Assignment-1 Example

Assignment-1 example

NANTCGNNACGAGGGGTAANGGAACTGGAATCCAAACTCTCTGAAGCTGAGAAGGAATTCATCGAAGGAGCACCAACACGTAGCAAACGATCACCATCCGAGTGGATACCAAGGCCACCCGAAAAATACAGTCTTACTGGGCACAGAGCTCCTATCAACAGAGTTATTTTCCATCCGGTCTTTAGTCTTATAGTATCTGCCAGCGAAGATGCCACTATCAAGGTGTGGGACTTCGAGAGCGGCGAATTCGAAAGAACGTTGAAGGGGCACACCGACAGCGTGCAGGACGTTTCCTTCGACGTCTCCGGGAAACTGTTAGTCTCATGCAGTGCGGACATGTCTATTAAGTTATGGGACTTTCACCAGTCATTCGCCTGCGTGAAAACCATGCACGGACATGATCACAGTGTCAGCTCTGTCGCATTTGTGCCACAAGGGGATTTCGTAGTGAGCGCCTCTAGGGATAAGACCATCAAAATATGGGAAGTAGCGACAGGGTATTGTGTCAAAACGTTAACGGGGCACAGAGAATGGGTACGGATGGCCAGAGTCAGTCCTTGTGGAGAATTAATAGCTAGTTGCTCGAACGATCAAACAGTACGGGTTTGGCACGTGGCAACAAAGGAAACGAAGGTCGAACTCAGAGACCACGAACACGTAGTGGAGTGTATCGCATGGGCACCGGACAGTGCAAGAGCATCGATCAACGCTGCTGCAGGGGCGGACAATAAGGGAGCCCATGAAGGACCTTTCCTCGCATCTGGCTCGCGAGACAAAGTAATTCGTGTATGGGATGTCGGTGCCGGTGTTTGTCTCTTCGCCCTATTGGGCCACGACAACTGGGTTCGCGGCATCGTCTTCCATCCTGGTGGCAAGTTCATCGTCAGTGNCTCTGACGACAAGANCCTGCGAGTATNGGANACGCGCAACANANGGGTAATGAAAACCCTCNAAGCGCACGTCCACTTCTGCNCCTCCNTTGATTTCACAAAAGCCATCCTTACGTGGTCNCCGGTAGTGNNNATCAAACGGNNAANN The sequences clipped from the file are indicated in bold. ATG underlined (the N is a T in other EST sequences).

Honeybee gene: The insert size of the cDNA clone sequenced was approximately 1,500 bases; therefore, 1006 bases of DNA sequence could be clipped out for analysis. MegaBLAST of the honeybee reference genome identified three regions of sequence similarity on linkage group/chromosome 6 corresponding to exon 4, 5 and 6 of the gene LOC408869. Using BLASTn, the last 37 nucleotides aligned with exon 7 (Fig-1). The DNA sequence was used to search the refRNA database for the honeybee genome. The EST aligned well with the refRNA sequence XM392399.3 starting a little downstream from the reference sequence in exon 2 and apart from the Ns in the sequence there was only one substitution and one nucleotide deletion noted. The EST in the clone is in the correct orientation because the numbering of the reference sequence and the EST ascended in the same direction. Thirty-three ESTs were identified in a megaBLAST search of the Honeybee EST database with my EST. All ESTs aligned well indicating that there is no evidence of alternative splicing. Analysis of the 5’ most EST sequences suggested that the 5’ untranslated region is approximately 400 bases long and the 3’ untranslated region was estimated to be approximately 700 bases. Blast analyses of the 5’EST sequences revealed the existence of 3 exons 1,2, 3 in the genome and all 5’ ESTs have these exons and none map to exon 1 of the model RNA in refRNA (Fig-1). Exon three must exist in an unsequenced gap of the genome sequence located between 17044 and 17045K because it does not align with honeybee genomic sequence. Therefore, the model refRNA is wrong because it includes an incorrect first exon and misses 3 exons found within the EST collection, and the first AUG is in exon 4 and at the beginning of my EST. Rather than covering 17,043-17,049K of LG6 as shown in NCBI, LOC408869 covers 17,031-17,049K. The gene is expressed in the head and brain of the honeybee adult. LOC408869 encodes a protein similar to Lissencephaly-1 CG8440-PA, isoform A.

Function in other organisms: Lissencephaly is a human condition with the phenotype of a smooth (Liss) brain (encephaly). Classical or Type 1 Lissencephaly can be caused by mutations in the Platelet-activating factor acetylhydrolase, isoform B1, alpha subunit (LIS1) gene (Lo Nigro et al., 1997). The LIS1 gene is haploinsufficient as deletions or point mutations in one copy of the LIS1 gene result in a smooth small brain associated with a number of neurological conditions including jerks, spasms and seizures. The severity of the condition depends on the position and type of DNA change, and in the most sever cases, death is due to respiratory problems or sepsis early in life. In Drosophila, Lis1 regulates metaphase spindle orientation in neuroblasts (Siller and Doe, 2008). Involvement of Lis1 in spindle function has been observed in C. elegans and mammalian cells; therefore, Lis1 has an evolutionaryly conserved role in eukaryotic cell division. The smooth small brain observed in humans with Lissencephaly is due to defects in neural migration. Mutation of one copy of the LIS1 homologue in mice results in brain developmental defects also due to delayed neural migration (Hirotsune et al., 1998).

The human LIS1 gene and conserved homologues encode a protein containing eight WD 40 conserved repeats. WD40 repeats are found in a number of eukaryotic proteins that function in a wide array of cellular processes from signal transduction to mRNA processing. The WD40 repeats found in LIS1 proteins are a subfamily called Lissencephaly type 1 homology motif. The LIS1 protein is found in a range of eukaryotic organisms and is involved in regulating the formation of the microtubule cytoskeleton and spindle orientation. The primary function of LIS1 is regulating the function of the microtubule motor dyein by binding to two distinct sites: within the N-terminal stem and near the P loop of the AAA domain (Tai et al., 2002). Therefore, the impaired neural migration defects of lissencephaly may arise due to altered dynein function.

Structure of the LIS1 protein: The crystal structure of mouse LIS1 complexed with the alpha subunit of platelet activating factor acetylhydrolase (PAF-AH) has been determined (Tarricone et al., 2004). The N-terminal region of LIS1 mediates dimerization of LIS1, and the WD40 repeat region of LIS1 interacts with PAF-AH. Another protein Nde1 competes with PAF-AH for binding LIS1. LIS1 is known also to interact with dynein, a microtubule motor molecule. Mutational changes that result in lissencephaly map to the region of the LIS1 protein that bind Nde1 and dynein suggesting that these mutational changes disrupt the interaction with Nde1 or dynein. From the crystal structure of LIS1:PAH-AH and competitive interactions with Nde1, a model for how LIS1, Nde1 and dynein interact has been proposed (Fig-2).

Figure 1. Structure of the LOC408869 gene. The boxes indicate exons, and the filled boxes indicate exons with coding sequence. The alignment of the EST is shown above the exons. The gap in the genomic sequence is indicated as a bracket. The relative position of the gene on linkage group 6 is indicated below the gene.

Figure 2. Proposed structure for the complex between LIS1, Nde1 and Dynein (Taken from Tarricone et al., 2004). Nde1 (green) interacts with LIS1 along the N-terminal arms of the two LIS1 molecules. One monomer of LIS1 (blue) interacts by AAA of dynein (yellow) and the other monomer interacts with the stem.
References:

Hirotsune, S. et al., 1998. Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality. Nature Genet. 19: 333-339.

Lo Nigro, C. et al. 1997. Point mutations and an intragenic deletion in LIS1, the lissencephaly causative gene in isolated lissencephaly sequence and Miller-Dieker syndrome. Hum. Molec. Genet. 6, 157-164.

Siller, K and Doe, C. Q. 2008. Lis/dynactin regulates metaphase spindle oreintation in Drosophila neuroblasts. Dev. Biol. 319, 1-9.

Tai, C. Y. et al. 2002. Role of dynein, dynactin, and CLIP-170 interactions LIS1 kinetochore function. J. Cell Biol. 156, 959-968.

Tarricone, C. et al. 2004. Coupling PAF signaling to dynein regulation: structure of LIS1 in complex with PAF-acetylhydrolase. Neuron 44, 809-821.