Comparative Genomics: Myosin Heavy Chains - Essay Example

COMPARATIVE GENOMICS al Affiliation) Key words: Genomics, Genes Myosin Heavy Chains de d as MyHCs are majorly committed ubiquitous actin motor proteins that normally drive various motile processes found in eukaryotic cells. Encoding of Myosin Heavy Chains isoforms found in skeletal muscles is done by families that are multi-gene that are clustered on human, rabbit, and rat chromosomes found in syntonic regions. To understand the genomic arrangement of skeletal Myosin Heavy Chains genes and its impact on the function, molecular genetics, and regulation of the multi-gene family, the paper has developed a high resolution map for human, rabbit, and rat loci with the help of a PCR market content mapping of artificial chromosome clones. The encoding of genes that isoforms Myosin Heavy Chains have been developed according to their transcriptional orientations and linear order within the set kb of 350 in human, rat, and rabbit. From the maps it is noted that transcriptional orientation, relative intragenic distances, and order of the genes are conserved between the species. Unlike other gene families that are clustered, the order does not portray the temporal patterns of those genes. Conversely, the gene organization, conservation from the divergence of the genes shows that the organization of the genes can be important for their function and regulation. Introduction Myosin Heavy Chains converts chemical energy from the hydrolysis of ATP to form a mechanical force that moves the motile processes like cytokines, cellular locomotion, and vesicular transport in eukaryotic cells. Myosin Heavy Chains are subdivided into class comprising of 9 to 11 classes. The conventional Myosin Heavy Chains include the sarcomere Myosin Heavy Chains that associates itself to form a function enzymatically and filaments in promoting contraction in striated muscles. Muscle myosin consists of double Myosin Heavy Chains and two associated dissimilar myosin light chains pairs. The seven Myosin Heavy Chains isoforms that dominate the mammalian skeletal muscles are 2 developmental isoforms, Myosin Heavy Chains embryonic and Myosin Heavy Chains perinatal; 3 adult skeletal muscles, Myosin Heavy Chains-Ha, Myosin Heavy Chains-IIb, Myosin Heavy Chains-IIx/d, and Myosin Heavy Chains-ß/slow which is outlined in the cardiac muscles. The Myosin Heavy Chains isoforms are regulated differently in response to diverting stimuli that include mechanical, physiological, hormonal, and other signals. The activity of ATPase conferred by the Myosin Heavy Chains in a muscle correlated the contraction speed, thus Myosin Heavy Chains are the main determinants of muscles functional properties where they are expressed. Recently, the adult fast IIb and IIa inactivation has shown that the gene is distinct in terms of their functionality and needed for conventional muscle function (Wolf, 2000). The sequence comparison of rat, rabbit, and human Myosin Heavy Chains CDNA series shows that the Myosin Heavy Chains are conserved highly and the orthologous isoforms that are encoded by genes are not complete and the importance of the grouped nature of sarcomere Myosin Heavy Chains is yet to be determined. In comparative genomics for the human, rat, and rabbit 6 skeletal Myosin Heavy Chains genes are used. The genes include the Myosin Heavy Chains-embryonic, Myosin Heavy Chains-Ha, Myosin Heavy Chains-perinatal or exoc Myosin Heavy Chains-IIb, Myosin Heavy Chains-IIx/d, Myosin Heavy Chains-IIb, and Myosin Heavy Chains-extra ocular relates to Myosin Heavy as8, Myosin Heavy Ha, Myosin Heavy? For the hybridization screening of PACs, high density filters that contain human, rabbit, and rat segments underwent screening by a process of hybridization with Myosin Heavy Chains specific probes. The filters of human were screened using Myosin Heavy Chains consensus probe of LL1000 producing a 28 positive. Similar probe was hybridized on a slot blot of 28 clones. The filters of rabbit and rat were probed using 8 Myosin Heavy Chains gene PCR products which enabled identifying 30 Myosin Heavy Chains positive rat and rabbit PAC clones. PACs for human, rat, and rabbit identified through the process of hybridization underwent screening by PCR with the primers of STS. The human gene primers for the STSs were developed and designed from their Myosin Heavy Chains sequences. The STSs used include the 5 Myosin Heavy Chains- Embryonic and 3 X13988, Myosin Heavy Chains-perinatal, Myosin Heavy Chains-IIa, and Myosin Heavy Chains-IIx/d. The human Myosin Heavy Chains-IIa primers, and Myosin Heavy Chains-IIb, and Myosin Heavy Chains-extra ocular were developed from the genomic sequence and CDNA that correspond to the genes. The rat and rabbit Myosin Heavy Chains gene specific were designed from the sequences that are published, including Myosin Heavy Chains-emb, Myosin Heavy Chains-pn, Myosin Heavy Chains-IIa, Myosin Heavy Chains-IIx/d, Myosin Heavy Chains-eo, Myosin Heavy Chains-IIa and Myosin Heavy Chains-IIx/d. The primers for rat and rabbit STS Myosin Heavy Chains-IIb were developed from the promoter sequence of IIb (Tanigawa, 2001). PCR was done in twenty to fifty µl using the 100 ng every primer, dNTPs, MgCl2, and buffer II prepared from the alkaline lysis. The PAC End Rescue sequences were used in generating additional STSs for rat, rabbit, and human contigs. Additionally, the sequences of PAC end were used in establishing that cluster of human PAC contains similar market content; right end group and left end groups comprising of unique clones of PAC. The STSs end rescue is named for the end of PAD from their derivatives. The litigation mediated bubble/vectoreter PCR was carried out on all rabbits, rat, and human PAC. The vectorette comprised of the primers; PCR vectorette was done employing the primer internal to vectorette, Sp6 from the PAC vector. The PCR products that contain PAC end underwent purification using the purification kit. The PAC sizes were determined from vectors through digestion with resolved and Noti through pulse electrophoresis to find out the sizes. The insert sizes of human were confirmed through the process of hybridization employing random primed DNA of total human. In intragenic distances determination PCR used primers Myosin Heavy Chains-IIa, Untranslated Region sense primers, and antisense primers were employed with templates of PAC in determining the intergenic distances between Myosin Heavy Chains-IIx/d and Myosin Heavy Chains-IIb of human. The rat and rabbit PCR primers Myosin Heavy Chains-IIa-F, Myosin Heavy Chains-IIx/d and Myosin Heavy Chains-IIx/d-F and Myosin Heavy Chains-IIb were used with the templates of PCR in determining the intergenic distance between the rabbit and rat Myosin Heavy Chains-IIa and Myosin Heavy Chains-IIa and Myosin Heavy Chains-IIx/d and Myosin Heavy Chains-IIx/d and Myosin Heavy Chains-IIb respectively. In hybrid mapping of radiation, screening was done in duplication by PCR with STS Myosin Heavy Chains-embryos of humans and the score were electronically submitted for evaluation (Royner, 2006). Comparing the order, number, and transcriptional orientation for the Myosin Heavy Chains multi-gene clusters in rabbit, rat, and human, the market mapping was used in assembling the PAC contigs for rat, rabbit, and human loci. The clones of PAC, which are difficult to combine have very small insert sizes, and generally offer more mapping information that the clones of YAC were employed in ensuring resolution of the closely connected linkages. Human The conservation of orientation, order, and general spacing of rat, rabbit, and human skeletal Myosin Heavy Chain genes supports the belief that their loci are formed from duplication of genes that prior to the divergence of the species a long time ago. It is suggested that the order of genes and the spacing on the locus may be crucial in the functioning of the gene family. Other common illustrations of the clustered families include the homeobox genes and globin where the linear order can be connected to the expression patterns regulation. Additionally, the T-Cell receptor organizations which are organized on clusters that are conserved in rabbit, rat, and human are crucial in regulating the recombination essential and splicing for the maturation of T-cell. The encoding of genes of single family members of various multi-gene family protein and contractile proteins like the actin, myosin light chains, and troponin do not undergo clustering. In this connection, the arrangement of the conserved linked skeletal Myosin Heavy Chain cluster is kind of intriguing. It is attractive in hypothesizing that the Myosin Heavy Chain arrangement of genes has undergone maintenance for so long since it is important for the regulation, function, and molecular evolution of the expanded gene family (“Myosin heavy chain composition and insulin stimulated glucose uptake in skeletal muscle: effects of growth hormone”, 2008). The spatial organization of Myosin Heavy Chain genes does not portray the temporal expression patterns compared to the homeobox genes and globin. Basically, the Myosin Heavy Chain-embryonic and Myosin Heavy Chain-perinatal are divided by three genes. This arrangement portrays the conservation of sequence among the sequences that are human coding. The Myosin Heavy Chain-embryonic and Myosin Heavy Chain-extra ocular genes are divergences in the series. Conversely, the 3 isoforms that are closely connected are also the same to each other. The three linked genes are the same, despite their difference in regulation; they have expressions that are overlapping in mammalian skeletal muscles. Therefore, the gene clustering for the three mammals may portray same functional roles, than the timing if developmental expression. The greater the divergence of Myosin Heavy Chain-embryonic and Myosin Heavy Chain-extra ocular the more they are peripherally located and higher patterns that are specialized. Myosin Heavy Chain-embryonic expressions are confined temporarily to early stages of embryonic process and the Myosin Heavy Chain-extraocular expression id confined to a special collection of muscles that work in eye movement. Since the comparing the available skeletal Myosin Heavy Chain thus portray that there is an apparent maintenance of series conservation within the isoforms of Myosin Heavy Chain orthologous. The paper therefore anticipates the relationship sequence’ among the coding of rabbit and rat to be the same to those that are described in human (Korfage, 2004). Although various transacting factors that involve transcription of muscle specific and some binding tools have been generated, the strategies that dictate the patterns of complex expression of individual skeletal Myosin Heavy Chain genes, which are controlled independently, are yet to be identified. The most celebrated progress has happened in the characterization and identification of the muscle specific and basal regulatory elements in the promoters of rabbit and rat Myosin Heavy Chain-IIb gene. The observation that the homozygous of rats and rabbits are null for Myosin Heavy Chain-IIX/d or Myosin Heavy Chain-IIb shows the compensation by the genes of Myosin Heavy Chain-IIa and Myosin Heavy Chain-IIx/d shows that they are independent. Further research shows that the animals share elements if muscle-specific regulations and has elements that promoted the expression patterns of gene specific. Additionally, their linear order does not leave their regulation by the locus control region. The locus control region offers a chromatin structure that ensures states that are permissive to transcribe, with the likelihood that the expression patterns are determined by the promoter regions. The conservation levels among the three genes shows strongly that the events of gene conversion have played a crucial role in the evolution of molecules of the genes. The Myosin Heavy Chain is the main components of muscles of contractile apparatus and their biochemical features are crucial determinants of muscle function and physiology. The complex and diverse expression arrangements may reflect the features of regulation that are differentiated from contractile protein genes. The outcomes that the physical patterns of the genes are extremely conserved, shows that the comparative researches of the loci will improve our understanding of the molecular genetics and regulation of multi-gene families. The genes’ proximity would make it highly possible that the intergenic regions have important elements of the regulations. Based on the characteristics of the amino acids, most sequences of fish results to monophyletic clades that are closely linked to the skeletal Myosin Heavy chain from the humans and chicken. Genes from the clade are in the form of skeletal muscle, but their cardiac expressions are also seen. The 6 tallest sequences, each having 38 exons after the beginning site of a translation, have similar exon-intron patterns as the genes of human skeletal MyHC3 and MyHC13. The 12 partial sequences have exon patterns that are consistent with the organization. MyHC3 and 13 differs from the human genes in the intron Exon 40. All fish genes have intron 40, meaning their absence in the human skeletal gene subset is a condition that is derived. The intron stage of sequences for fish skeletal is similar to the human skeletal genes. The genes of fish MyHC have shorter intron, and are generally shorter that those genes of human. Fish has MyHC4 with 63580bp, human have 9465-12769 bp while chicken have 23 kb. There are variations in the skeletal genes for fish, chicken, and human (Ketchum, 2001). The variation in the results is due to the loss or gain of the codons especially on the end of exon. Generally, data from fish is consistent with the belief that vertebrate ancestors’ had a skeletal MyHC gene that are differentiated and that their descendants have retained similar exon-intron pattern. The teleocast genes for fish are related to the cardiac genes of chickens and humans. The cardiac MyHC genes for the humans are shown in the heart and in the twitch skeletal fibers together with the cardiac genes of fish. Where the cardiac genes in fish miss the introns 18 and 36. The chicken cardiac gene has fewer exons than fish and humans. This means that those cardiac ancestors and the skeletal genes have the exon-intron pattern observed in normal skeletal MyHC genes and mammalian and teleost cardiac genes have lost introns independently. Generally, the data support the belief that vertebrate ancestor has differentiated cardiac gene, which was duplicated in the fish and mammal lineage but retained the cardiac function. There is a difference among the cardiac genes in the exons, length, but the phase of introns is similar in all skeletal and cardiac genes. The cardiac genes are longer than those in skeletal genes that ranges from 11210 -18775 but are shorter than the genes of mammalian MyHC. The variation in the length of exon among the cardiac genes and skeletal genes shows that the deletion/insertions happen commonly in shorter timescales. Contrary to the amino acid consistency and the evolution of intron of cardiac clades and skeletal, there appeared to have an evolution that is independent of intron position and head against the sequence of rod amino acid among the genes in the analysis. Two genes are external other vertebrate and urochord ate genes in the phylogenies. Meaning that the vertebrate and urochordates both confined the two MyHC lineages (Epp, 2003). The primary structure of MyHC and subunits of light chain has been found through protein sequencing and through cloning of the DNA. For the heavy chain, the structures have been reported for the chicken, human and others. The HMM portions’ sequence is compared with that of chicken, human, and fish. The Sis of fish have the same functional domain like those of chicken. The size of the genome encoding of the MyHC is 12kbp. The size is half that of chicken. In the case of human, it is indicated that the myosin heavy chain of smooth muscles and striated are developed from a single gene through splicing, additionally, each portion sequence in the myosin indicated that the substitution of amino acids in fish and certain higher vertebrate are very many. Conversely, the junction of the domains is not the same. The substitutions are taken to be the outcome of body temperature adoption in the facilitation of the ATP hydrolysis and the interaction with the actin at minimal body temperature (Domellöf, 2001). The substitutions lead to thermal stability differences of the myosin. The myosin stability results experiences a proper relationship to the temperature of the animal as later described. The different isoform expressions of the light chain and heavy chain are seen in human due to outcomes of temperature acclimation resulting to differences in the stability and activity of the myosin. Therefore, the comparison of the sequences in amino acid is shown below. Position Fish Chicken Human Total (1-1287) 83 79 79 SI (1-839) 79 76 76 ATP binding site I 100 95 95 ATP binding site II 97 94 94 ATP binding site III 96 96 93 Actin binding site I 93 93 100 Actin binding site II 87 87 83 Actin binding site III 83 92 92 RLC binding site 82 100 100 SHI-SH2 region 95 95 95 RLC-binding site 100 94 94 ELC binding site 78 57 52 24K-51K junction 27 33 33 49K-19K junction 60 57 63 S2 (841-1286) 90 83 83 Reference Domellöf, F. 2001 . Expression of myosin heavy chain isoforms in rat muscle spindles: an immunocytochemical study. Umeaa: University of Umeaa. Epp, T. A. 2003 . Characterization of the human cardiac @-myosin heavy chain gene. Ottawa: National Library of Canada = Bibliothèque nationale du Canada. Ketchum, A. S. 2001 . Molecular analysis of the Drosophila melanogaster cytoplasmic myosin heavy chain. New York: New York. Korfage, J. A. 2004 . Myosin heavy chain composition of the human jaw muscles. S.l.: s.n.]. Myosin heavy chain composition and insulin stimulated glucose uptake in skeletal muscle: effects of growth hormone. 2008 . Copenhagen: Copenhagen Muscle Research Centre, August Krogh Institute. Rovner, A. S. 2006 . Characterization of myosin heavy chain heterogeneity in smooth muscle. Norwood: New York University Press. Tanigawa, G. R. 2001 . Hypertrophic cardiomyopathy: mutations in the cardiac myosin heavy chain genes. New York: New York University Press. Wolf, W. A. 2000 . Nonmuscle myosin regulatory light chain and its role in myosin function and regulation. New York: North Atlantic Treaty Organization, Advisory Group for Aerospace Research and Development. Read More