Maintenance of transposon-free regions throughout vertebrate evolution-5

<p><b>Copyright information:</b></p><p>Taken from "Maintenance of transposon-free regions throughout vertebrate evolution"</p><p>http://www.biomedcentral.com/1471-2164/8/470</p><p>BMC Genomics 2007;8():470-470.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241635.</p><p></p> the non-genic 13 kb TFR hs11.145 (red bar). Thick blue bars indicate blocks of sequence that are alignable to the orthologous zebrafish TFR dr25.92. Small purple bar indicates the position of the human miRNA mir-129-2. (B) A close up view of 130 bp around mir-129-2, thick purple bar indicates the mature miRNA, thin purple line indicates pre-miRNA hairpin. Blue conservation plot is based on the alignment of 17 vertebrate species and green plot based on pairwise alignment of human and zebrafish that shows a conservation profile consistent with the presence of a miRNA conserved in each species [38]. (C) Syntenic region of the zebrafish genome (20 kb chr25:31,421,001–31,441,000) including the TFR dr25.92. Thick blue bars indicate blocks of sequence that are alignable to the orthologous human TFR hs11.145. Although there are currently no genes annotated in this region, the conservation profile suggests that an ortholog of mir-129-2 resides within the TFR. All images are modified screen shots taken from the UCSC genome browser [31]

Maintenance of transposon-free regions throughout vertebrate evolution-1

<p><b>Copyright information:</b></p><p>Taken from "Maintenance of transposon-free regions throughout vertebrate evolution"</p><p>http://www.biomedcentral.com/1471-2164/8/470</p><p>BMC Genomics 2007;8():470-470.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241635.</p><p></p> the non-genic 13 kb TFR hs11.145 (red bar). Thick blue bars indicate blocks of sequence that are alignable to the orthologous zebrafish TFR dr25.92. Small purple bar indicates the position of the human miRNA mir-129-2. (B) A close up view of 130 bp around mir-129-2, thick purple bar indicates the mature miRNA, thin purple line indicates pre-miRNA hairpin. Blue conservation plot is based on the alignment of 17 vertebrate species and green plot based on pairwise alignment of human and zebrafish that shows a conservation profile consistent with the presence of a miRNA conserved in each species [38]. (C) Syntenic region of the zebrafish genome (20 kb chr25:31,421,001–31,441,000) including the TFR dr25.92. Thick blue bars indicate blocks of sequence that are alignable to the orthologous human TFR hs11.145. Although there are currently no genes annotated in this region, the conservation profile suggests that an ortholog of mir-129-2 resides within the TFR. All images are modified screen shots taken from the UCSC genome browser [31]

Maintenance of transposon-free regions throughout vertebrate evolution-2

<p><b>Copyright information:</b></p><p>Taken from "Maintenance of transposon-free regions throughout vertebrate evolution"</p><p>http://www.biomedcentral.com/1471-2164/8/470</p><p>BMC Genomics 2007;8():470-470.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241635.</p><p></p>ue describes the subset of TFRs that have an orthologous TFR in human larger than 10 kb, the area in red have an orthologous TFR in human larger than 5 kb. (B) Histogram of the GC content of human TFRs. Area indicated in blue describes the subset of TFRs that have an orthologous TFR in zebrafish larger than 10 kb, the area in red have an orthologous TFR in zebrafish larger than 5 kb. (C) Scatter plot of the GC content of orthologous pairs of zebrafish and human TFRs ≥ 10 kb in both species. Points in red indicate TFR pairs with a difference of absolute GC% greater than 20

Maintenance of transposon-free regions throughout vertebrate evolution-3

<p><b>Copyright information:</b></p><p>Taken from "Maintenance of transposon-free regions throughout vertebrate evolution"</p><p>http://www.biomedcentral.com/1471-2164/8/470</p><p>BMC Genomics 2007;8():470-470.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241635.</p><p></p>he UCSC genome browser. Horizontal red bars indicate TFRs and brown ticks indicate transposons. (A) Zebrafish (chr3:24,391-24,451 kb, March 2006) including the 25.9 kb TFR dr3.89. Human proteins mapped to the zebrafish genome by chained tBLASTn are indicated in blue. (B) Human (chr7:23,450-23,510 kb, March 2006) including the 13.7 kb TFR hs7.101. Human RefSeq genes are indicated in blue. (C) Mouse (chr6:49,114-49,174 kb, February 2006) including the 9.3 kb TFR mm6.309. Mouse RefSeq genes are indicated in blue. (D) Opossum (chr8:296,183-296,243 kb, January 2006) including the 16.4 kb TFR md8.376. Human RefSeq genes mapped to the opossum genome with BLAT are indicated in blue. (E) Frog (scaffold_56:3,208-3,268 kb, August 2005) including a 14 kb region that contains no transposons (red box). Human proteins mapped to the frog genome with tBLASTn are indicated in blue

Presence of Transcription between Adjacent cDNAs

<p>PCR was carried out with and without reverse transcription (RT[+] and RT[−], respectively) using midbrain total RNA and the corresponding primer pairs (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020037#pgen-0020037-st003" target="_blank">Table S3</a>). PCR using genomic DNA was also carried out as a control. A DNA ladder (Promega; <a href="http://www.promega.com" target="_blank">http://www.promega.com</a>) was used as a size marker. The amplified fragments were confirmed as the expected ones by analyzing digestion pattern using several restriction enzymes. The lower band, observed in the RT(+) lane of the amplified fragment C, seems to be nonspecific, because it was amplified using only the right primer and because it showed a digestion pattern with restriction enzymes quite different from that of the upper band and the band of the genomic DNA (unpublished data).</p

Northern Blot Analysis of ENOR Transcripts

<p>Mouse whole brain total RNA (10 μg/lane) was used for the analysis except for ENOR2 and ENOR61, where mouse thymus total RNA was used. DNA fragments without any predicted repeated sequences were PCR-amplified from cDNAs in ENORs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020037#pgen-0020037-st003" target="_blank">Table S3</a>), labeled with ³²P-dCTP (Amersham Biosciences), and then used as probes. RNA size was estimated with an RNA ladder (Invitrogen). ENORs are listed in increasing order based on the estimated length of each region.</p

Discovery Pipeline for ENORs

<p>FANTOM and public transcripts were clustered into 37,348 TUs by grouping any two or more transcripts that shared genomic coordinates. Then, the following procedures were applied. (1) Protein-coding TUs were excluded by removing any whose transcripts had an open reading frame of either 150 amino acids or more (RIKEN/MGC cDNAs) or one amino acid or more (non-RIKEN/MGC cDNAs). (2) TUs wholly encompassed within introns of protein-coding TUs were excluded to avoid possible pre-mRNA intronic transcripts. (3) Intron-containing TUs were excluded to select for unspliced transcripts. (4) TUs lacking adjunct adenine-rich regions or containing polyA signals were excluded to select for internally primed transcripts. (5) Remaining UNA TUs that mapped within 100 Kb of one another on the mouse genome (mm5) were clustered together, provided they did not overlap the genomic coordinates of a protein-coding TU/NCBI RefSeq/Ensembl gene model with a CDS of 150 amino acids or more or a noncoding TU with a polyA signal within 100 bp of the 3′ end and without an adjunct adenine-rich region. (6) Reliably expressed UNA TU clusters were selected by identifying those with at least ten supporting ESTs. (7) Selected UNA TU clusters were then manually screened and separated based upon evidence of possible internal transcription state sites (based upon CpG islands, CAGE tags, and EST clusters), resulting in the identification of 66 ENORs.</p

Snapshots of the GEV Showing Transcription

<div><p>(A) The <i>Air</i>/<i>Igf2r</i> locus (Chromosome 17: 12,091,531–12,258,195).</p> <p>(B) The <i>Xist</i>/<i>Tsix</i> locus (X chromosome: 94,835,096–94,888,536).</p> <p>(C) The dystrophin <i>(Dmd)</i> locus (X chromosome: 76,500,000–76,754,601).</p> <p>For the transcripts, cDNA sequences from the RIKEN and public databases are shown, and are colored in brown and purple depending upon their chromosomal strand of origin. Predicted genes from Ensembl, NCBI, and RefSeq databases are shown in gray. CpG islands as defined by the UCSC Genome Browser are shown. Blue circles indicate unspliced, noncoding RIKEN cDNAs with adjunct adenine-rich regions. Red circles indicate RIKEN imprinted cDNA candidates [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020037#pgen-0020037-b038" target="_blank">38</a>].</p></div

qRT-PCR Analysis

<p>Analysis of (A) <i>Air,</i> (B) ENOR28, and (C) ENOR31 loci. Above in each panel, screen shots of the GEV featuring the loci around <i>Air,</i> ENOR28, and ENOR31 are shown. The orange bars indicate the regions for <i>Air,</i> ENOR28, and ENOR31. cDNA sequences from the RIKEN and public databases are shown. Sequences mapped on the plus strand and minus strand are brown and purple, respectively. Predicted genes from Ensembl, NCBI, and RefSeq databases are shown in gray. For RIKEN imprinted transcripts, imprinted cDNA candidates identified previously [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020037#pgen-0020037-b038" target="_blank">38</a>] are shown. CpG islands as defined by the UCSC Genome Browser are shown. Positions of primer pairs are marked by small vertical arrows. Below in each panel, qRT-PCR results for midbrain, hippocampus, thalamus, striatum, and testis using the corresponding primer pairs are shown.</p

Localization of ENOR Transcripts

<p>qRT-PCR was carried out using total and cytoplasmic RNA from mouse whole brain and the corresponding primer pairs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020037#pgen-0020037-st003" target="_blank">Table S3</a>). ENORs are listed in increasing order based on the estimated length of each region. Apart from the results shown, we also examined the localization of other mRNAs <i>(β-actin</i> and <i>GAPDH)</i> and additional regions of <i>Rian</i> and other ENORs, and these results were consistent with the rest (unpublished data).</p