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Genomic structure of the gene for mouse germ-cell nuclear factor (GCNF). II. Comparison with the genomic structure of the human GCNF gene.

Süsens U, Borgmeyer U - Genome Biol. (2001)

Bottom Line: The comparison reveals that the shorter human protein results from skipping the 45 base-pair third exon.Three different human isoforms - GCNF-1, GCNF-2a and GCNF-2b - are generated by differential usage of alternative splice acceptor sites of the fourth and the seventh exon.All human GCNF cDNAs identified so far are, however, derived from mRNAs generated by splicing the fourth to the second exon.

View Article: PubMed Central - HTML - PubMed

Affiliation: Zentrum für Molekulare Neurobiologie, Universität Hamburg, Martinistrasse 52, D-0246 Hamburg, Germany.

ABSTRACT

Background: Germ-cell nuclear factor (GCNF, NR6AI) is an orphan nuclear receptor. Its expression pattern suggests it functions during embryogenesis, in the placenta and in germ-cell development. Mouse GCNF cDNA codes for a protein of 495 amino acids, whereas the four reported human cDNA variants code for proteins of 454 to 480 amino acids. Apart from this size difference, there is sequence conservation of up to 98.7%. To elucidate the genomic structure that gives rise to the different human GCNF mRNAs, the sequence information of the human GCNF locus is compared to the previously reported structure of the mouse locus.

Results: The genomic structures of the mouse and human GCNF genes are highly conserved. The comparison reveals that the shorter human protein results from skipping the 45 base-pair third exon. Three different human isoforms - GCNF-1, GCNF-2a and GCNF-2b - are generated by differential usage of alternative splice acceptor sites of the fourth and the seventh exon.

Conclusion: By homology with the mouse gene, 11 GCNF coding exons can be defined on human chromosome 9. All human GCNF cDNAs identified so far are, however, derived from mRNAs generated by splicing the fourth to the second exon. Although the genomic sequence is highly conserved, the analysis suggests that alternative splicing generates a higher complexity of human GCNF isoforms compared with the situation in the mouse.

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Comparison of the exons of the mouse and human GCNF genes. The upper line of nucleotide sequence shows the murine protein-coding exons and their flanking sequences (AF254815-AF254821). Protein-coding nucleotides are underlined. The lower line of nucleotide sequence shows the corresponding human sequence. Identical nucleotides are highlighted by bold letters. The AG/GT splice signals are shown in italics. mGCNF indicates the deduced mouse protein sequence; hGCNF indicates the deduced human protein sequences; GCNF indicates identical protein sequences. (a) The second exon and its flanking regions. Forty-one out of 42 nucleotides are identical and the flanking splicing signals are conserved. (b) The third exon and its flanking regions. A homologous sequence coding for identical amino acids was found in the human genomic sequence. No human isoform containing this sequence has been reported. The splice donor site shows the typical pyrimidine-rich sequence followed by the sequence 5'-NCAG in both sequences, but the comparison reveals several base transitions. (c) A single splice donor site in the fourth exon coding for the DNA-binding domain is used in all mouse-derived cDNAs described so far. For the human isoform GCNF-2, the corresponding splice site is used, giving rise to a protein containing the sequence ISVSDD instead of the VSVPDD in mouse. Usage of an alternative splice site located 12 bp further downstream gives rise to the shorter isoform GCNF-1. An asparagine (N) is underlined because one of the human cDNA clones codes for a lysine in this position (U64876/NM_001489). (d) Sequences of the fifth exon coding for the carboxy-terminal extension of the DNA-binding domain are highly conserved. (e) The DNA sequence of the sixth exon is highly conserved. An arginine in hGCNF-2b instead of serine results from alternative splice donor sites of the seventh exon. (f) The comparison of the seventh exon reveals three positions where the mouse and the human isoforms diverge. Isoform hGCNF-2b is generated by using a splice donor site located three nucleotides further downstream. The exons coding for the putative α-helices 3 to 6 (g), 7 and 8 (h), 9 and 10 (i), 11 and 12 (j) in the ligand-binding domain are highly conserved. The comparison of the last coding exon in (j) was extended up to the end of the human cDNA sequence of S88309.
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Figure 3: Comparison of the exons of the mouse and human GCNF genes. The upper line of nucleotide sequence shows the murine protein-coding exons and their flanking sequences (AF254815-AF254821). Protein-coding nucleotides are underlined. The lower line of nucleotide sequence shows the corresponding human sequence. Identical nucleotides are highlighted by bold letters. The AG/GT splice signals are shown in italics. mGCNF indicates the deduced mouse protein sequence; hGCNF indicates the deduced human protein sequences; GCNF indicates identical protein sequences. (a) The second exon and its flanking regions. Forty-one out of 42 nucleotides are identical and the flanking splicing signals are conserved. (b) The third exon and its flanking regions. A homologous sequence coding for identical amino acids was found in the human genomic sequence. No human isoform containing this sequence has been reported. The splice donor site shows the typical pyrimidine-rich sequence followed by the sequence 5'-NCAG in both sequences, but the comparison reveals several base transitions. (c) A single splice donor site in the fourth exon coding for the DNA-binding domain is used in all mouse-derived cDNAs described so far. For the human isoform GCNF-2, the corresponding splice site is used, giving rise to a protein containing the sequence ISVSDD instead of the VSVPDD in mouse. Usage of an alternative splice site located 12 bp further downstream gives rise to the shorter isoform GCNF-1. An asparagine (N) is underlined because one of the human cDNA clones codes for a lysine in this position (U64876/NM_001489). (d) Sequences of the fifth exon coding for the carboxy-terminal extension of the DNA-binding domain are highly conserved. (e) The DNA sequence of the sixth exon is highly conserved. An arginine in hGCNF-2b instead of serine results from alternative splice donor sites of the seventh exon. (f) The comparison of the seventh exon reveals three positions where the mouse and the human isoforms diverge. Isoform hGCNF-2b is generated by using a splice donor site located three nucleotides further downstream. The exons coding for the putative α-helices 3 to 6 (g), 7 and 8 (h), 9 and 10 (i), 11 and 12 (j) in the ligand-binding domain are highly conserved. The comparison of the last coding exon in (j) was extended up to the end of the human cDNA sequence of S88309.

Mentions: The comparison of the genomic sequences of exons 2 to 11 was extended by 100 bp of intronic sequence in both directions (Figure 3). During the preparation of this manuscript all sequence information was made available by the International Human Genome Project collaborators at the NCBI database and included in the contig NT_008491. Sequences of the 5'-untranslated region and of exon 7 obtained with a genome walking approach did not diverge from the sequence at the NCBI.


Genomic structure of the gene for mouse germ-cell nuclear factor (GCNF). II. Comparison with the genomic structure of the human GCNF gene.

Süsens U, Borgmeyer U - Genome Biol. (2001)

Comparison of the exons of the mouse and human GCNF genes. The upper line of nucleotide sequence shows the murine protein-coding exons and their flanking sequences (AF254815-AF254821). Protein-coding nucleotides are underlined. The lower line of nucleotide sequence shows the corresponding human sequence. Identical nucleotides are highlighted by bold letters. The AG/GT splice signals are shown in italics. mGCNF indicates the deduced mouse protein sequence; hGCNF indicates the deduced human protein sequences; GCNF indicates identical protein sequences. (a) The second exon and its flanking regions. Forty-one out of 42 nucleotides are identical and the flanking splicing signals are conserved. (b) The third exon and its flanking regions. A homologous sequence coding for identical amino acids was found in the human genomic sequence. No human isoform containing this sequence has been reported. The splice donor site shows the typical pyrimidine-rich sequence followed by the sequence 5'-NCAG in both sequences, but the comparison reveals several base transitions. (c) A single splice donor site in the fourth exon coding for the DNA-binding domain is used in all mouse-derived cDNAs described so far. For the human isoform GCNF-2, the corresponding splice site is used, giving rise to a protein containing the sequence ISVSDD instead of the VSVPDD in mouse. Usage of an alternative splice site located 12 bp further downstream gives rise to the shorter isoform GCNF-1. An asparagine (N) is underlined because one of the human cDNA clones codes for a lysine in this position (U64876/NM_001489). (d) Sequences of the fifth exon coding for the carboxy-terminal extension of the DNA-binding domain are highly conserved. (e) The DNA sequence of the sixth exon is highly conserved. An arginine in hGCNF-2b instead of serine results from alternative splice donor sites of the seventh exon. (f) The comparison of the seventh exon reveals three positions where the mouse and the human isoforms diverge. Isoform hGCNF-2b is generated by using a splice donor site located three nucleotides further downstream. The exons coding for the putative α-helices 3 to 6 (g), 7 and 8 (h), 9 and 10 (i), 11 and 12 (j) in the ligand-binding domain are highly conserved. The comparison of the last coding exon in (j) was extended up to the end of the human cDNA sequence of S88309.
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Related In: Results  -  Collection

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Figure 3: Comparison of the exons of the mouse and human GCNF genes. The upper line of nucleotide sequence shows the murine protein-coding exons and their flanking sequences (AF254815-AF254821). Protein-coding nucleotides are underlined. The lower line of nucleotide sequence shows the corresponding human sequence. Identical nucleotides are highlighted by bold letters. The AG/GT splice signals are shown in italics. mGCNF indicates the deduced mouse protein sequence; hGCNF indicates the deduced human protein sequences; GCNF indicates identical protein sequences. (a) The second exon and its flanking regions. Forty-one out of 42 nucleotides are identical and the flanking splicing signals are conserved. (b) The third exon and its flanking regions. A homologous sequence coding for identical amino acids was found in the human genomic sequence. No human isoform containing this sequence has been reported. The splice donor site shows the typical pyrimidine-rich sequence followed by the sequence 5'-NCAG in both sequences, but the comparison reveals several base transitions. (c) A single splice donor site in the fourth exon coding for the DNA-binding domain is used in all mouse-derived cDNAs described so far. For the human isoform GCNF-2, the corresponding splice site is used, giving rise to a protein containing the sequence ISVSDD instead of the VSVPDD in mouse. Usage of an alternative splice site located 12 bp further downstream gives rise to the shorter isoform GCNF-1. An asparagine (N) is underlined because one of the human cDNA clones codes for a lysine in this position (U64876/NM_001489). (d) Sequences of the fifth exon coding for the carboxy-terminal extension of the DNA-binding domain are highly conserved. (e) The DNA sequence of the sixth exon is highly conserved. An arginine in hGCNF-2b instead of serine results from alternative splice donor sites of the seventh exon. (f) The comparison of the seventh exon reveals three positions where the mouse and the human isoforms diverge. Isoform hGCNF-2b is generated by using a splice donor site located three nucleotides further downstream. The exons coding for the putative α-helices 3 to 6 (g), 7 and 8 (h), 9 and 10 (i), 11 and 12 (j) in the ligand-binding domain are highly conserved. The comparison of the last coding exon in (j) was extended up to the end of the human cDNA sequence of S88309.
Mentions: The comparison of the genomic sequences of exons 2 to 11 was extended by 100 bp of intronic sequence in both directions (Figure 3). During the preparation of this manuscript all sequence information was made available by the International Human Genome Project collaborators at the NCBI database and included in the contig NT_008491. Sequences of the 5'-untranslated region and of exon 7 obtained with a genome walking approach did not diverge from the sequence at the NCBI.

Bottom Line: The comparison reveals that the shorter human protein results from skipping the 45 base-pair third exon.Three different human isoforms - GCNF-1, GCNF-2a and GCNF-2b - are generated by differential usage of alternative splice acceptor sites of the fourth and the seventh exon.All human GCNF cDNAs identified so far are, however, derived from mRNAs generated by splicing the fourth to the second exon.

View Article: PubMed Central - HTML - PubMed

Affiliation: Zentrum für Molekulare Neurobiologie, Universität Hamburg, Martinistrasse 52, D-0246 Hamburg, Germany.

ABSTRACT

Background: Germ-cell nuclear factor (GCNF, NR6AI) is an orphan nuclear receptor. Its expression pattern suggests it functions during embryogenesis, in the placenta and in germ-cell development. Mouse GCNF cDNA codes for a protein of 495 amino acids, whereas the four reported human cDNA variants code for proteins of 454 to 480 amino acids. Apart from this size difference, there is sequence conservation of up to 98.7%. To elucidate the genomic structure that gives rise to the different human GCNF mRNAs, the sequence information of the human GCNF locus is compared to the previously reported structure of the mouse locus.

Results: The genomic structures of the mouse and human GCNF genes are highly conserved. The comparison reveals that the shorter human protein results from skipping the 45 base-pair third exon. Three different human isoforms - GCNF-1, GCNF-2a and GCNF-2b - are generated by differential usage of alternative splice acceptor sites of the fourth and the seventh exon.

Conclusion: By homology with the mouse gene, 11 GCNF coding exons can be defined on human chromosome 9. All human GCNF cDNAs identified so far are, however, derived from mRNAs generated by splicing the fourth to the second exon. Although the genomic sequence is highly conserved, the analysis suggests that alternative splicing generates a higher complexity of human GCNF isoforms compared with the situation in the mouse.

Show MeSH