Limits...
Human glycolipid transfer protein (GLTP) genes: organization, transcriptional status and evolution.

Zou X, Chung T, Lin X, Malakhova ML, Pike HM, Brown RE - BMC Genomics (2008)

Bottom Line: In human cells, single-copy GLTP genes were found in chromosomes 11 and 12.Active transcription was found for 12q24.11 GLTP but 11p15.1 GLTP was transcriptionally silent.A solid foundation has been established for future identification of hereditary defects in human GLTP genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA. xzou@hi.umn.edu

ABSTRACT

Background: Glycolipid transfer protein is the prototypical and founding member of the new GLTP superfamily distinguished by a novel conformational fold and glycolipid binding motif. The present investigation provides the first insights into the organization, transcriptional status, phylogenetic/evolutionary relationships of GLTP genes.

Results: In human cells, single-copy GLTP genes were found in chromosomes 11 and 12. The gene at locus 11p15.1 exhibited several features of a potentially active retrogene, including a highly homologous (approximately 94%), full-length coding sequence containing all key amino acid residues involved in glycolipid liganding. To establish the transcriptional activity of each human GLTP gene, in silico EST evaluations, RT-PCR amplifications of GLTP transcript(s), and methylation analyses of regulator CpG islands were performed using various human cells. Active transcription was found for 12q24.11 GLTP but 11p15.1 GLTP was transcriptionally silent. Heterologous expression and purification of the GLTP paralogs showed glycolipid intermembrane transfer activity only for 12q24.11 GLTP. Phylogenetic/evolutionary analyses indicated that the 5-exon/4-intron organizational pattern and encoded sequence of 12q24.11 GLTP were highly conserved in therian mammals and other vertebrates. Orthologs of the intronless GLTP gene were observed in primates but not in rodentiates, carnivorates, cetartiodactylates, or didelphimorphiates, consistent with recent evolutionary development.

Conclusion: The results identify and characterize the gene responsible for GLTP expression in humans and provide the first evidence for the existence of a GLTP pseudogene, while demonstrating the rigorous approach needed to unequivocally distinguish transcriptionally-active retrogenes from silent pseudogenes. The results also rectify errors in the Ensembl database regarding the organizational structure of the actively transcribed GLTP gene in Pan troglodytes and establish the intronless GLTP as a primate-specific, processed pseudogene marker. A solid foundation has been established for future identification of hereditary defects in human GLTP genes.

Show MeSH

Related in: MedlinePlus

Methylation Status of Human Intronless GLTP Gene. Vertical bars show location of CpG dinucleotides. BstUI restriction sites, consisting of adjacent CpG dinucleotides, are shown by scissors. Individual clones are shown by horizontal rows of circles. Filled circles represent methylated CpG dinucleotides; unfilled circles represent nonmethylated CpG dinucleotides. Primers for RT-PCR (255 base product) are indicated by dotted arrows. The ATG start codon represents nucleotides +1, +2, and +3. B. & C. Combined bisulfite restriction analysis (COBRA) of intronless GLTP gene methylation status. B. Agarose gel electrophoresis patterns obtained before (Lane 1) and after (Lane 2) BstUI restriction digestion of bisulfite-treated PCR products for intronless GLTP gene (GLTPi) in human blood cell genomic DNA. C. COBRA analysis of intronless GLTP methylation status in different cell types. Lane 1 = pooled PCR products before BstUI restriction digestion; Lanes 2–9 shows action of BstUI restriction digestion. Lane 2 = glioma cells; Lane 3 = human skin fibroblasts; Lane 4 = human HBL100 breast cancer cells; Lane 5 = Gaucher cells; Lane 6 = human T47D breast cancer cells; Lane 7 = human IMR32 neuroblastoma cells; Lane 8 = human HTB126 breast cancer cells; Lane 9 = human Caov3 ovarian cancer cells. Because of the absence of BstUI restriction sites in amplified PCR products of the CpG island of the 5-exon/4-intron region, COBRA analysis could be used to verify the absence of methylation detected by direct sequencing and served only as a negative control for BstUI action shown Panel C. Std = molecular weight standards.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2262070&req=5

Figure 4: Methylation Status of Human Intronless GLTP Gene. Vertical bars show location of CpG dinucleotides. BstUI restriction sites, consisting of adjacent CpG dinucleotides, are shown by scissors. Individual clones are shown by horizontal rows of circles. Filled circles represent methylated CpG dinucleotides; unfilled circles represent nonmethylated CpG dinucleotides. Primers for RT-PCR (255 base product) are indicated by dotted arrows. The ATG start codon represents nucleotides +1, +2, and +3. B. & C. Combined bisulfite restriction analysis (COBRA) of intronless GLTP gene methylation status. B. Agarose gel electrophoresis patterns obtained before (Lane 1) and after (Lane 2) BstUI restriction digestion of bisulfite-treated PCR products for intronless GLTP gene (GLTPi) in human blood cell genomic DNA. C. COBRA analysis of intronless GLTP methylation status in different cell types. Lane 1 = pooled PCR products before BstUI restriction digestion; Lanes 2–9 shows action of BstUI restriction digestion. Lane 2 = glioma cells; Lane 3 = human skin fibroblasts; Lane 4 = human HBL100 breast cancer cells; Lane 5 = Gaucher cells; Lane 6 = human T47D breast cancer cells; Lane 7 = human IMR32 neuroblastoma cells; Lane 8 = human HTB126 breast cancer cells; Lane 9 = human Caov3 ovarian cancer cells. Because of the absence of BstUI restriction sites in amplified PCR products of the CpG island of the 5-exon/4-intron region, COBRA analysis could be used to verify the absence of methylation detected by direct sequencing and served only as a negative control for BstUI action shown Panel C. Std = molecular weight standards.

Mentions: CpG dinucleotides in the 5' end regions of transcriptionally active chromosomal alleles generally are unmethylated. Conversely, methylation of CpG islands is a known indicator of transcriptional silencing [31-33]. In human GLTPi, a 255 base sequence including the 5' UTR (-128 bases) and initial portion (20%) of the potential ORF (see additional file 1, Figure S2), met the criteria of a CpG island [31-34]. To assess methylation status, human blood genomic DNA was treated with bisulfite to convert unmethylated cytosines to uracils while leaving any methylated cytosines unaltered. Primers containing no CpG dinucleotides were used for PCR to avoid bias between DNA templates based on their original methylation status [34,35]. The results obtained by sequencing, after clonal amplification using pGEM-T, are shown in Figure 4. The 23 CpG dinucleotides, localized in the CpG island of GLTPi, were highly methylated in all clones (Figure 4A, filled circles), consistent with transcriptional silencing.


Human glycolipid transfer protein (GLTP) genes: organization, transcriptional status and evolution.

Zou X, Chung T, Lin X, Malakhova ML, Pike HM, Brown RE - BMC Genomics (2008)

Methylation Status of Human Intronless GLTP Gene. Vertical bars show location of CpG dinucleotides. BstUI restriction sites, consisting of adjacent CpG dinucleotides, are shown by scissors. Individual clones are shown by horizontal rows of circles. Filled circles represent methylated CpG dinucleotides; unfilled circles represent nonmethylated CpG dinucleotides. Primers for RT-PCR (255 base product) are indicated by dotted arrows. The ATG start codon represents nucleotides +1, +2, and +3. B. & C. Combined bisulfite restriction analysis (COBRA) of intronless GLTP gene methylation status. B. Agarose gel electrophoresis patterns obtained before (Lane 1) and after (Lane 2) BstUI restriction digestion of bisulfite-treated PCR products for intronless GLTP gene (GLTPi) in human blood cell genomic DNA. C. COBRA analysis of intronless GLTP methylation status in different cell types. Lane 1 = pooled PCR products before BstUI restriction digestion; Lanes 2–9 shows action of BstUI restriction digestion. Lane 2 = glioma cells; Lane 3 = human skin fibroblasts; Lane 4 = human HBL100 breast cancer cells; Lane 5 = Gaucher cells; Lane 6 = human T47D breast cancer cells; Lane 7 = human IMR32 neuroblastoma cells; Lane 8 = human HTB126 breast cancer cells; Lane 9 = human Caov3 ovarian cancer cells. Because of the absence of BstUI restriction sites in amplified PCR products of the CpG island of the 5-exon/4-intron region, COBRA analysis could be used to verify the absence of methylation detected by direct sequencing and served only as a negative control for BstUI action shown Panel C. Std = molecular weight standards.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2262070&req=5

Figure 4: Methylation Status of Human Intronless GLTP Gene. Vertical bars show location of CpG dinucleotides. BstUI restriction sites, consisting of adjacent CpG dinucleotides, are shown by scissors. Individual clones are shown by horizontal rows of circles. Filled circles represent methylated CpG dinucleotides; unfilled circles represent nonmethylated CpG dinucleotides. Primers for RT-PCR (255 base product) are indicated by dotted arrows. The ATG start codon represents nucleotides +1, +2, and +3. B. & C. Combined bisulfite restriction analysis (COBRA) of intronless GLTP gene methylation status. B. Agarose gel electrophoresis patterns obtained before (Lane 1) and after (Lane 2) BstUI restriction digestion of bisulfite-treated PCR products for intronless GLTP gene (GLTPi) in human blood cell genomic DNA. C. COBRA analysis of intronless GLTP methylation status in different cell types. Lane 1 = pooled PCR products before BstUI restriction digestion; Lanes 2–9 shows action of BstUI restriction digestion. Lane 2 = glioma cells; Lane 3 = human skin fibroblasts; Lane 4 = human HBL100 breast cancer cells; Lane 5 = Gaucher cells; Lane 6 = human T47D breast cancer cells; Lane 7 = human IMR32 neuroblastoma cells; Lane 8 = human HTB126 breast cancer cells; Lane 9 = human Caov3 ovarian cancer cells. Because of the absence of BstUI restriction sites in amplified PCR products of the CpG island of the 5-exon/4-intron region, COBRA analysis could be used to verify the absence of methylation detected by direct sequencing and served only as a negative control for BstUI action shown Panel C. Std = molecular weight standards.
Mentions: CpG dinucleotides in the 5' end regions of transcriptionally active chromosomal alleles generally are unmethylated. Conversely, methylation of CpG islands is a known indicator of transcriptional silencing [31-33]. In human GLTPi, a 255 base sequence including the 5' UTR (-128 bases) and initial portion (20%) of the potential ORF (see additional file 1, Figure S2), met the criteria of a CpG island [31-34]. To assess methylation status, human blood genomic DNA was treated with bisulfite to convert unmethylated cytosines to uracils while leaving any methylated cytosines unaltered. Primers containing no CpG dinucleotides were used for PCR to avoid bias between DNA templates based on their original methylation status [34,35]. The results obtained by sequencing, after clonal amplification using pGEM-T, are shown in Figure 4. The 23 CpG dinucleotides, localized in the CpG island of GLTPi, were highly methylated in all clones (Figure 4A, filled circles), consistent with transcriptional silencing.

Bottom Line: In human cells, single-copy GLTP genes were found in chromosomes 11 and 12.Active transcription was found for 12q24.11 GLTP but 11p15.1 GLTP was transcriptionally silent.A solid foundation has been established for future identification of hereditary defects in human GLTP genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA. xzou@hi.umn.edu

ABSTRACT

Background: Glycolipid transfer protein is the prototypical and founding member of the new GLTP superfamily distinguished by a novel conformational fold and glycolipid binding motif. The present investigation provides the first insights into the organization, transcriptional status, phylogenetic/evolutionary relationships of GLTP genes.

Results: In human cells, single-copy GLTP genes were found in chromosomes 11 and 12. The gene at locus 11p15.1 exhibited several features of a potentially active retrogene, including a highly homologous (approximately 94%), full-length coding sequence containing all key amino acid residues involved in glycolipid liganding. To establish the transcriptional activity of each human GLTP gene, in silico EST evaluations, RT-PCR amplifications of GLTP transcript(s), and methylation analyses of regulator CpG islands were performed using various human cells. Active transcription was found for 12q24.11 GLTP but 11p15.1 GLTP was transcriptionally silent. Heterologous expression and purification of the GLTP paralogs showed glycolipid intermembrane transfer activity only for 12q24.11 GLTP. Phylogenetic/evolutionary analyses indicated that the 5-exon/4-intron organizational pattern and encoded sequence of 12q24.11 GLTP were highly conserved in therian mammals and other vertebrates. Orthologs of the intronless GLTP gene were observed in primates but not in rodentiates, carnivorates, cetartiodactylates, or didelphimorphiates, consistent with recent evolutionary development.

Conclusion: The results identify and characterize the gene responsible for GLTP expression in humans and provide the first evidence for the existence of a GLTP pseudogene, while demonstrating the rigorous approach needed to unequivocally distinguish transcriptionally-active retrogenes from silent pseudogenes. The results also rectify errors in the Ensembl database regarding the organizational structure of the actively transcribed GLTP gene in Pan troglodytes and establish the intronless GLTP as a primate-specific, processed pseudogene marker. A solid foundation has been established for future identification of hereditary defects in human GLTP genes.

Show MeSH
Related in: MedlinePlus