Human Molecular Genetics, 2001, Vol. 10, No. 13 1335-1346
© 2001 Oxford University Press
Differential expression of the actin-binding proteins, 
-actinin-2 and -3, in different species: implications for the evolution of functional redundancy
1Neurogenetics Research Unit, Children’s Hospital at Westmead, Sydney, NSW, Australia, 2Department of Paediatrics and Child Health, University of Sydney, Sydney, NSW, Australia, 3Oncology Research Unit, Children’s Hospital at Westmead, Sydney, NSW, Australia, 4Genetics Division, Children’s Hospital, Harvard Medical School, Boston, MA, USA and 5Centre for Bioinformation Science, John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia
Received February 19, 2001; Revised and Accepted April 12, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The
-actinins are a multigene family of four actin-binding proteins related to dystrophin. The two skeletal muscle isoforms of -actinin (ACTN2 and ACTN3) are major structural components of the Z-line involved in anchoring the actin-containing thin filaments. In humans, ACTN2 is expressed in all muscle fibres, while ACTN3 expression is restricted to a subset of type 2 fibres. We have recently demonstrated that -actinin-3 is absent in 
18% of individuals in a range of human populations, and that homozygosity for a premature stop codon (577X) accounts for most cases of true -actinin-3 deficiency. Absence of -actinin-3 is not associated with an obvious disease phenotype, raising the possibility that ACTN3 is functionally redundant in humans, and that -actinin-2 is able to compensate for -actinin-3 deficiency. We now present data concerning the expression of ACTN3 in other species. Genotyping of non-human primates indicates that the 577X null mutation has likely arisen in humans. The mouse genome contains four orthologues which all map to evolutionarily conserved syntenic regions for the four human genes. Murine Actn2 and Actn3 are differentially expressed, spatially and temporally, during embryonic development and, in contrast to humans, -actinin-2 expression does not completely overlap -actinin-3 in postnatal skeletal muscle, suggesting independent function. Furthermore, sequence comparison of human, mouse and chicken -actinin genes demonstrates that ACTN3 has been conserved over a long period of evolutionary time, implying a constraint on evolutionary rate imposed by continued function of the gene. These observations provide a real framework in which to test theoretical models of genetic redundancy as they apply to human populations. In addition we highlight the need for caution in making conclusions about gene function from the phenotypic consequences of loss-of-function mutations in animal knockout models. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The
-actinins are a family of actin-binding proteins related to dystrophin and the spectrins (1). In non-muscle cells, the cytoskeletal isoforms, -actinin-1 and -4, are found along microfilament bundles where they mediate membrane attachment at adherans-type junctions in a dynamic manner regulated by calcium binding (2,3). In skeletal muscle, -actinin-2 and -3 are major structural components of sarcomeric Z-lines where they function to anchor actin-containing thin filaments in a constitutive manner (4). Recent studies suggest additional roles for the -actinins in skeletal muscle. Sarcomeric -actinins bind to other thin filament and Z-line proteins including nebulin, myotilin, CapZ and myozenin (5–7), the intermediate filament proteins, synemin and vinculin (8,9), and the sarcolemmal membrane proteins, dystrophin and ß1 integrin (10,11). These binding studies suggest that the -actinins play a role in thin filament organization and the interaction between the sarcomeric cytoskeleton and the muscle membrane. In addition, sarcomeric -actinin binds phosphatidylinositol 4,5-bisphophate (12), phosphatidylinositol 3-kinase (13) and PDZ-LIM adaptor proteins (14,15), suggesting a role in the regulation of myofibre differentiation and/or contraction.
In humans, the -actinin-2 gene, ACTN2, is expressed in all skeletal muscle fibres, whereas expression of ACTN3, encoding -actinin-3, is limited to a subset of type 2 (fast) fibres (16). We have recently demonstrated that -actinin-3 is absent in 18% of individuals in a range of human populations and that homozygosity for a premature stop codon (577X) accounts for all cases of true -actinin-3 deficiency identified to date. An additional polymorphism (523R) occurs in linkage disequilibrium with 577X, but does not appear to exert a deleterious effect when expressed in the heterozygous state in coupling with 577R. Absence of -actinin-3 is not associated with an obvious disease phenotype, suggesting that ACTN3 is redundant in humans (17).
Functional redundancy occurs when two genes perform overlapping functions so that inactivation of one of the genes has little or no effect on the phenotype (reviewed in 18). -actinin-2 expression in human skeletal muscle completely overlaps -actinin-3. ACTN2 and ACTN3 are 80% identical and 90% similar (4). In addition, -actinin-2 and -actinin-3 form heterodimers in vitro and in vivo, suggesting structural similarity and lack of significant functional differences between the two skeletal muscle -actinin isoforms (19). On this basis we hypothesize that -actinin-2 is able to compensate for the absence of -actinin-3 in type 2 (fast) fibres in humans.
Despite the apparent functional redundancy of ACTN3 in humans, sequence comparison of human and chicken skeletal muscle -actinin genes suggest that human ACTN2 and ACTN3 have both evolved very slowly since their divergence more than 300 million years ago, implying strong functional conservation (17). Here we demonstrate that ACTN2 and ACTN3 are differentially expressed, spatially and temporally, during mouse embryonic development. Unlike humans and non-human primates, ACTN2 expression does not completely overlap ACTN3 in postnatal mouse skeletal muscle, suggesting that ACTN3 may not be functionally redundant in the mouse. We provide further evidence for the evolutionary conservation of the ACTN3 gene and demonstrate that the null mutation has likely arisen in humans, with marked variation in frequency among different human populations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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There is marked population variation in the allelic frequency of 577R and 577X
We have previously demonstrated that the stop codon polymorphism (577X) is present in different ethnic populations (17). To determine the population frequency and distribution of each allele at positions 523 and 577, we genotyped 485 DNA samples (970 chromosomes) derived from anonymous individuals whose DNA was collected for other purposes, including the four major human groups (from Asia/Americas, Australasia, Africa and Europe; Table 1). The 577X allele is most frequent in Eurasia (0.51) and least frequent in Africa (0.16). Frequency of the 577X allele in the African Bantu sample is significantly lower than that of all other samples. The 577X allele frequencies in African Americans and Aboriginal Australians are also significantly lower than the Asian and European groups.
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The null mutation (577X) likely arose in humans
To determine the origin of the 577X allele (and the 523R allele, which occurs in strong linkage disequilibrium with 577X) (17), we genotyped 36 unrelated baboons (diverged from human lineage
25 x 106 years ago) and 33 unrelated chimpanzees (diverged from human lineage 5 x 106 years ago). All 69 non-human primates were homozygous for the ‘wild-type’ alleles in exons 15 (523Q) and 16 (577R), suggesting that the polymorphisms originated after the separation of the human and chimpanzee lineages, or that they have a very low frequency in non-human primates.
Identification of mouse Actn genes
To determine the number and identity of -actinin genes in the murine genome, the chromosomal localizations for mouse orthologues of the four human ACTN genes were identified using the Jackson Laboratory interspecific backcross mapping panel C57BL/6JEi x SPRET/Ei)F1 x SPRET/Ei (Jackson BSS) (20). PCR primers were designed based on human exon sequences to amplify across small murine introns that would presumably vary between Mus musculus and Mus spretus. Each PCR product was sequenced and shown to represent the murine orthologue of a human ACTN gene, confirming that the mouse genome contains a single orthologous locus corresponding to each human gene. SSCP analysis of these PCR products on the backcross mapping panel revealed that Actn1 is at 36.2 cM on mouse chromosome 12, Actn2 is at 2.1 cM on chromosome 13, Actn3 is in the most proximal (centromeric) group of loci on chromosome 19 and Actn4 is at 5.3 cM on chromosome 7 (Fig. 1). These localizations are supported by the observation that there are genes with human orthologues linked to the corresponding human ACTN genes at each of these locations (http://www.ncbi.nlm.nih.gov/ Homology/index.html).
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Cloning of mouse Actn2
We determined the full-length coding sequence of mouse Actn2 by screening a mouse diaphragm cDNA library (GenBank accession no. AF248643). The mouse Actn3 coding sequence is available from GenBank (accession no. AF093775). The similarity between mouse Actn2 and Actn3 is the same as between human ACTN2 and ACTN3, i.e. 88% similar and 79% identical. The mouse proteins are colinear and have the same functional domains as the human proteins—an N-terminal actin-binding domain, four central repeat domains and C-terminal EF-hands (4).
ACTN3 is conserved during evolution
We have previously shown strong sequence conservation between human and chicken skeletal muscle -actinin genes (17). There is only one skeletal muscle ACTN gene in the chicken (4), whereas we have now demonstrated that the mouse genome contains four orthologues which all map to evolutionarily conserved syntenic regions for the four human genes. Sequence comparison between mouse and human ACTN2 and ACTN3 suggests that the evolution of the -actinins has been slow relative to other genes (Table 2). The nucleotide substitution rate at non-synonymous sites between the human and mouse ACTN3 genes (0.016 substitutions/site) is substantially lower than the average rate for a sample of more than 1000 genes (0.072 substitutions/site). It is also lower than the mean rate of ubiquitously expressed genes (0.029 substitutions/site), which generally evolve more slowly than genes with tissue-specific patterns of expression (21). The nucleotide substitution rate at synonymous sites between the human and mouse ACTN3 genes (approximately 0.45 substitutions/site) is similar to the average rate for the same sample of more than 1000 genes (approximately 0.49 substitutions/site), implying that the low rate of substitution in ACTN3 is not due to an intrinsically low mutation rate in this gene.
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Tissue-specific expression of the skeletal muscle
-actinin isoforms-Actinin-2 is more widely expressed than -actinin-3.Northern blot analysis was used to assess mRNA transcript expression in mouse tissues. For
-actinin-2, a 3.5 kb transcript was detected in skeletal and cardiac muscle and in the brain but was not expressed in smooth muscle, kidney, liver or lung. The membrane was stripped and reprobed to detect a 3.4 kb transcript representing -actinin-3. A transcript for -actinin-3 was detected only in skeletal muscle (data not shown).
We further determined the tissue-specific expression of the sarcomeric -actinins using western blot analysis. In the mouse, antibodies directed against -actinin-2 identified a 96 kDa protein band in skeletal and cardiac muscles, the diaphragm and brain. -Actinin-3 antibodies recognized a band of similar size in skeletal muscle and diaphragm, but not in cardiac muscle or brain. In humans, -actinin-2 was present in skeletal, cardiac and extraocular muscles and in the brain, with greater amounts in grey matter than in white matter, but not in liver or lung. -Actinin-3 was detected in limb skeletal muscle and at very low levels in the brain (grey matter) (Fig. 2). The results of western blot analysis of baboon tissue were identical to human, although brain samples were not available for analysis.
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To further define the expression of
-actinin isoforms in the brain, we stained serial sections of mouse brain from postnatal day 3 (P3) mice with antibodies to -actinin-2, -actinin-3 and desmin (as a negative control). -Actinin-2 stained many structures including pyramidal cells in the hippocampus, choroid plexus, meninges and cells of the olfactory bulb (Fig. 3). Staining was enriched in the pyramidal cell dendrites and puncta of the dentate gyrus. These puncta may represent synaptic interactions of the perforant pathway with the granule cells. -Actinin-3 also stained pyramidal cells of the hippocampus, meninges and cells of the olfactory bulb. Positive staining was present in the dendritic processes of pyramidal and olfactory bulb cells. There was negative staining with antibodies to desmin and with secondary antibody alone.
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Expression of
-actinin-2 does not completely overlap -actinin-3 in the mouse skeletal muscleWe performed immunocytochemistry on sections of skeletal muscle from control human quadriceps muscle. As noted previously,
-actinin-2 is expressed in all muscle fibres and -actinin-3 is expressed in type 2 fibres (16). Fibre type expression of the -actinins in baboon skeletal muscle demonstrates the same staining patterns as for humans (Fig. 4B).
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Type 2 (fast) fibres are the predominant fibre type in the mouse (80% of fibres, compared with 40–50% in humans). Three muscles from the mouse were analysed; extensor digitorium longus (EDL, predominantly fast fibres), soleus (predominantly slow fibres) and crural (mixed). Quantitation of the mouse skeletal muscle
-actinin isoforms demonstrated that -actinin-2 is expressed in all type 1, 2A and 2X fibres (Fig. 4A). -Actinin-2 was also expressed in a subset of type 2B fibres, but the proportion varied between different muscles and between different areas of the same muscle. In general, 60% of type 2B fibres positively stain with -actinin-2. -Actinin-3 expression is restricted to type 2B fibres. Consequently, there is some co-expression of -actinin-2 and -3. For example, in soleus (80% type 1 and 20% type 2A fibres), all fibres express -actinin-2 but not -actinin-3. In the EDL (50% type 2A and 50% type 2B fibres), 60% of fibres stained positively for -actinin-2 and >95% of fibres stained positively for -actinin-3, with variable expression of -actinin-2 in different areas of the muscle. Three separate areas of the crural were analysed. In areas predominantly composed of type 2B fibres, all fibres stained positive for -actinin-3 and 60–70% expressed -actinin-2, i.e. there was high co-expression of the two -actinin skeletal muscle isoforms (60% of fibres). However, in areas of the same specimen with high fibre type 1 expression, -actinin-3 expression is low (17%) and subsequently, co-expression of -actinin-2 and -3 is also low (15%) (Fig. 4B). Thus, although there is some co-expression of -actinin-2 and -3 in mouse skeletal muscle, unlike humans and non-human primates, there are many fibres in the mouse that express -actinin-3 only.
Developmental expression of sarcomeric -actinins in mouse and human
ACTN2 is expressed before ACTN3 with different tissue-specific patterns of expression.
To determine the developmental expression of -actinin-2 and -3 in mouse embryos, we performed immunocytochemistry on sections of whole mouse embryos at embryonic days (E) 11.5, 12.5 (period of organogenesis), 14.5 and 16.5 (fetal growth and development). The first skeletal muscle isoform expressed is -actinin-2. At E11.0, -actinin-2 staining was localized to the mesenchyme next to the first brachial arch and cardiac tissue. -Actinin-3 staining was negative. At E12.5, -actinin-2 was expressed in cardiac muscle, and also in intercostal muscles, in neuroepithelial and laryngopharyngeal tissues and in the intestinal region, particularly the duodenum. -Actinin-3 was not expressed at E12.5.
At E14.5, -actinin-2 was the predominant isoform expressed, with positive staining of the intercostal muscles, diaphragm, throat, gut and a smooth muscle layer around the stomach. At high power, -actinin-2 stained in a striated pattern consistent with localization at the Z-line. At this stage, -actinin-3 was present in skeletal muscle only, at a very low level compared with -actinin-2 (Fig. 5). The pattern of staining in skeletal muscle is similar at E16.5 and, in addition, both -actinin-2 and -3 were detected in the skin. -Actinin-3, but not -actinin-2, was expressed in hair shafts of E16.5 whiskers, whereas -actinin-2 was localized to muscle fibres at the base of the hair shaft (Fig. 5). An actin complex which contains -actinin has been identified in human epithelial hair follicles, and may function to maintain hair follicle structure (22). By P1, -actinin-2 and -3 have approximately equal staining intensities in mouse skeletal muscle. -Actinin-3 is the predominant skeletal muscle isoform by P30 (Fig. 4B).
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Western blot analysis with antibodies to
-actinin-2 and -3 was performed on fetal human muscle protein lysates. The specimen ages ranged from 16 weeks gestation to newborn samples. Both -actinin-2 and -3 were expressed from the earliest age studied in human muscle and were expressed before fast myosin isoforms (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have identified two
-actinin genes in the mouse (Actn2 and Actn3) which correspond to human ACTN2 and ACTN3, allowing us to directly compare the expression of genetically distinct isoforms in different species. In humans, the potential for -actinin-2/-3 functional redundancy is indicated by complete overlapping expression of -actinin-3 by -actinin-2 at the protein level, and confirmed by the presence of an ACTN3 null variant with no detectable phenotypic effect. The proteins are structurally and functionally similar, and it is likely that -actinin-2 can compensate for -actinin-3 deficiency. We have shown that the ACTN3 null allele (577X) probably results from a single mutation that occurred after the divergence of humans and chimpanzees. The variant occurs with moderate frequency in human populations from Africa, Europe, Asia and Australasia. It does not appear to contribute to major forms of muscular disease.
In contrast, we have shown that -Actinin-3 may not be functionally redundant in mouse skeletal muscle. -Actinin-3 is the predominant isoform in postnatal mouse muscle and, unlike human and baboon, murine -actinin-2 expression does not completely overlap that of actinin-3. -Actinin-2 and -3 are differentially expressed, spatially and temporally, during embryonic development, suggesting independent transcriptional regulation and different roles during myogenesis. The tissue distribution of the sarcomeric -actinins is wider than previously reported as both -actinin-2 and -3 are expressed in mouse and human brain. Wyszynski et al. (23) have previously demonstrated expression of -actinin-2, but not -actinin-3, in rat cerebellum and suggested that -actinin-2 may provide structural support or regulation of cytoskeletal remodelling of the NMDA receptors.
In other mammals, such as rabbits and pigs, there are also fast- and slow-muscle-specific isoforms of -actinin, although the gene(s) responsible have not been isolated (24,25). The presence of two sarcomeric -actinin genes may, however, be restricted to mammals. Sarcomeric -actinins are encoded by a single ACTN gene in Drosophila (26) and in chickens (27), with specific fast and slow muscle isoforms in avian skeletal muscle generated by alternate splicing.
Gene duplication is an important process in the evolution of complexity, although most duplicated genes in eukaryote genomes are silenced within a few million years (28). Duplicate genes are likely to be functionally redundant immediately following duplication. When one copy is not silenced, redundancy is usually lost, either as one gene acquires a new function (29), or through ‘subfunctionalization’, as the function of the parent gene is partitioned between the duplicated genes (30).
Despite the apparent lack of two genes in birds, the mammalian ACTN2 and ACTN3 genes appear to have arisen from a gene duplication that occurred substantially earlier than the divergence of birds and mammals (17). This implies that one copy of the gene has been lost or has evolved a new function in birds. In mammals both copies of the gene have survived, and our comparison of the human and mouse ACTN2 and ACTN3 sequences shows that the genes have been highly conserved throughout mammalian evolution. We propose that ACTN2 and ACTN3 may have had only partially overlapping patterns of expression during most of mammalian evolution, as they do in mice. The complete overlap of ACTN3 function by ACTN2, apparent in humans, may have arisen relatively recently, although the similarity of expression patterns between humans and baboons suggests that it is at least as old as the time of separation of these species (>20 x 106 years). Long-term preservation of redundancy is theoretically possible under certain restricted conditions (18,31) as has been observed for duplicated genes in the mouse [e.g. pax2 and pax5 (32); and Hoxa-10 and Hoxa-11 (33)].
The apparent functional redundancy of ACTN3 in humans allows us to test theoretical models of genetic redundancy as they apply to the human genome. The principle issue is whether Darwinian selection is the basis for maintaining expression of genes with redundant functions. Complete overlap of function can be evolutionarily stable if there is an associated fitness advantage. This may operate through a ‘dosage effect’, i.e. two genes are maintained because individuals with two genes have an advantage over those with only one gene (more gene product is better) (31). However, the absence of -actinin-3 in a substantial portion of the human population with no apparent phenotype suggests that gene dosage does not affect fitness, and hence maintenance of the redundancy. It is possible, however, that the fitness difference between individuals with one and two copies of the ACTN genes existed in the past but has been lost during recent human evolution.
In an alternate model, redundancy can be maintained when the function of one gene completely overlaps, but extends beyond that of another gene, as long as the gene whose function is completely overlapped performs the common function of the two genes more effectively (18,31). According to this model, -actinin-2 and -3 may perform the same role in type 2 (fast) fibres, but -actinin-3 performs the role more effectively. In this situation, the presence or absence of -actinin-3 in humans has functional (and fitness) implications only in some environments or under extreme conditions that are not immediately evident. The force-generating capacity of muscle fibres at high velocity and the capacity of the individual to adapt to exercise training are all strongly genetically influenced (34). Since actively contracting striated muscle is the most energy-demanding tissue in the body, efficiency of skeletal muscle contraction coupled with resistance to fatigue is crucial to survival in evolutionary terms.
Under this model, -actinin-3 would be conserved because it functions more effectively in type 2 fibres. -Actinin-2 would be conserved because of its more general function. The functional overlap of the genes would eventually be lost through mutation, as individuals with genes of overlapping function have no advantage over those in which the genes functioned independently. It is difficult to explain the spread of the ACTN3 null allele under this model, since the model implies that individuals with ACTN3 have an advantage over those that lack the gene. However, it is possible that the advantage conferred by ACTN3 has been lost during recent human evolution. The occurrence of the allele at intermediate frequencies in different human populations suggests a relatively early origin of the allele, probably >100 000 years ago. Any change in physiology, lifestyle or environment that might have caused the loss of a fitness difference between individuals with and without the ACTN gene would have to have persisted for at least this long.
The persistence of -actinin-3 redundancy in humans may be better explained by a model put forward by Gibson and Spring (35), who suggest that there is no need to invoke active selection for redundant gene expression in vertebrate development. They propose that, following gene duplication in multidomain proteins, selection operates to reject deleterious point mutations in redundant genes because the majority of coding point mutations have a ‘dominant-negative’ effect on the protein relative to its normal function in the cell. As a result the redundant gene is maintained with a high degree of evolutionary conservation. In contrast, mutations that result in complete loss of gene expression may occur with little effect on phenotype or fitness because of the redundancy. The redundancy itself is a historical consequence of either polyploidy in the vertebrate common ancestor (36) or subsequent gene duplication. Redundancy is preserved because the rate of mutations leading to complete loss of function is much lower than the rate of (usually deleterious) mutations leading to altered function. Redundancy cannot be maintained indefinitely in this way and eventually it is lost as rare loss-of-function mutations occur. Under this model, the intermediate frequency of the ACTN3 577X null allele represents a transitory stage in the eventual loss, by genetic drift, of the highly conserved but redundant ACTN3 gene. The complete loss of the gene is unlikely to occur unless the human population is drastically reduced in size, since the mean time to fixation of a neutral mutation is four times the effective population size (37). As long as the human population is extremely large, the null allele will remain at its current intermediate frequency.
ACTN3 577X is the human equivalent of a ‘gene knockout’ or homozygous null mutation of single genes in mice and other experimental species that produce no phenotype or a subtle phenotype compared with double knockouts for more than one member of a multigene family (38,39). In humans, as in experimental species, there is a need for caution in drawing inferences about function from the phenotypic consequences of loss-of-function mutations. No disease-associated ACTN3 mutations have been reported to date, but we predict that, despite the absence of any detectable phenotypic effect of the loss-of-function 577X allele, ACTN3 missense mutations may have dominant-negative phenotypes (40).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ACTN3 genotyping
Human genomic DNA was isolated from blood by phenol–chloroform extraction following cell lysis with Triton X-100 and digestion with Proteinase K, or from muscle (
7 mg of tissue) using QIAamp tissue kits (Qiagen). Exons 15 and 16 of ACTN3 were amplified from genomic DNA. The primers for exon 15 were: forward, 5'-GGTGGGTAGGTGGGTGAGGC-3'; reverse, 5'-GAGTGTACCAGCCACACGTCC-3'. The primers for exon 16 were: forward, 5'-CTGTTGCCTGTGGTAAGTGGG-3'; reverse, 5'-TGGTCACAGTATGCAGGAGGG-3', corresponding to adjacent intronic sequences. The PCR reaction cycle for both sets of primers was: 94°C for 5 min, 94°C for 30 s and 70°C for 60 s, for 35 cycles, with a final extension of 72°C for 10 min. The Q523R alleles (codons CAG and CGG, respectively) can be distinguished by the presence (523R) or absence (523Q) of an MspI restriction site (C
CGG). 523Q PCR products have 118 and 74 bp fragments. 523R PCR products have 21, 74 and 97 bp fragments. The R577X alleles (codons CGA and TGA, respectively) can be distinguished by the presence (577X) or absence (577R) of a DdeI restriction site in exon 16. 577R PCR products have 205 and 85 bp fragments; 577X PCR products have 108, 97 and 86 bp fragments. Digested PCR fragments were separated by 10% polyacrylamide gel electrophoresis and stained with ethidium bromide. Genomic DNA was also isolated from 36 unrelated baboons and 33 unrelated chimpanzees. For chimpanzees, ACTN3 exons 15 and 16 were amplified using primers designed for the human sequence (see above). To amplify baboon DNA, we designed primers complementary to human ACTN3 sequence within exons. The forward primer for exon 15 was 5'-CACCTC- TAGCGGATGGAGAA-3' and the reverse primer was 5'-CGAGTCCTCCACAGAGTG-3'. The forward primer for exon 16 was 5'-CCAGAGCCTGCTGACAGC-3' and the reverse primer was 5'-TCCCACTTGGTGTTGATGTC-3'. PCR fragments were digested with MspI and DdeI as detailed above. PCR fragments of exons 15 and 16 from two baboons and one chimpanzee were sequenced to confirm amplification of the appropriate sequence and the results of restriction enzyme analysis.
Chromosomal localization of mouse Actn genes
The chromosomal localizations for the four mouse Actn genes were identified using the Jackson Laboratory interspecific backcross panel (C57BL/6JEi x SPRET/Ei)F1 x SPRET/Ei (Jackson BSS) as described by Rowe et al. (20). PCR was performed using primers based on human cDNA sequences and designed to amplify across small introns in the four murine Actn genes as follows: Actn1 forward, 5'-CAGATGAATGAGTTCCGGGCCTCCTTCAAC-3'; Actn1 reverse, 5'-TAACCCAAGCTGATGAGGCAGGCTT-3'; Actn2 forward, 5'-TTCCAATCCTTCATCGACTTCATGAC-3'; Actn2 reverse, 5'-GGCATCCTCTTGATGCAGTACTGGGCCT-3'; Actn3 forward, 5'-AACGAGTTCCGAGCATCCTTCAACC-3'; Actn3 reverse, 5'-CTTCCCCCAGGTCATAGCCCATGGAGAT-3'; Actn4 forward, 5'-CTGCACCCTGGGCCCCAGCGGACC-3'; Actn4 reverse, 5'-ATGAAGGCTTGGAAGGTCACAAAG-3'.
PCR reaction cycles for Actn1 and Actn4 primers were 30 cycles of: 94°C for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C for 60 s, with a final extension of 72°C for 10 min. Actn2 and Actn3 were amplified under similar conditions, but the annealing temperature was 60°C. Several primer pairs amplified multiple murine loci that segregated in the Jackson BSS panel; however, the correct -actinin loci were easily identified by DNA sequencing of the PCR products as, in each case, the amplified coding regions of each murine locus were identical to the orthologous human gene. Variation between the different mice breeds, M.musculus and M.spretus, were detected by separating radiolabelled products on MDE SSCP gels following the manufacturer’s protocols (FMC Bioproducts). Segregation of the C57BL/6JEi alleles four genes was determined in the 94-backcross offspring and the map locations were determined by the Jackson Laboratory Backcross Mapping Group. Missing typings were inferred from surrounding data where assignment was unambiguous. Map locations in cM are given relative to the most centromeric marker on the Jackson BSS map as of January 2001. Raw data from The Jackson Laboratory are available for these loci at http://www.jax.org/resources/documents/cmdata.
Cloning and sequencing cDNA of mouse Actn2
PCR amplification of mouse Actn2 was performed using primers designed from human cDNA sequence. The primers used were: forward, 5'-GCCATGAACCAGATAGAGCC-3' (from –3 to 17 bp), covering the ATG start codon at position 1; reverse, 5'-TCCATCTTGTTCTGGATCCAG-3' (from 1953 to 1973 bp). The PCR product was sequenced and used to screen a mouse diaphragm muscle cDNA library. Two clones were isolated from the library, sequenced and compared with human ACTN2 sequence.
Sequence and statistical analysis
Sequences were aligned using GDE (Genetic Data Environment) 2.2 (41). Substitution rates at synonymous and non-synonymous sites were estimated by the method of Li (42), with correction for multiple substitution using the two-parameter method of Kimura (37). Allele frequency differences among population groups were assessed by the method of Raymond and Rousset (43) implemented in the computer package ‘Arlequin’ (http://anthropologie.unige.ch/arlequin). Haplotype frequencies were obtained using the Expectation-Maximization algorithm (44) implemented in Arlequin. Linkage disequilibrium was estimated by the method of Slatkin and Excoffier (45) implemented in Arlequin.
Northern blot analysis
RNA was isolated from frozen mouse (C57Black) tissue via guanidine thiocyanate extraction (46) with some modifications. Approximately 750 mg of tissue was crushed in a liquid-nitrogen-chilled mortar and pestle and prepared with denaturing solution (4 M guanidium thiocyanate, 25 mM sodium citrate pH 7.0, 0.5% sarcosyl and 0.1 M 2-mercaptoethanol), precipitated with isopropanol and finally dissolved in 0.1% diethyl pyrocarbonate water.
RNA was prepared for electrophoretic separation by the method described by Lehrach et al. (47). Transfer of RNA to Hybond N+ membrane was performed following the protocol recommended by the manufacturer (Amrad Pharmacia Biotech). The probe for hybridization was prepared using Gigaprime DNA labelling kit (Bresatec). Hybridizations were performed in 50% formamide, 5x SSPE (3.6 M NaCl, 0.2 M Na2HPO4, 0.02 M Na2EDTA, pH 7.7), 1x Denhardt’s solution (2% BSA, 2% Ficoll 400 and 2% polyvinylpyrrolidine) and 10 mg/ml denatured salmon sperm DNA. The membrane was washed in 2x SSC at room temperature and 0.1x SSPE at 65°C. The membrane was exposed to X-ray film (Kodak) overnight at –70°C.
Western blot analysis
Mouse and human protein samples were prepared as described by Hoffman et al. (48), with some modifications. Approximately 50 mg of tissue was cut from frozen samples maintained at –70°C and crushed in a liquid-nitrogen-cooled mortar and pestle. Samples were transferred to a microcentrifuge tube and homogenized and vortexed in sample buffer (62.5 µM Tris–PO4, 0.1% glycerol, 1 mM EDTA, 1.5 mg/ml aprotonin, 1.25 mg/ml leupeptin, 1.0 mg/ml pepstatin, 0.04 mg/ml PMSF, 2.5% SDS and 0.05 M DTT). Before electrophoresis, samples were boiled for 5 min and centrifuged. Protein concentrations of muscle lysates were estimated using the amido black protein estimation protocol (49).
Supernatants containing 50 µg of protein sample were loaded into wells of SDS–polyacrylamide gel (5% stacking gel and 7.5% separating gel) and were electrophoresed for 1 h at 200 V. Transfer onto prepared PVDF membrane (Amersham Pharmacia Biotech) was performed overnight at 40 V using Mini Protean II gel electrophoresis and transfer unit (Bio-Rad Laboratories). Membranes were blocked in 5% non-fat milk powder in TBST [0.01 M Tris–HCl, 0.05% (v/v) Tween-20 and 9% (w/v) NaCl]. Primary antibodies (see below) were applied for 2 h. The membrane was washed and alkaline phosphatase-conjugated secondary antibody was applied to the membrane for 1 h. BCIP-NBT tablets (Sigma) (one tablet in 10 ml of deionized water) were applied to the membrane for 10 min or until bands were detected.
Immunocytochemistry
Frozen P1 mouse brains were stained according to the procedure detailed by Weinberger et al. (50) with some modifications. After dewaxing, sections were blocked in 10% goat serum before adding the primary antibodies, -actinin-2 (1/500) and -actinin-3 (1/200) and the negative control, desmin (1/800). Images were taken with an Olympus microscope with digital SPOT camera and software.
Gastrocnemius (including soleus) and EDL muscle from 1- and 6-month-old mice were removed, stretched to prevent contracture, frozen in tissue-freezing medium (ProSciTech) and stored at –70°C. Quadriceps muscle from baboon and human samples were also analysed. Five micron sequential sections were mounted on gelatin-coated slides, fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) on ice for 2 min, rinsed in PBS, incubated in ethanolamine for 10 min and rinsed again before blocking with 10% fetal calf serum (FCS). Primary antibodies used were anti--actinin-2 and -3 (16,19), and fibre typing was performed using antibodies directed against fast myosin isoforms 2A (SC-71) and 2B (BF-F3) (51), and slow myosin (Chemicon). All antibodies were diluted in 10% FCS and applied to sections. In mice, those fibres that remained unstained with the above-mentioned fibre typing antibodies were classified as type 2X fibres. For baboon and human muscle, antibodies directed against fast myosin isoforms (MY32; Sigma) were also used to assess fibre typing. After rinsing with PBS, secondary antibodies conjugated to cyanine 3 were applied. Sections were mounted and viewed using the Olympus fluorescent microscope and images captured using SPOT digital imaging software.
Preparation and histochemistry of polyester-embedded mouse embryos
Embryonic tissue was prepared according to the procedure outlined by Sheppard et al. (52). Briefly, tissue was fixed in 5% (v/v) glacial acetic acid in absolute ethanol for 6 h at 4°C, followed by dehydration and embedding in polyester wax (BDH). Sections were cut at 4 µm thickness. Slides were stored at 4°C and dewaxed in 50% xylene/50% histolene and rehydrated with decreasing grades of ethanol. Sections were blocked using 10% FCS in PBS and stained according to the procedure outlined above.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr Peter Jeffrey (Children’s Medical Research Institute) for providing the mouse embryonic tissue, David Fribourg for
-actinin PCR primer design, and Lucy Rowe and Mary Barter for outstanding assistance with mouse backcross mapping. We also thank Professor Peter Gunning, Dr Sandra Cooper and Ms Biljana Ilkovski for their thoughtful reviews of the manuscript. Baboon blood and tissue was obtained from the Baboon colony facility, Wallacia, NSW. This work was supported by an RACP Glaxo Wellcome Australia Fellowship to K.N.N. and generous support from the Ramaciotti Foundation, the Australian Brain Foundation (K.N.N.) and the Joshua Frase Foundation, the Muscular Dystrophy Association of USA, and National Institutes of Health (NIAMS) grants R01 AR44345 and K02 AR02026 to A.H.B.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Clinical Sciences Building, Children’s Hospital at Westmead, Locked Bag 4001, Westmead, Sydney, NSW 2145, Australia; Tel: +61 2 9845 3011; Fax: +61 2 9845 3082; Email: kathryn@chw.edu.auThe authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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REFERENCES |
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