atquang's blog

The overall objective of our proposal is to create a phylogenetic tree to determine the reliability of HOXC genes as an indicator of phylogeny. By aligning the sequence, the genes will become easy to compare and allow for the creation of a phylogenetic tree, as proposed. The sequencing data can also be used to determine how conserved the HOXC gene is. By understanding the evolutionary and genetic differences of HOXC genes between different species, the function of HOXC genes, which are currently unknown, can be better understood. The creation of a phylogenetic tree will allow for the determination of reliability of using HOXC genes as an indicator of phylogeny. If the phylogenetic tree proves to be reliable, this would be a phylogenetic tree of vertebrates that can be used in order to trace the evolutionary history of vertebrates. If new species were to be found, its HOXC gene can be sequenced to determine its phylogeny accurately.

Pigmentation in dogs and other mammals (including you) is caused by the relative amounts and types of two classes of pigment: eumelanin and phaeomelanin. The eumelanins are the black and brown pigments, and the phaeomelanins are red and yellow. Both eumelanins and phaeomelanins are synthesized in pigment-producing cells called melanocytes. First, the enzyme tyrosinase converts the amino acid tyrosine to a chemical called dopaquinone. If the enzyme called tyrosinase-related protein 2 (TRP-2) is present, it converts the dopaquinone to a version of eumelanin that has a brown color, Cocoa's pigment. If the enzyme called tyrosinase-related protein 1 (TRP-1) is present, it converts the brown version of eumelanin into the final, black pigment.

The objective of our proposal is to create a phylogenetic tree examining the possibility of using the HOXC gene as an indicator of the phylogeny. One specific aim of the proposal is to identify highly conserved genes that are present in vertebrates. In preliminary work, we found that one example of a highly conserved gene is the BOP1 gene present in ribosomal DNA. Further research done on HOX genes has noted that the duplication of HOX clusters is shared by all vertebrae (Ruddle et al. 1994). The statement motivated our group to propose that the HOX genes express little variation in vertebrae, and test the statement by knowing how consistent this data would be if we were to expand our subject of interest to a whole kingdom, rather than specific subjects used for their evidence (chicken, zebrafish, Xenopus, and newt). We will now look into HOX genes to see how similar or different expression of the HOX gene is throughout the Animalia kingdom.

The HOX gene is highly conserved, and as a result, HOX genes, in general, are a reliable gene to use in creating a phylogenetic tree with a wide variety of species. It has been established in many studies done in the past that HOX genes are highly conserved throughout evolution (Gaunt, 1994). Highly conserved genes are genes that do not often change due to their vital role in the body or the cell. Because the highly conserved genes do not change often, they are especially useful in determining the phylogeny of evolutionarily distant species. Studies have already established that HOXC genes are highly conserved and a good indicator of phylogeny in early vertebrates, specifically in cartilaginous fish (Koraku, 2011).

The third specific aim is to determine the reliability of HOXC genes as an indicator of the phylogeny. While molecular phylogenetic determination is a reliable way of determining the phylogeny of organisms, Because there are only 4 possible nucleotides, there is a chance that the phylogenetic tree may be incorrect. To determine whether the tree is accurate, the bootstrap value of the tree will be calculated. By comparing the tree, the overall objective of determining the use of HOXC as an indicator of phylogeny can be achieved. 

The objective of our proposal is to create a phylogenetic tree examining the possibility of using the HOXC gene as an indicator of the phylogeny. One specific aim of the proposal is to identify highly conserved genes that are present in vertebrates. In preliminary work, we found that one example of a highly conserved gene is the BOP1 gene present in ribosomal DNA. Further research done on HOX genes has noted that the duplication of HOX clusters is shared by all vertebrae (Ruddle et al. 1994). The statement motivated our group to propose that the HOX genes express little variation in vertebrae, and test the statement by knowing how consistent this data would be if we were to expand our subject of interest to a whole kingdom, rather than specific subjects used for their evidence (chicken, zebrafish, Xenopus, and newt). We will now look into HOX genes to see how similar or different expression of the HOX gene is throughout the Animalia kingdom.

The overall objective of our proposal is to identify a subject that contains a highly conserved gene that can be characterized by the amount of expression. One specific aim the proposal has is to identify highly conserved genes that are present in the Animalia kingdom. In preliminary work, we found that one example of a highly conserved gene is the BOP1 gene present in ribosomal DNA. Further research done on HOX genes has noted that “[the duplication of HOX clusters] are shared by all vertebrae.”1 The statement motivated our group to propose that the HOX genes express little variation in vertebrae, and test the statement by knowing how consistent this data would be if we were to expand our subject of interest to a whole kingdom, rather than specific subjects used for their evidence (chicken, zebrafish, Xenopus, and newt). We will now look into HOX genes to see how similar or different expression of the HOX gene is throughout the Animalia kingdom.

The overall objective of our proposal is to identify a subject that contains a highly conserved gene that can be characterized by the amount of expression. One specific aim the proposal has is to identify highly conserved genes that are present in the Animalia kingdom. In preliminary work, we found that one example of a highly conserved gene is the BOP1 gene present in ribosomal DNA. Further research done on HOX genes has noted that “[the duplication of HOX clusters] are shared by all vertebrae.”1 The statement motivated our group to propose that the HOX genes express little variation in vertebrae, and test the statement by knowing how consistent this data would be if we were to expand our subject of interest to a whole kingdom, rather than specific subjects used for their evidence (chicken, zebrafish, Xenopus, and newt). We will now look into HOX genes to see how similar or different expression of the HOX gene is throughout the Animalia kingdom.

The restriction digests for Agouti and TYRP1 are then analyzed by running an agarose gel. Agouti and TYRP1 restriction digests are performed in separate gels. Gel electrophoresis is performed by first pouring 3.0 g high-resolution agarose into a prepared gel box. A comb is immediately inserted into the gel and removed when the gel has solidified. The gel is then covered with 1x TAE buffer until the top of the box is covered. The ladder is premade and given by Assistant Gregory Teicher. Restriction digest products are prepared by adding 3 µL of SmartGlow (a dye). 12 µL of digests and ladders are loaded in any order, being sure each lane is labeled and given the correct solution. The settings for the gel are set to no timer (infinite) and a voltage of 115 V. The gel is stopped when looked Assistant Teicher approves of the gel.

Analysis of canine MC1R sequences is performed when MC1R sequencing from GeneWiz is received. First, the quality of MC1R gene sequences is checked to see if there is useable data. Sequencing files are modified using the sequencing program “4Peaks” to cut off unreliable ends of the files. Potential heterozygous sequences (two peaks on top of one another) are noted. BLAST results are retrieved to compare known sequences in the NCBI database with our dog sequence, noting Query cover, E-value, and percent identity of the top 5 hits. Our MC1R genome is then compared with Tasha the Boxer’s MC1R genome (the first dog to have its full genome sequenced) using EMBOSS Needle. Single nucleotide polymorphisms (SNPs) were noted and compared to specific MC1R SNPs identified in Nowacka-Woszuk, Salamon, Gorna, and Switonski’s paper (2012).