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Identification of three prominin homologs and characterization of their messenger RNA expression in Xenopus laevis tissues.

Han Z, Papermaster DS - Mol. Vis. (2011)

Bottom Line: Two of these homologs are likely to be the X. laevis orthologs of mammalian prominin-1 and 2, respectively, while the third homolog is likely to be the X. laevis ortholog of prominin-3, which was only found in nonmammalian vertebrates and the platypus.Similar to mammalian prominin-1, we found that exons 26b, 27, and 28a of the X. laevis prominin-1 gene are alternatively spliced, and that the splice isoforms of mRNA show tissue-specific expression profiles.Our results suggest that the mRNAs of prominin homologs are expressed in many tissues of X. laevis, but differ in their expression levels and mRNA splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT

Purpose: Prominin is a family of pentaspan transmembrane glycoproteins. They are expressed in various types of cells, including many stem/progenitor cells. Prominin-1 plays an important role in generating and maintaining the structure of the photoreceptors. In this study, we identified three prominin homologs in Xenopus laevis, a model animal widely used in vision research, and characterized their messenger RNA (mRNA) expression in selected tissues of this frog.

Methods: Reverse-transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) were used to isolate cDNAs of prominin homologs. Semiquantitative RT-PCR was used to measure the relative expression levels of mRNAs of the three prominin homologs in four X. laevis tissues, specifically those of the retina, brain, testis, and kidney. Sequences of prominin homologs were analyzed with bioinformatic software.

Results: We isolated cDNAs of three prominin homologs from X. laevis tissues and compared their sequences with previously described prominin-1, 2, and 3 sequences from other species using phylogenetic analysis. Two of these homologs are likely to be the X. laevis orthologs of mammalian prominin-1 and 2, respectively, while the third homolog is likely to be the X. laevis ortholog of prominin-3, which was only found in nonmammalian vertebrates and the platypus. We identified alternatively spliced exons in mRNAs of all three prominin homologs. Similar to mammalian prominin-1, we found that exons 26b, 27, and 28a of the X. laevis prominin-1 gene are alternatively spliced, and that the splice isoforms of mRNA show tissue-specific expression profiles. We found that prominin-1 was the most abundant homolog expressed in the retina, brain, and testis, while prominin-3 was the most abundant homolog in the kidney. The expression level of prominin-2 was the lowest of the three prominin homologs in all four examined tissues of this frog.

Conclusions: Our results suggest that the mRNAs of prominin homologs are expressed in many tissues of X. laevis, but differ in their expression levels and mRNA splicing. Prominin-1 is the most abundant of the three prominin homologs expressed in the frog retina.

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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.
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f3: 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.

Mentions: We performed semiquantitative RT–PCR with mRNAs isolated from the X. laevis retina, brain, testis, and kidney to analyze the expression profiles of xlProminin-1, 2, and 3, because these four tissues are mRNA rich and differ significantly in their function and metabolism. The results are presented in Figure 3. Precautions were taken to ensure specificity of the PCR reaction and accuracy of the analysis, including ensuring that measurements were consistent when using two completely different primer pairs for each prominin homolog. We also used relatively large PCR products (>1.4 kb), such that small nonspecific products could easily be recognized and disregarded. For the primer design, we carefully chose primers that only complement the desired homolog. We found that mRNAs of three xlProminin homologs were differentially expressed in these tissues: xlProminin-1 was the predominant form found in the retina, brain, and testis, while x1Promnin-3 was the predominant form found in the kidney. The expression level of xlProminin-2 was the lowest of the three prominin homologs in all examined tissues in this frog.


Identification of three prominin homologs and characterization of their messenger RNA expression in Xenopus laevis tissues.

Han Z, Papermaster DS - Mol. Vis. (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108902&req=5

f3: 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.
Mentions: We performed semiquantitative RT–PCR with mRNAs isolated from the X. laevis retina, brain, testis, and kidney to analyze the expression profiles of xlProminin-1, 2, and 3, because these four tissues are mRNA rich and differ significantly in their function and metabolism. The results are presented in Figure 3. Precautions were taken to ensure specificity of the PCR reaction and accuracy of the analysis, including ensuring that measurements were consistent when using two completely different primer pairs for each prominin homolog. We also used relatively large PCR products (>1.4 kb), such that small nonspecific products could easily be recognized and disregarded. For the primer design, we carefully chose primers that only complement the desired homolog. We found that mRNAs of three xlProminin homologs were differentially expressed in these tissues: xlProminin-1 was the predominant form found in the retina, brain, and testis, while x1Promnin-3 was the predominant form found in the kidney. The expression level of xlProminin-2 was the lowest of the three prominin homologs in all examined tissues in this frog.

Bottom Line: Two of these homologs are likely to be the X. laevis orthologs of mammalian prominin-1 and 2, respectively, while the third homolog is likely to be the X. laevis ortholog of prominin-3, which was only found in nonmammalian vertebrates and the platypus.Similar to mammalian prominin-1, we found that exons 26b, 27, and 28a of the X. laevis prominin-1 gene are alternatively spliced, and that the splice isoforms of mRNA show tissue-specific expression profiles.Our results suggest that the mRNAs of prominin homologs are expressed in many tissues of X. laevis, but differ in their expression levels and mRNA splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT

Purpose: Prominin is a family of pentaspan transmembrane glycoproteins. They are expressed in various types of cells, including many stem/progenitor cells. Prominin-1 plays an important role in generating and maintaining the structure of the photoreceptors. In this study, we identified three prominin homologs in Xenopus laevis, a model animal widely used in vision research, and characterized their messenger RNA (mRNA) expression in selected tissues of this frog.

Methods: Reverse-transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) were used to isolate cDNAs of prominin homologs. Semiquantitative RT-PCR was used to measure the relative expression levels of mRNAs of the three prominin homologs in four X. laevis tissues, specifically those of the retina, brain, testis, and kidney. Sequences of prominin homologs were analyzed with bioinformatic software.

Results: We isolated cDNAs of three prominin homologs from X. laevis tissues and compared their sequences with previously described prominin-1, 2, and 3 sequences from other species using phylogenetic analysis. Two of these homologs are likely to be the X. laevis orthologs of mammalian prominin-1 and 2, respectively, while the third homolog is likely to be the X. laevis ortholog of prominin-3, which was only found in nonmammalian vertebrates and the platypus. We identified alternatively spliced exons in mRNAs of all three prominin homologs. Similar to mammalian prominin-1, we found that exons 26b, 27, and 28a of the X. laevis prominin-1 gene are alternatively spliced, and that the splice isoforms of mRNA show tissue-specific expression profiles. We found that prominin-1 was the most abundant homolog expressed in the retina, brain, and testis, while prominin-3 was the most abundant homolog in the kidney. The expression level of prominin-2 was the lowest of the three prominin homologs in all four examined tissues of this frog.

Conclusions: Our results suggest that the mRNAs of prominin homologs are expressed in many tissues of X. laevis, but differ in their expression levels and mRNA splicing. Prominin-1 is the most abundant of the three prominin homologs expressed in the frog retina.

Show MeSH
Related in: MedlinePlus