Han Z , Papermaster DS .

R01EY006891 NEI NIH HHS , R01 EY006891 NEI NIH HHS

	Figure 1. Alignment of the predicted protein sequences of three X. laevis prominin homologs, showing characteristic features of prominins, including the pentaspan transmembrane topology, a conserved cysteine rich domain, conserved leucine residues and N-glycosylation sites. We designated the three X. laevis prominin homologs as xlProminin-1, 2, and 3, respectively. Identical and conserved residues are indicated by differentially shaded boxes. Predicted transmembrane domains are marked with M#. The predicted signal peptides of xlProminin-1 and 2 are boxed. No signal peptide was predicted for xlProminin-3. Positions of alternatively spliced exons are indicated by arrows and the sequences of the alternatively spliced exons are given. Note that the N- and C-termini of the three X. laevis prominin homologs are less similar than other regions. Cysteine residues at the junction of M1 and the first intracellular domain (I1) are marked with yellow. Conserved leucine residues of the three xlProminin homologs are marked with red. Conserved glycosylation sites are marked with green.
	Figure 2. Phylogenic analysis of prominin homologs from several species. This phylogenetic tree was constructed to show the existence of multiple prominin homologs in metazoan animals from fly to human and their evolutionary relationship. Three X. laevis prominin homologs, designated as xlProminin-1, 2, and 3, are placed in branches of the consensus phylogenetic tree with prominin-1, 2, and 3 from other species. The numbers at each node are the percentage bootstrapping values of 100 replicates. The evolutionary distance of any two sequences is represented by the length of the branches. Sequences used in the phylogenetic analysis are excerpted from: H. sapien Prom-1 (NP_006008), P. troglodytes Prom-1 (XP_517115), M. mulatta Prom-1 (NP_001070888), R. norvegicus Prom-1 (NP_068519), M. musculus Prom-1 (NP_032961), C. familiaris Prom-1 (XP_850831), B. taurus Prom-1 (HQ159409), E. caballus Prom-1 (XM_001498729), D. rerio Prom-1a (NP_001108615), T. rubripes Prom-1a (HQ159405), D. rerio Prom-1b (NP_932337), T. rubripes Prom-1b (HQ159406), O. anatinus Prom-1 (HQ159408), G. gallus Prom-1 (XP_001232165), X. laevis Prom-1 (XM_001163920), X. tropicalis Prom-1 (HQ159400), H. sapien Prom-2 (NP_653308), P. troglodytes Prom-2 (XP_001143498), C. familiaris Prom-2 (XP_854455), B. taurus Prom-2 (XP_599188), E. caballus Prom-2 (XP_001494215), R. norvegicus Prom-2 (AAN63818), M. musculus Prom-2 (NP_620089), X. laevis Prom-2 (NP_001163922), X. tropicalis Prom-2 (HQ159402), X. laevis Prom-3 (NP_001163921), X. tropicalis Prom-3 (HQ159403), D. rerio Prom-3 (XP_684527), T. rubripes Prom-3 (HQ159407), D. melanogaster Prominin (ABH07113), D. melanogaster Prominin-like protein AF127935 (NP_647770), C. elegans protein F08B12 (NP_509907), C. elegans protein M28.8 (NP_496294), C. elegans protein M28.9 (NP_496296).
	Figure 3. Semiquantitative reverse-transcription PCR analysis of xlProminin-1, 2, and 3 performed on the retina, brain, testis, and kidney. A: Aliquots (20 μl) of the PCR reaction were taken at the end of the 27th, 31st, 35th, and 39th cycles, separated and visualized on a 1% agarose gel. Two independent primer pairs (see Table 2) were used to amplify each xlProminin homolog in each tissue to ensure the specificity and efficiency of the PCR reaction. A fragment of Xenopus laevis β-actin cDNA was also amplified from equivalent starting material of total RNA to serve as a control. The Invitrogen 1KB PLUS DNA (Cat. No. 10787–018) ladder was used for size standards. B: Integrated signals in areas occupied by individual DNA bands were quantified and plotted to compare the relative expression level of xlProminin-1, 2, and 3 in four X. laevis tissues: the retina, brain, testis, and kidney. xlProminin-1, 2 and 3 are expressed in all four tissues. Among the three prominin homologs, prominin-1 is predominantly expressed in the retina, brain, and testis, while prominin-3 is predominantly expressed in the kidney. The expression level of prominin-2 is the lowest of the three prominin homologs in all examined tissues, and is barely detectable in the kidney.
	Figure 4. Exon organization of xlProminin-1, 2, and 3. Homologous exons of xlProminin-1, 2, and 3 are aligned. Genes of all three xlProminins are alternatively spliced and their exon organization is evolutionarily conserved. Constitutive exons are marked in black. Alternatively spliced exons are marked orange. Spliced forms identified in cDNA clones are indicated by joining lines. Translation start sites (ATG) are marked with green lines and the positions of translational stop codons are marked with white lines. Note the complex splicing of exons 26b, 27, and 28a, which generates several distinct isoforms of xlProminin-1. Positions of primers used in reverse-transcription (RT)-PCR for semiquantitative analysis of mRNA expression and for alternative splicing are indicated on the diagram.
	Figure 5. Analysis of the xlProminin-1 alternatively spliced isoforms in four tissues: the retina, brain, testis, and kidney. We found that profiles of the xlProminin-1 alternative splicing are different in these tissues. A: Products of reverse-transcription PCR (RT–PCR) from a region of extensive alternative splicing on the xlProminin-1 gene (exon 20 to 28) were separated on a 1% agarose gel and visualized with ethidium bromide staining. Five discrete PCR products were excised from the gel, cloned, and sequenced. Their exon compositions were determined and each product was designated with a Greek letter (α, β, γ, δ, and ε) for identification when used in the quantitative analysis shown in panel B. The predominant isoforms of xlProminin-1 expressed in the retina lack the alternatively spliced exons 26b, 27, and 28a, whereas the predominant isoform of xlProminin-1 expressed in the kidney retains these exons. The predominant isoforms of xlProminin-1 expressed in the brain and kidney retain exon 28a. Exon 27 was retained only when exon 26b was retained as well. B: Quantification of the xlProminin-1 alternatively spliced isoforms in four tissues: the retina, brain, testis, and kidney. The resolved RT–PCR products were linearly scanned and the optical densities were plotted. Green arrows indicate peaks that represent isoforms resulting from alternative splicing. The major isoform of xlProminin-1 expressed in the retina, ε, lacks the alternatively spliced exons 26b, 27, and 28a, whereas the major form of xlProminin-1 expressed in the kidney, α, retains all possible exons. The majority of xlProminin-1 expressed in the brain and kidney retains exon 28a.
	Figure 6. Comparison of exon organization of prominin-1 gene from X. laevis, mouse, and human. It appears that the gene structure of prominin-1 is evolutionarily conserved in these animals. Alternative splicing of the prominin-1 gene is seen in all three animals, however, with considerable differences in the choices of alternative exons and the splicing patterns. Homologous exons of xlProminin-1 and mouse and human prominin-1 are aligned for comparison of their exon organization. Constitutive exons are marked in black. Alternatively spliced exons are marked in orange. Spliced forms identified in cDNA clones are indicated by jointed lines. Note that homologous exons are assigned with the same number to maintain consistency with preexisting nomenclature [2,31]. Exons 26b and 27 are conserved and alternatively spliced in all three species. Alternative exons 3a, 8a, 11a, and 28a of xlProminin-1 are not found in mouse or human prominin-1. Splicing of alternative exons 4a results from using an alternative 3′ splice site in exon 4, and is only observed in mouse prominin-1 [31]. Alternative exon 19 of xlProminin-1 is alternatively spliced in mouse prominin-1, but no evidence has been found that this exon is alternatively spliced in human prominin-1. Alternative exon 25 of human prominin-1 is not found in prominin-1 from the mouse or X. laevis. The alternative exon 26a of mouse prominin-1 and the alternative exons 25 and 26a of human prominin-1 are not found in xlProminin-1. Exons 3, 9, and 28 of mouse prominin-1 are alternative, but appear to be constitutive in X. laevis. The region encompassing exons 24 to 28 of xlProminin-1 and homologous sequences of the prominin-1 gene from the mouse and human are regions of extensive alternative splicing. Diagrams of translated proteins are aligned with their coding sequences. Divisions of proteins by predicted transmembrane domains are marked.

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