Figure 1.
Gene structure of FLJ33706, a human-specific de novo protein-coding gene.
Data for the tracks ‘Spliced Human EST’ and ‘Human mRNA’ was extracted and assembled from UCSC Genome Browser. We re-sequenced all available mRNAs and spliced ESTs, shown in the track ‘Re-sequenced ESTs/mRNAs’. On the basis of these data, we inferred gene structure for this novel gene, with six exons marked as ‘1∼6’ in the track ‘FLJ33706 Gene Structure’. The exons partially derived from re-sequenced data were highlighted in green. An ORF with two short coding exons located at exon 3 and exon 4 was identified to encode a 194-amino-acid-long peptide (track ‘Open Reading Frame (ORF)’). Newly inserted transposable elements, especially Alu sequences, contributed substantially to the formation of the first coding exon and six standard splicing junctions on the branch leading to human and chimpanzee, marked as ‘a∼f’ in the track ‘FLJ33706 Gene Structure’. All repeat elements in this region were shown in track ‘RepeatMasker’, extracted from UCSC Genome Browser. Coding exons in tracks ‘Spliced Human EST’, ‘Human mRNA’ and ‘FLJ33706 Gene Structure’ were represented by higher vertical bars, while UTR regions and intronic regions were represented by lower vertical bars. Size scales were added in the figure to give benchmarks for gene sizes. Tracks with different size scales were separated by horizon lines.
Figure 2.
Syntenic alignments of five flanking splicing junctions of FLJ33706.
(Top) The syntenic alignments of five flanking splicing junctions of FLJ33706 among 13 species were shown, in which intron regions were highlighted in blue boxes and splicing sites were highlighted in red. Lineage-specific insertions, unalignable bases in the gap region and uncertain regions were were marked as ‘-’, ‘ = ’ and ‘N’, respectively. (Bottom) The splicing sites from 8 species were shown in the context of the phylogeny. Red ‘Y’ represents presence of the splicing signal and blue ‘N’ represents absence. Exons and introns are not drawn to scale.
Figure 3.
Alignment of human FLJ33706 ORF with orthologous genomic sequences in four other primates.
For each position in human FLJ33706 ORF, the corresponding orthologous genomic sequences in chimpanzee, gorilla, orangutan and rhesus monkey were aligned to human reference to identify the types of variations. Only amino acid sites with at least one variation among “Human-Human” (SNP), “Human-Chimpanzee”, “Human-Gorilla”, “Human-Orangutan” or “Human-Rhesus” were shown. Identical sites were shown as black dots while divergent sites were shown in red (non-synonymous mutations), green (synonymous mutations) and blue (SNP). Two human-specific mutations that escaped stop codons were highlighted by black frames. Amino acids with non-synonymous variations were highlighted in red while synonymous variations in blue. All sequencing data in this study were traced and manually checked to ensure reliability.
Figure 4.
FLJ33706 mRNA expression in peripheral tissues and brain regions.
FLJ33706 mRNA levels were measured in eight peripheral tissues and eight brain regions using TaqMan-based Real-Time PCR system. Relative quantity was calculated using expression means of human leukocyte as references (Fold = 1.0). FLJ33706 had relatively higher expression levels in brain regions (highlighted in red) than in peripheral tissues (shown in white). The expression levels of human BDNF1 and BDNF4 (shown in blue bars) in cortex (BDNF1_CTX, BDNF4_CTX) were also compared with those of FLJ33706 using also leukocyte FLJ33706 expression as a reference.
Table 1.
population genetics statistics of FLJ33706.
Figure 5.
Western-blot assays to determine the protein expression levels of FLJ33706.
(A) A specific band with molecular mass of about 22 kDa in SDS-PAGE, which was consistent with the predicted molecular weight of FLJ33706 putative proteins, was detected in the Western-blot assay. The band could not be detected in pre-immune control and peptide competition control. Pre: pre-immune reaction assay; Ab: FLJ33706 antibody assay; Pep: peptide competition assay. (B) E. coli samples before and after the transformation of FLJ33706 recombination plasmids were assayed by Western blot using (a) His-tag specific antibody and (b) anti-FLJ33706. FLJ33706 expression in human cortex was shown in (c) as the control. Before: E. coli samples before the plasmid transformation; After: E. coli samples after the plasmid transformation. (C) The specific band can be detected in all human brain regions, but not in mouse brain regions used as controls. H-CTX, M-CTX: human/mouse cortex; H-MID, M-MID: human/mouse midbrain; H-CER, M-CER: human/mouse cerebellum; (D) FLJ33706 expression can be detected in different human individuals. 384, 994, 275, 995, 705, 710, 1277: individual IDs. 22KD: theoretical molecular weight of FLJ33706 protein; 42KD: molecular weight of beta-actin protein as endogenous control. In A and C, antibodies for FLJ33706 and endogenous control were mixed in Western assays.
Figure 6.
Immunohistochemistry studies of FLJ33706 protein in human brain.
Results from confocal immunofluorescence imaging to visualize FLJ33706 protein (red) in normal human cortex were shown. Neurons were marked with beta-tubulin-III (green). The nucleus was also stained with DAPI (blue). The optimal dilution of FLJ33706 antibody was optimised based on the detection of cytoplasmic signal in brain cells. The three rows showed results from three independent experiments.