Comparative Genomics - Essay Example

COMPARATIVE GENOMICS Comparative Genomics Materials Reagents Human, rat, rabbit, chicken, rat samples 2. Iso-pentane 3. Liquid nitrogen 4. Gel electrophoresis reagents Equipments 1. Gel electrophoresis equipment 2. Qualitative RT-PCR 3. Computer 4. Fluorescence analyzer soft ware. Procedure Sequencing of the cDNA of humans, chicken and fish were carried out by blasting against the NCBI DATABASES by retrieving the sarcomeric MYH cDNA of our sample organisms (Saccone and Pesole, 2005). The fish sample was screened for MYH genes using several primers which were selected from the NCBI gene bank. Samples that contained the target gene were sub cloned for future sequencing in the polymerase chain reactions. The sub-cloned samples were loaded in the 96 wells of the PCR plate and the original primers for the first screening used (Ciccarelli, and Miklods, 2009). Sequencing of the positive clones was later done. The heart of the fish was used to construct cDNA pools that were later amplified using the smart cDNA library construction kit. The MYH primers were used as the probes. The PCR products were cloned, sequenced and named depending on the organ of origin which was majorly the heart (cardiac). In fish, the boundaries of introns and exons were identified using the Genscan. Results were verified by screening their sequence structure of the introns splicing based on the flanking regions of the exons. The length of the genes was estimated from the sites where the first start codon (AUG) and the third stop codon (UGA) were identified. The inter-gene distance was determined from the first nucleotide from the stop codon and the last neighboring nucleotide before the next start codon. The untranslated region therefore, will lead to overestimation of the inter-gene distance and underestimation of the gene length. Qualitative comparison of the fish and human intron-exon organization was done. Fish, Human and Chicken Genomics The nucleotide sequences of our samples were analyzed using the public data base from the NCBI. The sequences were biologically aligned using the BLAST program that is present in the NCBI data bases too. The gene organization details were obtained from the NCBI an the Ensembl Genome Browser data bases (Rossi et al., 2009). For the muscle samples, rat samples were reared in a convectional colony under controlled conditions of 25oc, 12 hours day light and night each and 50% relative humidity. Water and foods were provided under the veterinary regulations and guide lines. After three months, the rats were killed using the CO2 gas asphyxiation by skilled personnel. Muscles were quickly removed and stored under liquid nitrogen at a temperature of -80oc. The human muscle biopsy was obtained from the neuromuscular tissue bank with all the ethics approved (Bourque and Mabrouk, 2006). Similarly, the extra ocular eye muscle samples were obtained from Veneto eye bank foundation with all the ethics observed. They were frozen in the iso-pentane coupled with the liquid nitrogen and stored at -80oc until use (Parrington and Coward, 2007). For the qualitative RT-PCR, the RNA from the frozen muscles was extracted using the Promega SV RNA extraction kit on following the manufacturer’s guidelines. Integration of the invitrogen superscript III was coupled with the cDNA and later ran for a RT-PCR on an ABIprism. Primers used were obtained from the NCBI data base. The miR-499 real time PCR was done using the TaqMan miRNA assay kit with all the protocols fully followed (Rossi et al., 2009). Quantitative RT-PCR Promega RNA isolation kit was used to extract RNA from the muscles. The cDNA was analyzed through real time RT-PCR using the ABI 7000 Prism. The mir-499 was performed using the Taqman miRNA kit that was supplied by the applied bio-systems (Rossi, 2009). Antibodies Generation Peptides corresponding to the terminal sequences of the rat MYH14 and MYH15 WERE used to produce polyclonal antibodies from a rabbit. They were purified by use of affinity chromatography using insolubilized immunogens (Rossi, 2009). Immuno-Blotting Protein samples were separated using 10% polyacrylamide gel which had high concentration of glycerol to allow separation of the MYH. Moreover, polyclonal antibodies were obtained from the chicken serum. .later immune-fluorescence was done (Rossi, 2009). Results Amino acids sequences of the fish revealed that most of the sequences matched with those of humans and chicken. The teleost six sequence of the fish had 38 exons from the start of the translation site giving a similar pattern with the human skeletal genes MYH3 and MYH13.b the fish gene gave an intron 40 which was absent in the human skeletal muscles. This was due to the fact that human beings have an interrupting exon instead of the intron in the exon 40. Intron phase of the fish and the human were identical (Schmid and Nanda, 2004). Human cardiac genes were located in the skeletal slow twitch fibers a similar scenario in fish cardiac genes. Sequence of the fish cardiac gave 37 out of 39 exons that were observed in skeletal muscles. However, fish lacked intron 18 and 36. The human cardiac lacked introns 13 and 17 making it have fewer exons in comparison to fish and chicken (Fulton and Alftine, 1997). Variation were noted in exons 7, 16, 17 and 41 lengths despite possessing the same intron phase in both cardiac and skeletal muscles therefore, cardiac muscles were longer than skeletal genes. However, still they were shorter than human MYH. Variations in exons length in both cardiac and skeletal genes are an indication that deletions or insertion mutations were the causal for the variations. MYH14 orthologues were found in fish. This was an indication that the gene had the same origin in comparison with other vertebrates (human and rat). Moreover, the miR-499 gene is contained in the MYH14 gene that was common in all the species. MYH15 that common in humans was the shortest of the sarcomeric MYH genes. This is a clear indication that the Human MYH14 and MYH15 genes had a similar origin with the fish MYH14. However overtime, the human MYH15 gene experienced a structural remodeling due to divergent evolution. Moreover, the divergent evolution is supported in that the chicken MYH14 and MYH15 were similar to that of human beings (Rossi, 2009). Quantitative Reverse Transcriptase Polymerase (RT-PCR) was used in analysis of the expression patterns of both MYH14 and MYH15. The MYH14 and the miR-499 transcripts were present in the rat’s heart besides its skeletal muscles and the extra ocular muscles. In addition the MYH15 were found in high quantities in the extra ocular muscle but absent in the heart and leg muscles. In human beings, the MYH14 and MYH15 were found in the extra ocular muscles (Rossi, 2009). MYH14 was found to be the orthologue if the chicken myosin 2 and it is present in both the heart and the skeletal muscles. This was based on the anti-chicken serum which gave positive results of agglutination on mixing. The MYH15 gene was the orthologue of the chicken ventricular MYH which is solely located in the extra ocular muscles (Rossi, 2009). Based on evolution, the MYH 14 gene has much resemblance in amphibians and avian skeletal muscles. The difference is that in mammals such as human beings, the gene is located in the extra ocular muscles (Sineo and Stanyon, 2006). The presence of the miR-499 is assumed to have contributed to the conservation of the gene in both mammals and fish (Rossi, 2009). Myosin’s composes of a family of ARP dependent protein motors that are used in muscles contractions and other forms of eukaryotic actin-based motilities. It is known that all eukaryotes have several isoforms of their myosis. However, some of the myosins are specialized hence located in specific organs only. The function and structure of myosins is so conserved in various species such that they can bind in other species (Rossi et al., 2009). Analysis of the results showed that the fish homologues in mammals are present in the skeletal MYH while the human cardiac homologues in the fish are found in the heart but they have an overlap (Kocher and Kole, 2008). Gene conversion and the tender, duplication are associated with the evolution iof the skeletal and cardiac MYH genes in fish. Variation in the regulation and sequence of the MYH multi gene family contributes to the various forms of muscle properties. Class II myosins are ancient hence found in all eukaryotic organisms exclusive of plants. The myosinsds II genes are believed to have evolved in a homogenous rate across the species that is the reason why their traces are common among species. It is assumed that the deutrosome had two sarcomeric MYH genes. However, one is thought to have been lost as evolution continued in the vertebrates’ lineage. Divergence of the urochordates to vertebrates led to duplication of more genes but still it is unclear if the extra sarcomeric genes originated from a single gene. Our study was based on the fast skeletal muscles and the slow cardiac muscles. The ancestral cardiac and skeletal myosin seems to have diversified in an independent manner within the various classes of the vertebrates. Moreover, the unequal crossing over stipulated above is thought to have contributed to the different duplication rates in both skeletal and cardiac MYH in vertebrates. It is evident that both skeletal and cardiac myosins are tandemly arranged in mammals such as the human beings and chicken. Sequencing of fish transcrioptomes suggested that the myosins were of skeletal genes (Rossi et al., 2009; Kocher and Kole, 2008). Sarcomeric muscles of the teleosts were used due to considerations of several factors. The muscle fibers are spatially separated in the teleosts. The white fast-twitch muscles are known to make up the bulk of the fish body whiles the red slow-twitch fibers which are only found in the mid lateral band which is beneath the skin. Hyperplasia leads to continuous growth of fish muscles through out the life hence they can studied. Finally, the fish muscles are so plastic hence prone to changes into various MYH isoforms hence hey are not destroyed. Only 9 MYH genes have been discovered in human while fish has more than 29 of them (Rossi et al., 2009). Fish, Human and Chicken Based on the amino acids sequencing, most of the fish sequences had a monophyletic clade which was closely related to the humans and frog samples. The clade genes were from the skeletal muscles but cardiac muscles were observed too (Kocher and Kole, 2008). The slow red muscle in the teleost was in contrast to the mammalian pattern in the cardiac MYH gene which was in form of slow twitch fibers. Six teleost clade sequences had 38 exons counting started from the translation sote start. The same results were obtained in humans skeletal genes of MYH3 and MYH13 which differs from other human MYH (1, 2, 4 and 8) due to the presence of an intron interrupting the exon 40. All fish genes had the intron 40 which lacked in humans implying that the intron was a derived one. However, the intron phase of the fish skeletal muscles was similar to the humans skeletal; muscles. Similarly, the fish MYH genes had shorter introns in comparison to the humans and chicken. All in all, a slight difference was noted in exons length of genes 3, 7, 16, 17, 450 and 41 in both the human and skeletal muscle genes. Since there was no change of the intron phase, the variation in the exon length is attributed to gain or loss of codons at the 3’ of the exon sequence. The analysis above indicates that the hypothesis statement that the common ancestor of the vertebrates had a differentiated skeletal MYH gene. Moreover, the descendants of the MYH gene have retained the gene in that the exon-intron organization and expression is conserved. Human MYH13 had the closest relationship with the fish giving a hint that it may have same about from gene duplication. The MYH3 seemed to be older than the human and the chicken gene (Rossi et al., 2009). Cardiac fish cardiac genes lacked introns 18 and 36 besides the Dre A5 which lacked intron 20. The human cardiac genes had fewer exons in comparison to their skeletal muscle genes. However, the missing introns were not similar in humans and teleosts intron 37, 36, 18 and 13 were found present in the skeletal genes in all the teleosts, mammals and their affiliates. This too supports the theory that both cardiac and skeletal muscle genes have a conserved hence a well, organized intron-exon organization. Moreover, it was a proof that the mammals and teleosts lost their intron independently ion that there was no relationship between the lost intron. Therefore, the vertebrate ancestors had the same cardiac gene which differentiated cardiac gene which was independently duplicated in mammals and fish species (Rossi et al., 2009). Some differences are noted in the exons length in that cardiac genes are longer than the skeletal genes despite the same intron phase. In the same case, the exons lengths were shorter than the human MYH genes. The variations in the exon lengths are explained by the fact that those sections underwent deletion and insertion due to evolution as time passed. The cardiac clade had two ore genes namely Fru X and Dre X. the genes were orthologues to each other but they were distinct from the teleost cardiac genes. The two extra genes were intronless giving us the idea that due to differentiation in cardiac and skeletal genes, retro transposition retro-transposed genes are known to become pseudo genes in future due to lack of the regulatory elements. Discussion Comparative genomic hybridization is used to compare two genomes that are suspected to be closely related in that they have gained or lost a gene or the whole chromosome (Mushegian, 2010). The method involves isolation of DNA samples from the sources and labeling each sample with different fluorophore also known as fluorescent molecules of different colors manly red and green (Bergman, 2012). The DNA was first denatured to produce a single strand that was used for hybridization. Florescence microscope and computer software are applied in analysis. The different fluorescent signals are compared with the length of the chromosomes (Brown, 2007). A high intensity of the test sample is an indication that it is highly corresponding to the sample source. On the other hand, higher intensity of the reference sample is an indication that the test sample lost some material in that specific region of the test sample. A neutral color yellow is observed when there is no difference in the two samples provided that red and green colors were used as the fluorophores (Rossi, Mammucari, Carla, Reggiani, and Schiaffino, 2009). Isolation and sequencing of human cDNA coding for cardiac β-myosin heavy chain designated under the title MHYH7. The gene was found to be 22, 883 base pairs long with 1935 of its amino acids of the protein being encoded in 38 exons. The 5’intranslated region is 86bop long having been split by two introns. On the other hand, the 3, untranslated region is 114 bp long. Further analysis of the gene revealed three Alu repeats besides another one in the flanking region of the 3’ end (Jean, Haas, Lichter and Bach, 1990). On analysis, it is evident that the molecular organization of the gene functions in conservation of the coding ratio, pattern, number and positioning of introns a feature associated with the vertebrates’ sarcomemeric myosin heavy chain genes (Jean, 1990). Comparison of the human β-chains with homologous sequences of rabbit, and rats and the heterologus embryonic heavy chains sequences showed that the protein sub-regions responsible for actin and nucleotide binding are similar in both scenarios. The difference was that the mammalian β-myosin heavy chains were highly conserved in heterologous heavy chains (Jean, 1990). Myosins are highly conserved protein that is common in all eukaryotic cells (Weiss, Wertman, Krauter, and Leinwand, 1999). It provides motor functions in cytokinesis, muscle contractions and phagocytosis. The heavy chains have ATPases that provides with energy during the former named processes. Every myosin is known to compose an amino terminal head and a carboxyl tail. Myosins have divided into several classes based on their head domain properties. For example, the class II myosins are co posed of two headed myosins with filaments that are composed of two heavy chain subunits and four light chain sub units (Weiss and Leindwand, 1996). Cardiac genes of MyHC-α and MyHC-β have a 4.5kb inter-genic separation in chromosomes 14 in humans and 9-13 in mice and they are similar in rat and rabbit. Comparison of the sequences of human and rat cDNA showed that MyHCs are highly conserved with the skeletal clusters being maintained in all mammalian species (Weiss, Wertman, Krauter, and Leinwand, 1999). Fish homologues of mammalian skeletal MYH genes are similar in expression in mammals’ heart (Kocher and Kole, 2008). However, the difference is that in fish, the genes have an overlap in their domains overlap.Class II muosins are represented in all eukaryotes except the plants. Genes of mammals and chicken are tandemly arrayed. Analysis of the skeletal genes sequencing from fish gave a close relationship of the MYH genes form humans and chickens (Kocher and Kole, 2008). Fish has an intron 40 in their genes a feature that was lacking in humans (Kocher and Kole, 2008). However, the intron phase was identical to human beings skeletal genes. The fish MYH genes gave shortest introns on comparison of humans and the chicken. Mammalian genome has three ancient genomes of the sarcomeric MYH14, MYH15 and MYH16. MYH14 and MYH 15 have their orthologues present in birds such aw chicken coding for the chicken slow 2 and its ventricular MYH of the cardiac. However, only the MYH14 is present in fish. In all the sample species the MHY14 gene composed of microRNA. MYH16 are absent in chicken but present in fish (Kocher and Kole, 2008). MYH16 codes for the ventricular MYH in chicken but it is absent in fish (Rossi, Mammucari, Carla, Reggiani, and Schiaffino, 2009). MYH16 is common in all jaw muscles of primates, carnivores but it is in form of pseudo gene in humans. Nucleotide and amino acids sequences were carried out using the public databases and tools. BLAST was used to align the sequences and the mapping done using the NCBI data bases (Rossi, 2009). MYH14 of fish was similar to the chicken analysis. Different sequences were obtained in the mammals besides them being the shortest of all the sarcomeric myosins. The MYH14 and 15 of the vertebrates had a similar precursor with the fish MYH 14 implying that they had the same common precursor only that they underwent some remodeling due to divergent evolution. The chicken MYH 14 and 15 had same similarities to that of human beings (Rossi et al., 2009). Quantitative RT-PCR revealed that the MYH 14 and the miR-499 in mammals were found in the rat heart, the slow skeletal muscles and the extra ocular muscles with detection of low levels in the fast skeletal muscles. The MYH15 was present in high amounts in the extra ocular muscles but completely absent in the slow and fast muscles of then leg and the cardiac muscles. In humans, the MYH14 were solely detected in the extra ocular muscles (Rossi et al., 2009). Polyclonal antibodies that were raised against the amino terminal of the MYH 14 and 15 were run on a standard gel electrophoresis and later immunoblotted. The results revealed that the antibody against the MYUH 15 reacted in a selective mode. In conjunction, the striated muscles were completely uncreative in that no agglutination was noted. Western blot revealed that the band that was detected by the anti-MYH 15 have similarities with the β-MYH bands that were present in the slow muscles. Transverse sections had the MYH 15V present in all the orbital and the global layers of the extra ocular muscle fibers. The anti-chicken serum gave different results in that it was distributed in small quantities in both the orbital and global layers of the slow tonic muscles. Immunofluorescence staining of the MYH 15 A bands of the extra ocular longitudinal muscles revealed that they are a member of the sarcomeric myosin. Anti MYH 15 stains were rare in the fast and hindlimbs muscles with the intracapsular region totally uncreative (Rossi et al., 2009). MYH14 gave a strong agglutination reaction with the chicken antiserum in rat. However, in humans, the fibers were much more than in rats and they corresponded to the slow tonic muscles of the rat. Chicken and human MYH 14 muscles genes gave a lower electrophoretic mobility than in rat extra ocular muscles. The MYH 14 orthologues in of chicken was significantly detected in the cardiac and skeletal muscles at the transcript level in both the slow tonic extra ocular muscles and in muscle spindles of the bag fibers at the protein level. The MYH 14 was therefore concluding to be the coding gene in then mammalian slow tonic myosins. The MYH 15 orthologues of chicken ventricular myosins of the heart were located in restricted regions of the extra ocular muscles and in the muscle spindles extra capsular region of the bag fibers (Rossi et al., 2009). Immuno-cytochemical studies in chicken revealed that the chicken ventricular myosin is present at embryonic and adult heart but not in the adult skeletal muscles. External sources reveal that the MYH 15 gene is used to control pumping in amphibians and at the same time to control the eye ball movements in mammals (Rossi et al., 2009). The variations of the same gene functioning in a separate manner are an indication that the gene underwent functional specialization hence the remodeling of the gene. MYH 14 genes are dominant in mammals and avian extra ocular muscles and little amounts in the slow MYH and the slow twitch fibers. This shows that two distinct myosin type genes are found in slow-twitch and the slow-tonic fibers. The MYH 14 transcripts are known to be expressed in cardiac and skeletal muscles but their proteins are strictly expressed in little quantities in both extra ocular and muscle spindles. The scenario may be related to the presence of miR-499 which is also expressed in fish cardiac and skeletal muscles (Rossi et al., 2009; Kocher and Kole, 2008). The mammalian genome of the human beings composes of sarcomeric myosins heavy chains genes MYH14, 15 and 16. These genes are in addition to the skeletal and cardiac muscles. The MYH 16 is commonly found in the jaws of carnivorous animals. All in all the expression of then MYH14 and 15 is not well understood leaving room foe more studies. However, the MYH 14 and 15 orthologues of the human beings are found in frogs and birds such as the chicken. The MYH 14 codes for chickens’ slow myosin 2 while the MYH15 codes for the ventricular MYH. In contrast, the fish species was found to contain only the MYH 14 orthologues of human beings. Note that the MYH14 protein was only noted in the in the minor fibers of the extra ocular muscles with responding trait of the slow-tonic fibers and in the muscle spindle bag fibers. During development of the organisms, MYH14 is known to be present at the cardiac, skeletal and then extra ocular muscles. However, the concentration is known to decrease as aging continues with some remains of it being visible in the slow tonic fibers after birth (Rossi et al., 2009). A similar scenario was observed in MYH15 which is absent in embryonic and fetal muscles although its first detection is noted in the extra ocular muscles of the orbital layer. The expression pattern of the MYH 14 and 15 has led to identification of other MYH isoforms that are considered to be involved in the sarcomeric architecture of skeletal muscles. Therefore, this leads to no ambiguity in the analysis of the slow-tonic fibers in mammals such as human beings (Rossi et al., 2009). In mammals, sarcomeric myosin heavy chains have been identified in mammals and they are highly conserved in gene clusters. In humans, the chromosome 14 is known to code for the cardiac myosins α-MYH β-MYH with similar structures in the skeletal muscles. Chromosome 17 in human being too is known to code for a gene that on its expression leads to formation of the six skeletal myosins in conjunction of the 2A, 2B, 2X MYHs, developmental and neo-natal isoforms and the extra ocular isoform. Recent research in human genome project led to identification of additional sarcomric MYH genes. MYH 14 has been identified in chromosomes 20, MYH 15 in chromosome 3 and MYH 16 in chromosome 7. However, the three sarcomeric genes were found to be different from the other sarcomeric genes in that there were great variations in their exons-intron organization despite the same MYH ancestors. MYH 14 orthologues are found in the fish and chicken whereby they code for the slow 6yme myosins 2. Orthologues of MYH 16 are absent in the chickens but they are present in the fish. The MYH 15 orthologues are present in chicken coding for the ventricular MYH but no forms of it were noted in fish (Rossi et al., 2009; Shimizu, 2003). The three isoforms remains a mystery still in tat no deep information about them has yet been detected. However, MYH 16 is present in the jaw muscles of the carnivores and primates such as monkeys but it is in form of a pseudogene I the human beings. Failure of Garriock et al., 2005 ton detect the MYH15 mRNA using the RT-PCR in the mouse heart led to their conclusion that the gene is a form of pseudogene in the mammals (Sineo and Stanyon, 2006). This is because there is great similarity between the mouse and the human beings hence the organism is used in study of complex human systems. In mouse, the MYH 14 is known to compose of micro RNA-499 within the intron 19 of the mouse gene chromosome (Rossi et al., 2009). Conclusion It is now evident that both duplication and differentiation gave rise to different MYH genes of the skeletal and cardiac muscles. This is thought to have happened after the divergence evolution of the vertebrates from the urochordates in prior to actinopterygians and sarcopterygians. All in all fish, mammals (human) and birds (chicken) had close relationship of the MYH genes hence of the same descendants. Exon organization is consistent hence an indication that independent duplication in each of the chordates classes (Volff, 2006). The chicken genes were more related to human beings in comparison to the fish. This may be based due to physiological similarity in that both humans and chicken are homeotherms while the fish is a poikilotherm (Rossi et al., 2009). Therefore, from our analysis, it seems that MYH 15 is an orthologue of chicken ventricular myosins and it is a form of slow type myosin. Mammals’ myosins were found to be related as showed from the human, rats, chicken and rabbit. Significance differences were found to be present in fish which is a poikilotherm. Selective expression of the MYH 15 in the orbital layers of the extra ocular muscles may be related to the physiological function differences between the orbital and global layers. Therefore, our experiment was successful and errors that may have occurred are due to experimental errors in our machines. Bibliography Bergman, N, 2012. Comparative Genomics - Volume 2. New York: Humana Press. Bourque, ‎G. & Mabrouk, N. 2006. 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