles The neurological defects of dilute appear to result were produced in large-scale mutagenesis screens, ®rst in part from defects in smooth endoplasmic reticulum using ionizing radiations, which, in certain germ-cell (SER) transport. In both the dilute rat and the dilute stages, primarily make large deletions as well as other mouse, SER has been reported to be missing from the complex rearrangements, and later with chemicals such Dekker- dendritic spines of cerebellar Purkinje cells ( as ethylnitrosourea (ENU) which, when spermatogonia Ohno Takagishi et al. 1996; et al. 1996). Since SER is are treated, primarily make point mutations. The vast still present in the dendritic shaft, it has been proposed majority of these induced mutations [called dilute opis- that MyoVA is required only for the short-range trans- thotonus (dop)ordilute lethal (dl)] are homozygous lethal port of SER into the dendritic spine and that another and presumably represent null alleles (Russell 1971; Strobel et al. 1990). Four viable classes of alleles were also recovered and presumably represent hypomorphic Corresponding author: Nancy A. Jenkins, ABL-Basic Research Pro- alleles. The ®rst class, called dilute (d), produces a light- gram, P.O. Box B, Bldg. 539, Frederick, MD 21702-1201. Email: [email protected] ened coat color but no neurological defect. The second x 1Present address: University of California, Rm. 401 Barker Hall, #3202 class, called dilute intermediate (d ), is the most common Berkeley CA 94720-3202. class. The coat color of dx mice is intermediate between Genetics 148: 1951±1961 (April, 1998) 1952 J.-D. Huang et al. wild type and d mice, and dx mice are neurologically (Version 8) provided by Genetics Computer Group, University normal. The last two classes, called dilute neurological (dn) of Wisconsin (Madison). Point mutations, deletions and inser- xn tions in the cDNA can be easily detected by this method. and dilute intermediate neurological (d ), have a lightened Preparation of protein samples and electrophoresis: A small coat color and a neurological defect that either disap- amount of frozen brain or spleen was chipped off the organ pears as the mice age or is mild and persists throughout under liquid nitrogen and homogenized in 0.5 ml of 5% ice- life. These two classes are distinguished by coat color, cold trichloroacetic acid (TCA) with a hand-held homoge- which is like that of d (dn)ordx (dxn) mice, respectively. nizer. A 10 ml aliquot was removed and the protein concentra- n tion was determined by the BCA assay (Pierce, Rockford IL). In some cases, d mutations were originally classi®ed as The TCA precipitates were pelleted by centrifugation at op op viable d alleles. To avoid confusion with the lethal d 12k 3 g for 10 min, 48; the pellets were washed with water, alleles, we consider them all dn mutations in this report. respun and brought up in sample buffer (25 mm tris base, 38 In studies described here, we have used an RT-PCR- mm glycine, 5% SDS, 5% beta-mercaptoethanol, 50% glycerol, based sequencing approach to identify the mutations and z1 mg/ml bromophenol blue) for a ®nal protein concen- tration of 1 mg/ml. Samples were loaded at 20 mg/lane onto responsible for 17 viable dilute alleles. We hoped that, 5±20% mini-gradient SDS-PAGE gels (Laemmli 1970), and by determining the nature and position ofthe mutations transferred (Towbin et al. 1979) to PVDF membranes (Bio- responsible for each dilue allele and by correlating this Rad, Hercules CA). Blots were stained with antibodies directed information with mutation phenotype, we could gain against the head domain of myosin V (produced by F. S. Espindola new insights into the functional domains of MyoVA. ). This antibody was produced in rabbits using a bacterially expressed fusion protein of the chicken myosin V In these studies we focused primarily on ENU-induced head domain fused to maltose-binding protein (Esprea®co et alleles since they are most likely to be caused by point al. 1992) and puri®ed on an amylose af®nity column. Maltose- mutations, which are a very informative class of muta- binding protein reactivity was removed from the antisera by tions for structure-function studies. Members of all four absorption to a maltose-binding protein column and then viable classes of dilute alleles were sequenced. In the af®nity-puri®ed on a column constructed with the original n xn fusion protein. The ®nal antibody was used at 0.05 mg/ml. case of the d, d , and d alleles, some spontaneous and Blots were processed for chemiluminescence according to the radiation-induced alleles were also included since few manufacturer's directions (Boehringer Mannheim, Mann- ENU-induced alleles from these classes were available heim, Germany). For head alleles: In some experiments, anti- for study. Seven of the mutations mapped to the MyoVA body against the tail domain of myosin V (Esprea®co et al. head and are reported here. 1992) was also used at 1 mg/ml. Blots were stripped and stained for myosin VI (assumed to be unaffected by the MyoVA muta- tions) as a loading control with anti-myosin-VI antibody (Has- son and Mooseker 1994) used at 1 mg/ml. MATERIALS AND METHODS Quanti®cation of myosin V: Blots were scanned with a 600 Mice: The seven alleles reported in this study were gener- dpi, 8 bit greyscale scanner (Microtek Lab Inc., Torrance CA); the relative amount of myosins V and VI per lane was ated by mutagenesis of (101/Rl 3 C3H/Rl) F1 hybrid mice at Oak Ridge National Laboratory, Oak Ridge, TN. Six of the determined with Image software (National Institute of Health, seven alleles are extinct and only frozen tissues were available Bethesda, MD). In order to control for small differences in gel for analysis. The Myo5a2ENURcc allele is maintained at the Na- loading or transfer ef®ciency, the relative amount of myosin VI tional Cancer Institute-Frederick Cancer Research and Devel- for each lane of a given tissue was determined and used to opment Center by crossing carriers to C57BL/6J-d v se/d v se mice. normalize the amount of myosin V per lane. Northern analysis: Total RNA was prepared from brain, spleen and skin of C57BL/6J, 101/Rl, C3H/Rl and dilute mu- tant mice by the RNAzol method (Tel-Test, Inc, Friendswood, RESULTS TX). RNA was poly(A) selected once using the mRNA Puri®- cation kit from Pharmacia (Piscataway, NJ). For Northern analy- Mutant characterization: Each of the viable dilute al- sis, the RNA was electrophoresed through a 0.8% agarose gel leles sequenced in these studies was characterized as andthen transferred to a Hybond-N1 membrane (Amersham, follows.
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