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Keywords:

  • MHC-alpha;
  • aMHC;
  • vMHC;
  • MyH15;
  • synteny

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Myosin heavy chains (MHC) are cytoskeletal motor proteins essential to the process of muscle contraction. We have determined the complete sequences of the Xenopus cardiac MHC genes, α-MHC and ventricular MHC (vMHC), and have characterized their developmental expression profiles. Whereas α-MHC is expressed from the earliest stages of cardiac differentiation, vMHC transcripts are not detected until the heart has undergone chamber formation. Early expression of vMHC appears to mark the cardiac conduction system, but expression expands to include the ventricle and outflow tract myocardium during subsequent development. Sequence comparisons, transgenic expression analysis, and comparative genomic studies indicate that Xenopus α-MHC is the true orthologue of the mammalian α-MHC gene. On the other hand, we show that the Xenopus vMHC gene is most closely related to chicken ventricular MHC (vMHC1) not the mammalian β-MHC. Comparative genomic analysis has allowed the detection of a mammalian MHC gene (MyH15) that appears to be the orthologue of vMHC, but evidence suggests that this gene is no longer active. Developmental Dynamics 233:1287–1293, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In skeletal and cardiac muscle, the cellular machinery used to generate contraction is assembled into sarcomeric complexes containing actin thin filaments and myosin thick filaments. The thick filaments are composed of polymers of large motor proteins called myosin heavy chains (MHC) and smaller associated myosin light chains (MLC). The force of contraction is generated from the ATP-dependent movement of myosin motor proteins along the actin thin filaments (reviewed by Gregorio and Antin, 2000). Cardiac MHC genes have been studied extensively in mammals, and the structure, genomic organization, developmental expression, and transcriptional regulation of these genes has been well documented (reviewed by Morkin, 2000). Although a variety of different myosin motor proteins are found in diverse cell types, the thick filaments of cardiac muscle are composed entirely of conventional MHC proteins known as cardiac α-MHC and β-MHC (Mahdavi et al., 1982; Gordon et al., 2000).

During mammalian development, both cardiac α-MHC and β-genes are expressed in the heart from the time of initial differentiation of the myocardium (Morkin, 2000). In mouse for example, the cardiac (α-/β-) MHC genes are both expressed in the newly formed cardiac tube between 7.5 and 8 day post coitum. As the heart becomes chambered, β-MHC begins to be restricted to the ventricle, whereas α-MHC continues to be expressed throughout myocardial tissues of both chambers (Lompre et al., 1984; reviewed in Morkin, 2000). Although many aspects of gene regulation during heart development are evolutionarily conserved, expression of MHC genes may vary quite significantly between higher and lower vertebrates. For example, during chicken development, at least three myosin heavy chain genes, aMHC, vMHC1, and slow tonic MHC/ ssMHC-2, are expressed (Gonzalez-Sanchez and Bader, 1985; Bisaha and Bader, 1991; Yutzey et al., 1994; Machida et al., 2002). Whereas aMHC appears to be related to mammalian α-MHC, the relationship of vMHC and slow tonic MHC to mammalian MHCs is much less clear (Moore et al., 1993). During zebrafish development, two cardiac myosin heavy chains, aMHC and vMHC, are expressed from the onset of heart development (Yelon et al., 1999; Berdougo et al., 2003). Once again, it is not clear whether vMHC is related to mammalian β-MHC or whether it represents and independent gene.

In the frog, only a single cardiac myosin heavy chain gene (called α-MHC) has been characterized to date (Logan and Mohun, 1993). This gene is expressed at high levels in the developing heart from the onset of cardiac differentiation. As will be explained below, however, the precise relationship of this gene to the mammalian α- and β-MHC genes is quite unclear. To further investigate the relationship of lower vertebrate myosin heavy chain genes and those in mammals, we have determined the complete sequence of the frog α-MHC gene and have identified and characterized a second cardiac MHC gene, vMHC. We have determined the developmental expression profiles of these genes and used comparative genomic methods to examine their evolutionary relationship to mammalian MHC genes. We find that the α-MHC is closely related to chick aMHC and mammalian α-MHC. Frog vMHC on the other hand does not appear to be the orthologue of mammalian β-MHC but is most closely related to chicken vMHC and an inactive mammalian pseudogene, MyH15.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Examination of expressed sequence tags (ESTs) from the adult Xenopus heart showed high representation of α-MHC (Logan and Mohun, 1993) and a second MHC gene that we have named vMHC (see below). These two genes were represented by 74 and 126 hits, respectively, within heart ESTs, and no other MHC sequences were detected. We have determined the complete sequences of Xenopus α-MHC and vMHC. This accomplishment was achieved by assembly of overlapping EST entries from GenBank and by a combination of polymerase chain reaction (PCR) amplification and sequencing of any missing regions. The α-MHC gene encodes a peptide 1,928 amino acids long (accession no. AY913767). Pairwise comparisons of amino acid sequences showed that Xenopus α-MHC is 88% identical to human and mouse α-MHC. However, Xenopus α-MHC is also 87% identical to human and mouse β-MHC; therefore, it is not clear which mammalian cardiac MHC represents the orthologue of Xenopus α-MHC. To more critically examine the relationship of Xenopus α-MHC to the mammalian genes, we have focused on conserved regions of the proteins that are considered diagnostic for the α- and β-isoforms. Alignments of amino acid sequence from the myosin tail region of human and mouse cardiac α-MHC/β-MHC proteins (aa1072-1101) with Xenopus α-MHC shows that Xenopus α-MHC has amino acid sequences intermediate between α- and β-MHC. Comparisons to chick sequences showed that the chicken atrial MHC (aMHC) gene (accession no. NM_001001302) is most closely related to Xenopus α-MHC. We note that chick aMHC also has amino acid usage intermediate between mammalian α-MHC and β-MHC proteins (Fig. 1A).

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Figure 1. Alignment of selected protein domains from Xenopus, human, mouse, and chick cardiac myosin heavy chains (MHC) proteins. B: Region 86–115. A: Alignment of Xenopus α-MHC, human and mouse α-MHC and β-MHC chick atrial MHC in the region corresponding to residues 1072–1101 of the mouse protein. Xenopus α-MHC and chick aMHC display amino acid residues intermediate between mammalian α-MHC and β-MHC sequences (boxed regions). B: Alignment of Xenopus vMHC with chick vMHC and slow tonic MHC (ssMHC-2) proteins and with Xenopus α-MHC, human α-MHC and β-MHC, and chick atrial MHC. The region corresponds to residues 86–115 of the mammalian protein sequence. The alignment reveals amino acid residues that are conserved in ventricular vMHC genes from different species (boxed regions).

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The second Xenopus cardiac MHC gene (vMHC) encodes a protein of 1,938 amino acids (accession no. AY913766) that is closely related to the chicken ventricular cardiac MHC (vMHC1) and chicken slow tonic MHC (ssMHC-2; accession no. NM_204766 and AB057661, respectively). Alignment of the amino acid sequences showed that the Xenopus vMHC is 87% identical to chick vMHC1 and 80% identical to chick ssMHC-2 but is only 76% identical to either the human- or mouse-α and β-MHC proteins, which are the closest mammalian matches. Alignment of amino acid sequence from the N-terminal region of the Xenopus vMHC (amino acids 86-115) with a diagnostic region of the human α- and β-MHC sequences did not reveal of preferential relationship of Xenopus vMHC to either mammalian α- or β-MHC (Fig. 1B). The relationship of Xenopus vMHC to mammalian MHC genes will be described in more detail below.

We have carried out in situ hybridization analysis to determine the expression patterns of the Xenopus cardiac α-MHC and vMHC genes. We found that α-MHC was expressed strongly and specifically in the paired myocardial primordia, commencing at stage 26 (Fig. 2A–C). α-MHC expression continued in the myocardial precursors at stage 30 (Fig. 2D). We note that, unlike α-MHC, vMHC expression was not detected in the embryo at stage 30 (Fig. 2E). Commencing at approximately stage 35, α-MHC expression was detected in the lymph heart and jaw muscles in addition to the heart (Fig. 2F–H). Examination of the chambered and septated heart (stage 45) showed that α-MHC transcripts are present throughout the myocardium of the outflow tract, ventricle, and atria (Fig. 2I,J). The expression pattern of vMHC is distinctly different from that of α-MHC. Transcripts for vMHC were not detected until approximately stage 43 (data not shown), well after expression of most other myocardial marker sequences. At stage 45, by which time the atrial and ventricular chambers of the heart are highly developed, vMHC expression was detected in limited regions of myocardial tissue that ring the border between the ventricle and outflow tract and the ventricle and atria (Fig. 2K,L). In the adult heart, α-MHC was detected throughout the myocardium (Fig. 2M), whereas vMHC was detected in the outflow tract and ventricle but absent in the atria (Fig. 2N). These studies indicate that developmental expression of vMHC expression is quite different from that of either α- or β-MHC in mammals, both of which are expressed throughout myocardial tissues from the onset of differentiation. Adult expression of Xenopus vMHC, however, more closely resembles the pattern of mammalian β-MHC.

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Figure 2. In situ hybridization analysis of developmental expression of the Xenopus α-MHC and vMHC genes. A: Lateral view of stage 26 embryos showing α-MHC transcripts in the bisymmetric heart patches. B: Ventral view of embryo in A. C: Transverse section through the heart patch of stage 26 embryo. D: Lateral view of stage 30 embryo showing increased α-MHC expression in precardiac tissues (arrow). E: Lateral view of stage 30 embryo showing an absence of vMHC expression at this stage of development. F: Lateral view of stage 38 embryo showing α-MHC expression in the heart, muscles of the jaw (jm), and the lymph heart (lh). G: Transverse section through stage 36 heart shows that α-MHC expression is confined to the myocardium. H: Transverse section through the lymph heart (stage 38) stained for α-MHC. I,J: Whole-mount and section in situ hybridizations, respectively, of stage 45 heart showing that α-MHC is expressed in all chambers and outflow tract. K,L: Whole-mount and section in situ hybridizations, respectively, of stage 45 heart, showing restricted expression of vMHC in a ring around the outflow tract and the atria at the junction with the ventricle. M: Section in situ of adult Xenopus heart shows that α-MHC is expressed throughout the myocardium, including the atria (a) and outflow tract. N: Section in situ hybridization of adult Xenopus heart shows that vMHC is expressed in the myocardium of the outflow tract and ventricle but not the atria.

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Because the sequence and expression studies have not provided conclusive evidence showing whether Xenopus α-MHC is more closely related to mammalian α- or β-MHC, we have extended our investigation to the transcription regulatory regions of the genes. Comparison of the regulatory regions of mouse and human α-MHC revealed a high degree of sequence conservation. Similarly, mammalian β-MHC genes also show conservation of regulatory sequences. However, there is little or no conservation of regulatory elements between the α-MHC and β-MHC genes. We have isolated a ∼6-kb genomic fragment of the Xenopus α-MHC gene that is capable of recapitulating the endogenous pattern of α-MHC expression in the heart, lymph heart, and jaw muscles using a green fluorescent protein (GFP) reporter (Fig. 3A,D, data not shown). This construction contains ∼4 kb of the upstream promoter region plus the first intron of Xenopus α-MHC. Transgenic expression of GFP is first observed weakly in the early heart of stage 28 embryos (data not shown). Strong expression of GFP was observed in the heart of stage 37 embryos (Fig. 3A), similar to the endogenous pattern of expression of the α-MHC gene at stage 37 detected by whole-mount in situ hybridization (Fig. 3B). At stage 45, transgenic expression of the Xenopus α-MHC promoter was observed in the heart, the muscles of the jaw, and the lymph hearts (Fig. 3C,D and data not shown) identical to the pattern of the endogenous gene (Fig. 2F).

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Figure 3. Transgenic analysis of Xenopus α-MHC promoter activity. A 6-kb fragment containing the promoter and the first intron was sufficient to drive cardiac specific expression. A: Green fluorescent protein (GFP) fluorescence is detected in the heart (arrows) of a stage 37 transgenic embryo. Expression of GFP in the eye is an indicator of transgenesis from integration of γ-crystallin–GFP reporter. B: Whole-mount in situ hybridization of a stage 37 embryo shows the endogenous localization of α-MHC transcripts in the heart. C: Isolated heart from stage 45 embryo showing α-MHC transgene expression in all chambers of the heart. oft, outflow tract; a, atria, v, ventricle. D: In addition to the heart (ht), α-MHC transgene expression is also observed in the jaw muscles (jm). E: A transgene using a truncated α-MHC promoter, (855 bp) reiterates expression of the longer promoter and the endogenous gene. F: Comparison of the regulatory regions of Xenopus α-MHC with mouse and human α-MHC and β-MHC promoter regions. Note the conservation of regulatory elements between frog and mammalian α-MHC promoters but the lack of conservation with β-MHC promoter. MEF-2 (myocyte-specific enhancer-binding factor 2 site), M-CAT (CATTCCT motif), CArG (CArG box or serum response element), and TRE (thyroid hormone regulatory element), GATA (GATA factor binding element).

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Comparison of the conserved consensus binding sites in the regulatory regions of the mammalian α- and β-MHC genes and the Xenopus α-MHC promoter revealed that the frog promoter strongly resembled the mammalian α-MHC promoter (Fig. 3F). The conservation of the upstream regulatory region of α-MHC between Xenopus, mouse, and human was conspicuous as the promoters share similar spacing and sequence for the consensus binding sites of the cardiogenic transcription factors SRF (CArG), MEF-2, GATA, and Tef-1 (M-CAT; Fig. 3F). No significant conservation of upstream regulatory regions between Xenopus α-MHC and the mammalian β-MHC genes was observed. Because the conservation of cardiac regulatory elements between Xenopus, mouse, and human α-MHC genes are within an 855-bp region of the Xenopus promoter, we have tested whether this region alone is sufficient to drive correct developmental expression. Transgenic studies showed that the 855-bp promoter was sufficient to drive GFP expression in the heart, lymph heart, and facial muscles (Fig. 3E, data not shown), in a pattern identical to that of the longer construction (Fig. 3C,D). GFP expression, however, was significantly weaker with the 855-bp promoter, indicating that enhancer elements are probably missing from the truncated construction.

Comparative Genomic Analysis of Xenopus α-MHC and vMHC Sequences

Overall, our promoter studies provide strong evidence that Xenopus α-MHC is the orthologue of the mammalian α-MHC gene, but the relationship of vMHC to mammalian β-MHC remains uncertain. In the mouse and human genomes, the α- and β-MHC genes are located in a tandem arrangement, indicating that they arose through a duplication event (Mahdavi et al., 1984; Yamauchi-Takihara et al., 1989; Gulick et al., 1991). Using newly available Xenopus tropicalis genome sequences, we have examined the gene organization of the frog α-MHC region and a comparison of the syntenic regions of the frog, human, and mouse chromosomes is shown in Figure 4A. Unfortunately, the corresponding region of the chicken genome has not been assembled and so cannot be included in the comparison. The zebrafish genome contains a gene closely related to α-MHC, but the synteny of this region of the chromosome is not conserved. Our synteny studies show that the organization of genes surrounding the α-MHC locus is identical in the different species, indicating that evolutionarily related sequences are indeed being examined. Furthermore, we observe that no gene duplication event has occurred in the frog genome and that only a single gene, α-MHC, exists at this location. We conclude from this information that an orthologue of mammalian β-MHC gene is unlikely to exist in Xenopus and that vMHC is probably related to a different MHC gene.

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Figure 4. Synteny of cardiac myosin heavy chain (MHC) genes in evolutionarily distant genomes. A: The genomic organization of the frog (X. tropicalis) cardiac α-MHC gene is compared with the corresponding regions in the human and mouse genome. Genes located on chromosome 14 of mouse and human correspond to embryonal Fyn-associated substrate (Efs), chemokine-like factor superfamily 5 (CKLFSF5), α-MHC, β-MHC, putative peptide 1500001L15, and thiamine triphosphatase (THTPA). The order of X. tropicalis genes was determined from incompletely annotated genomic sequence (scaffold 60). The sequence of the chicken genome in this region is not available. B: The order of genes flanking vMHC in the X. tropicalis genome compared with the corresponding region of the chicken, human, and mouse genome. Genes located on chromosome 1 of chicken, chromosome 3 of human, and chromosome 16 of mouse correspond to huntington-interacting protein-1 (HIP-1), vMHC (also called vMHC1/MyH15), putative protein KIAA1524, and T-cell receptor interacting molecule (TRIM). The order of X. tropicalis genes was determined from incompletely annotated genomic sequence (scaffold 53). C: Attempted polymerase chain reaction detection of MyH15 transcripts in the mouse adult heart. A fragment of the MyH15 gene was amplified from genomic DNA but not from cDNA synthesized from adult heart RNA. Mouse α-MHC expression was used as a positive control for heart cDNA.

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Because conserved vMHC sequences are present in the Xenopus and chicken genomes and both are expressed during heart development, it seems likely that this gene is evolutionarily ancient and predates the divergence of the mammalian lineage. Therefore, we asked the question—Does the mammalian genome contain an orthologue of vMHC? Using genomic comparisons of the genes flanking vMHC in Xenopus and chicken, we located the syntenic regions of the mouse and human genomes (Fig. 4B). To our surprise, we identified mouse and human myosin genes (annotated within the human genome as MyH15) that appear to be the orthologues of the vMHC genes found in Xenopus and chicken. To test whether MyH15 may be expressed in mammals, we carried out a reverse transcriptase-PCR (RT-PCR) experiment using RNA isolated from the adult mouse heart (Fig. 4C). This analysis showed that the mouse MyH15 gene is not expressed in the adult heart (Fig. 4C). In addition, searches of human and mouse EST databases failed to detect any entries corresponding to MyH15 transcripts. Analysis of mouse genome sequences indicates that the putative mouse MyH15 and Xenopus vMHC proteins would be 65% identical at the protein level. This rate is significantly less than the degree of sequence conservation that is observed between the Xenopus and chicken vMHC proteins. Because the MyH15 gene appears to be inactive, it is possible that these sequence differences have arisen due to nonselected mutations in the pseudogene.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Our analysis suggests that only two cardiac MHC genes are expressed in the developing and adult heart of Xenopus. It is interesting that these two MHC genes do not appear to be the direct equivalents of the cardiac α-MHC and β-MHC genes expressed in mammals. Xenopus expresses a single gene that is highly related to mammalian α-MHC. Because the primary sequence of Xenopus α-MHC is apparently intermediate between the protein sequences of mammalian α- and β-MHC, this assignment is primarily based on similarities between the promoters of the Xenopus and mammalian α-MHC genes. The promoter regions of the Xenopus and mammalian α-MHC genes show conservation in the presence and spacing of several consensus binding sites of transcription factors. A 612-bp fragment of the upstream regulatory region plus a portion of the first intron is sufficient to drive cardiac specific expression in mice (Gupta et al., 1998), and the equivalent region of the Xenopus promoter can recapitulate the endogenous α-MHC pattern of expression (Fig. 3E). One notable difference between Xenopus and mammalian α-MHC promoters is the absence of thyroid hormone regulatory elements (TRE) in the frog promoter. In mammals, TRE element(s) are present in the regulatory region of the α-MHC gene and thyroid hormone modulates expression of α-MHC in the ventricular chambers of the heart (Lompre et al., 1984). The absence of TREs in the frog α-MHC promoter suggests that ventricular expression of α-MHC in the adult heart is independent of thyroid hormone regulation.

Our studies strongly suggest that the Xenopus genome does not contain a gene orthologous to mammalian β-MHC. Instead, Xenopus expresses a vMHC protein that is highly related (87% identical) to chicken vMHC1. The vMHC gene of Xenopus appears to function approximately equivalently to the mammalian β-MHC gene, because expression of both genes is largely restricted to the ventricle in the adult heart. Similarly, chicken vMHC is preferentially expressed in the ventricle (Bisaha and Bader, 1991; Machida et al., 2000). However, one major difference between Xenopus and chicken cardiac MHC expression is the observation that vMHC in chicken is expressed from the onset of cardiac differentiation (Yutzey et al., 1994), whereas vMHC expression in Xenopus is not observed until several days after cardiac differentiation first occurs.

Initial expression of Xenopus vMHC appears to mark the conduction system of the tadpole heart. The cardiac conduction system consists of differentially coupled myocytes that are usually histologically indistinguishable from the adjacent myocardium (reviewed by Pennisi et al., 2002). In Xenopus, electrical signals are conducted through the heart in a pattern consistent with the existence of a conduction system (Sedmera et al., 2003), and we observe vMHC in the myocardium that rings the openings to the ventricle at the outflow tract and at the atria. Unfortunately, there is no other characterized marker for conduction system tissue in Xenopus; therefore, it is not possible to confirm this assignment using an independent marker. However, the early pattern of frog vMHC expression at the atrioventricular junction closely resembles the location of developing conduction system in chicken as defined using a range of molecular markers (Sanders et al., 1986; Evans et al., 1988; Machida et al., 2000, 2002).

The increasing availability of whole genome sequences provides an extremely valuable resource for identification of orthologous genes between distant species. Rather than relying solely upon conservation of primary sequence, the localization of related genes to syntenic regions of chromosomes virtually ensures correct assignment of evolutionary relationships. We have used sequence information provided by the Xenopus genome project (http://genome.jgi-psf.org/Xentr3/Xentr3.home.html) plus information from chicken and mammalian genome databases (www.ensembl.org) to precisely identify the myosin heavy chains expressed during frog heart development. It has been determined previously that the mammalian α- and β-MHC genes arose through a tandem duplication event (Mahdavi et al., 1984; Yamauchi-Takihara et al., 1989). Our survey of Xenopus genome sequences indicates that this duplication event did not occur in frogs and that only a single MHC gene is located at this position in the frog genome (Fig. 4A). Further genome analysis allowed us to identify a previously uncharacterized coding region, MyH15, as the mammalian orthologue for the Xenopus and chicken vMHC genes (Fig. 4B). It seems very likely that the MyH15 gene is no longer active. Inspection of EST databases failed to show any entries for MyH15, and RT-PCR analysis of adult mouse heart RNA (Fig. 4C) did not reveal any detectable expression. At present, however, we cannot exclude the possibility that MyH15 might be expressed at low levels during mammalian heart development or in noncardiac tissues. It is possible that the duplication event that created the tandem α- and β-MHC genes in mammals may have facilitated the loss of activity of the MyH15 gene. We can speculate that, soon after the α-MHC duplication, early mammals may have expressed α-MHC, β-MHC, and vMHC (MyH15) in the heart. During subsequent mammalian evolution, the MyH15 gene became inactive, whereas the promoter of the β-MHC gene diverged to allow for preferential expression in the ventricular myocardium of the adult heart.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Whole-Mount In Situ Hybridization

Whole-mount in situ hybridization was carried out with antisense digoxigenin-labeled probes to α-MHC and vMHC, using a modification of the protocol by Harland, (1991). Plasmids were linearized with NotI and transcribed with T7 RNA polymerase using the MEGAscript kit (Ambion). For serial sections, embryos or whole hearts were post-fixed in 4% paraformaldehyde, embedded in Paraplast, and 10-μm transverse sections were prepared.

Isolation of the Xenopus α-MHC Promoter

A fragment corresponding to the 5′ end of Xenopus α-MHC mRNA was used to probe a Xenopus lambda genomic library (from Mike King). Approximately 106 phage were screened at high stringency (0.2 × standard saline citrate/0.1% sodium dodecyl sulfate at 65°). Positive phage were purified and phage DNA was isolated from liquid culture by cesium chloride gradient centrifugation, restriction digested with EcoRI, and subcloned for analysis. Restriction mapping, subcloning, and Southern blot analysis isolated an 6-kb fragment that contains 4 kb of 5′ flanking sequence and the complete first intron of the Xenopus α-MHC gene.

Generation of Transgenic Embryos

For Xenopus transgenic constructions, a 6-kb fragment containing approximately 3.8 kb of the Xenopus α-MHC promoter and all first intron sequences was amplified using Pfu polymerase, a T3 primer, and a 3′ primer containing an AgeI linker: 5′-TTACACCGGTCAGCAAAAGGACTAGAGAC-3′. PCR product was subcloned into the EcoRI/AgeI sites of a modified pEGFP-1 vector (Clontech). The 855-bp truncated α-MHC promoter construction was produced by subcloning a XhoI/PstI fragment of the transgenic construct into the XhoI/PstI sites of the modified pEGFP-1 vector. α-MHC full-length promoter plus GFP reporter sequences was excised from the plasmid vector by digestion with EcoRI and PmeI, and the α-MHC minimal promoter–GFP construct was excised with XhoI and PmeI. Transgenes were purified using the QIAEX II gel extraction kit (Qiagen) and used to generate transgenic embryos as previously described (Kroll and Amaya, 1996; Sparrow et al., 2000). Expression of GFP in the eye, driven by the γ-crystallin promoter, was used as a marker of transgenesis. GFP fluorescence was detected in live embryos using a Leica FL-III microscope and MagnaFire digital camera.

PCR Detection of Mouse MyH15 and α-MHC in the Adult Heart

Mouse α-MHC primers 5′-CCGCAATGATGGCTGAGGAGC-3′forward and 5′-CCGCTCGCTCTTCCTCATGCC-3′reverse, and mouse MyH15 primers 5′-AACATGTCAGAAGAGCTGAAG-3′forward, and 5′-CAAGCAAATGCCTACCTGGAT-3′reverse, were used to amplify cDNA prepared from adult mouse heart or from mouse genomic DNA.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

P.A.K. is the Allan C. Hudson and Helen Lovaas Endowed Professor of the Sarver Heart Center at the University of Arizona College of Medicine. P.A.K. was funded by the Sarver Heart Center and by the NHLBI of the NIH, and a subcontract from the University of Iowa, SCOR grant HL62178 (to Ronald Lauer).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES