Human and mouse alpha-synuclein genes: comparative genomic sequence analysis and identification of a novel gene regulatory element

Abstract

The human alpha-synuclein gene (SNCA) encodes a presynaptic nerve terminal protein that was originally identified as a precursor of the non-beta-amyloid component of Alzheimer's disease plaques. More recently, mutations in SNCA have been identified in some cases of familial Parkinson's disease, presenting numerous new areas of investigation for this important disease. Molecular studies would benefit from detailed information about the long-range sequence context of SNCA. To that end, we have established the complete genomic sequence of the chromosomal regions containing the human and mouse alpha-synuclein genes, with the objective of using the resulting sequence information to identify conserved regions of biological importance through comparative sequence analysis. These efforts have yielded approximately 146 and approximately 119 kb of high-accuracy human and mouse genomic sequence, respectively, revealing the precise genetic architecture of the alpha-synuclein gene in both species. A simple repeat element upstream of SNCA/Snca has been identified and shown to be necessary for normal expression in transient transfection assays using a luciferase reporter construct. Together, these studies provide valuable data that should facilitate more detailed analysis of this medically important gene.

Figure 1

Genetic map showing the position of Snca on mouse chromosome 6. Genetic distances as determined on the Jackson Laboratory Backcross DNA Mapping Panel are shown on the left in centiMorgans (cM). For clarity, not all markers mapped on the BSS backcross are shown for all of the loci. Underlined genes have mapped human homologs; the human map locations are shown on the right. Some of the human homologies were obtained from the Mouse Genome Database (http://www.informatics.jax.org/, July 1999). The extensive region of homology between MMU6 and HSA7 interrupts the region of shared synteny between MMU6 and HSA4q22.

Figure 2

Global alignment of human SNCA and mouse Snca. Numbers on the vertical axis represent the proportion of identical nucleotides in a 100-bp window for a point on the plot. Numbers on the horizontal axis indicate the nucleotide position from the beginning of the human genomic sequence. Peaks shaded in blue correspond to the SNCA/Snca coding regions. Peaks shaded in light blue correspond to SNCA/Snca mRNA untranslated regions. Peaks shaded in red correspond to evolutionary-conserved regions (ECRs), defined as areas where the average identity is >75%.

Figure 3

(A) Percentage identity plot (PIP) of human and mouse genomic sequence upstream of the first coding exon of SNCA and Snca. The location of SNCA noncoding and coding exons (orange and blue shading, respectively), introns (yellow shading), and the NACP-REP1 repeat (green shading; half bar) are indicated. A scale representing the degree of sequence identity, indicated as a percentage, is located at the right of each row. Human repetitive elements identified in the human sequence are indicated along the top of the homology window and are described in the key. (B) Alignment of the human NACP-REP1 (126 nt) and mouse (63 nt) repeat by pairwise comparison using the ALIGN program (http://dot.imgen.bcm.tmc.edu:9331/seq-search/alignment.html).

Figure 3

(A) Percentage identity plot (PIP) of human and mouse genomic sequence upstream of the first coding exon of SNCA and Snca. The location of SNCA noncoding and coding exons (orange and blue shading, respectively), introns (yellow shading), and the NACP-REP1 repeat (green shading; half bar) are indicated. A scale representing the degree of sequence identity, indicated as a percentage, is located at the right of each row. Human repetitive elements identified in the human sequence are indicated along the top of the homology window and are described in the key. (B) Alignment of the human NACP-REP1 (126 nt) and mouse (63 nt) repeat by pairwise comparison using the ALIGN program (http://dot.imgen.bcm.tmc.edu:9331/seq-search/alignment.html).

Figure 4

Fold expression of luciferase activity. 293 T cells were cotransfected with pGL 3-ASP, pGL 3-ASPdel, or pGL 3-Basic and pRL-TK. For each experiment, cells were plated into nine wells. Three wells at a time were independently transfected in parallel with three individually prepared aliquots of each of the three constructs in calcium phosphate. The relative activity with each pGL 3 plasmid was calculated by dividing the luminescence intensity of the firefly luciferase by that of the Renilla luciferase in each independent aliquot of cells and then averaging the three relative luciferase activities seen. The fold expression for pGL 3-ASP or pGL 3-ASPdel was then determined by dividing the average relative activity of each construct by that of the average obtained with pGL 3-Basic. Data shown here are the averages +1 SEM of three independent experiments performed on three separate days.