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Phylogenetic implications of the superfast myosin in extraocular muscles

Fred Schachat^* and Margaret M. Briggs

Department of Cell Biology, Division of Physiology, Duke University Medical School, Durham, NC 27710, USA

View larger version (16K): [in a new window]   Fig. 1. Physical map and exon organization of the fast/developmental myosin heavy chain genes. (A) The position, orientation and size of the MYH genes that compose the fast/developmental cluster. (B) The exon organization of each of the MYH genes. The position and exon organization of the fast/developmental MYH genes are based on analysis of the MYH region of human chromosome 17 using GeneQuest and the known cDNA sequences of the rat embryonic MYH gene (Strehler et al., 1986) and the rabbit MYH13 gene (Briggs and Schachat, 2000) to define exon boundaries. Only the positions of the coding exons (3-40 for the embryonic and MYH13 gene and 3-41 for the other members of the cluster) are presented. The predicted coding sequences exhibited greater than 99% identity with the cDNA sequences reported by Weiss et al. (1999b). Regions of homologous organization (exon—intron size and spacing) are indicated by distinctive colors. These structural homologies primarily reflect the order of gene duplication, although some, such as the two conserved patterns in the 5'-region of the MYH13 gene, are more probably the result of gene conversion events. This is readily evident for the segment from the beginning of exon 4 to the end of exon 5, which is identical to the corresponding sequence in the adjacent perinatal MYH gene.

View larger version (101K): [in a new window]   Fig. 2. Alignment of the fast/developmental and IIM MYH genes. To construct a sequence phylogeny, the amino acid sequences of the human embryonic, IIA, IIX, IIB, IIM, perinatal, cardiac and ß and MYH13 MYH gene sequence were aligned using Clustal X (Thompson et al., 1997). The high level and continuity of sequence conservation among striated muscle myosins made it possible to use low penalties for gap introduction and extension without introducing spurious gaps. Low values were necessary to properly align both the N- and C-terminal sequences. *, colon and stop mark positions with identical, strongly or weakly conserved amino acids, respectively. Color indicates the chemical characteristics of amino acids: light blue, hydrophobic; purple, acidic; dark blue, H and Y; green, N, Q, S and T; dark brown, R, K and C; light brown, G; yellow, P. A white background marks non-conserved residues.

View larger version (102K): [in a new window]   Fig. 2. Alignment of the fast/developmental and IIM MYH genes. To construct a sequence phylogeny, the amino acid sequences of the human embryonic, IIA, IIX, IIB, IIM, perinatal, cardiac and ß and MYH13 MYH gene sequence were aligned using Clustal X (Thompson et al., 1997). The high level and continuity of sequence conservation among striated muscle myosins made it possible to use low penalties for gap introduction and extension without introducing spurious gaps. Low values were necessary to properly align both the N- and C-terminal sequences. *, colon and stop mark positions with identical, strongly or weakly conserved amino acids, respectively. Color indicates the chemical characteristics of amino acids: light blue, hydrophobic; purple, acidic; dark blue, H and Y; green, N, Q, S and T; dark brown, R, K and C; light brown, G; yellow, P. A white background marks non-conserved residues.

View larger version (106K): [in a new window]   Fig. 2. Alignment of the fast/developmental and IIM MYH genes. To construct a sequence phylogeny, the amino acid sequences of the human embryonic, IIA, IIX, IIB, IIM, perinatal, cardiac and ß and MYH13 MYH gene sequence were aligned using Clustal X (Thompson et al., 1997). The high level and continuity of sequence conservation among striated muscle myosins made it possible to use low penalties for gap introduction and extension without introducing spurious gaps. Low values were necessary to properly align both the N- and C-terminal sequences. *, colon and stop mark positions with identical, strongly or weakly conserved amino acids, respectively. Color indicates the chemical characteristics of amino acids: light blue, hydrophobic; purple, acidic; dark blue, H and Y; green, N, Q, S and T; dark brown, R, K and C; light brown, G; yellow, P. A white background marks non-conserved residues.

View larger version (104K): [in a new window]   Fig. 2. Alignment of the fast/developmental and IIM MYH genes. To construct a sequence phylogeny, the amino acid sequences of the human embryonic, IIA, IIX, IIB, IIM, perinatal, cardiac and ß and MYH13 MYH gene sequence were aligned using Clustal X (Thompson et al., 1997). The high level and continuity of sequence conservation among striated muscle myosins made it possible to use low penalties for gap introduction and extension without introducing spurious gaps. Low values were necessary to properly align both the N- and C-terminal sequences. *, colon and stop mark positions with identical, strongly or weakly conserved amino acids, respectively. Color indicates the chemical characteristics of amino acids: light blue, hydrophobic; purple, acidic; dark blue, H and Y; green, N, Q, S and T; dark brown, R, K and C; light brown, G; yellow, P. A white background marks non-conserved residues.

View larger version (9K): [in a new window]   Fig. 3. Phylogenetic relationships among the striated MYH genes. The phylogenetic tree is based on neighbor-joining (N-J) analysis of the MYH amino acid sequences aligned in Fig. 2,Fig. 2,Fig. 2,Fig. 2. All gapped positions were omitted for the N-J analysis. Because it was the most divergent of the striated MYHs, the IIM gene was designated as the outlier. The number of times 1000 independently chosen subsequences yielded the same tree are indicated at the node points. This bootstrap analysis indicates that the tree is well supported. A partial human IIM sequence was assembled by analysis of the chromosome 7 DNA sequence using GeneQuest. Because the human IIM genomic sequence is currently incomplete, a full-length IIM sequence was generated by fusing the human sequence to amino acid residues 1-525 of the cat IIM sequence (Hoh et al., 1999). A frameshift we identified in the coding region of the human IIM gene probably explains the absence of IIM expression in human (Rowlerson et al., 1983).

View larger version (15K): [in a new window]   Fig. 4. Differences in selective pressures on the two functional domains of myosin. Sequence relatedness analysis using the entire amino acid sequence for the fast/developmental MYH genes generated different phylogenetic relationships for (A) the myosin head or motor domain, amino acid residues 1-841, and (B) the -helical coiled-coil rod or tail domain, residues 842 to approximately 1938. The motor sequences were more similar to the adult fast MYH genes, indicative of both the role of the MYH13 gene in the superfast contractions of extraocular muscle and the two regions of exon homology evident in Fig. 1. In contrast, the rod domain exhibited a closer relationship to the and ß cardiac sequences, reaffirming the inference that MYH13 diverged and specialized before the precursors of the slow/cardiac and fast/developmental clusters had significantly diverged. This is a modified version of a figure from Briggs and Schachat (2000). emb, embryonic; PN, perinatal.

View larger version (24K): [in a new window]   Fig. 5. MYH13 uses multiple transcription initiation sites and generates alternatively spliced mRNAs. The alternative splicing patterns of three full-length cDNAs of extraocular myosin are shown. Transcript A skips exon 2. Transcription of B and C begin downstream of A in exon 1, and they both include exon 2. The nucleotide sequence of the longest rabbit cDNA is aligned with the human gene sequence to show the predicted exon boundaries. Short stretches of the introns are shown in lower case, and the consensus splice sites are marked by underlining. The locations of the transcription start sites A, B and C are indicated by horizontal arrows, and the TATA boxes upstream of B and C are boxed. Only part of exon 3 is shown. From Briggs and Schachat (2000).

View larger version (6K): [in a new window]   Fig. 6. Comparison of the density of myogenic factor binding sites in the proximal promoter regions of the IIB and MYH13 genes. Phylogenetic footprinting shows that the extraocular (EO) MYH proximal promoter lacks most of the functionally important binding sites for myogenic factors identified in the IIB gene.

View larger version (5K): [in a new window]   Fig. 7. The position of potential MYH13 regulatory regions based on phylogenetic footprinting. Phylogenetic footprinting identifies several highly conserved upstream and intronic regions between the human and mouse extraocular (EO) MYH genes. Conserved regions are depicted by open boxes. The positions of exons 1, 2 and 3 are indicated as filled boxes. The mouse genomic sequence spanning the myh13 gene (AC019008) was identified using Blast, and the exon boundaries were determined using GeneQuest.

View larger version (5K): [in a new window]   Fig. 8. A model for the MYH13 gene chromosomal domain. Conserved sites identified by phylogenetic footprinting (see Fig. 7) and two bounding CpG island domains revealed by GeneQuest are shown on a map of the human chromosomal region from the end of the upstream perinatal gene to a region downstream of the MYH13 gene. Here, the sites are tentatively assigned functions as the proximal promoter, upstream regulatory elements (UREs) and a locus control region (LCR) by analogy with the model for gene organization presented by Blackwood and Kadonaga (1998). This region is drawn to scale.

View larger version (19K): [in a new window]   Fig. 9. Modeling the natural history of the muscle fast/developmental gene cluster. This model of gene duplication integrates the genomic organization (Fig. 1) and phylogeny of the fast/developmental MYH cluster.

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