the nuclear receptors comprise a family of transcriptional regulators involved in a wide variety of biological processes such as embryonic development , differentiation and homeostasis [ 1 , 2 ] .
the family includes ligand - dependent zinc - finger transcription factors for steroid hormones , estrogens , thyroid hormones , retinoids , vitamin d and other hydrophobic molecules .
in addition , several family members are ' orphan receptors ' for which ligands have yet to be identified .
nuclear receptors have been assigned to six subfamilies on the basis of evolutionary studies . as the first member of the sixth subfamily
, gcnf is also known by its systematic name nr6a1 . on the basis of homology and expression profile ,
the receptor has been given the alternative name of retinoic acid receptor - related testis - associated receptor ( rtr ) .
transfection experiments reveal that gcnf can act as a constitutive repressor when binding as a homodimer to promoters containing a direct repeat dna element 5'-aggtcaaggtca-3 ' ( dro ) [ 7 , 8 , 9 , 10 ] .
the mouse receptor ( mgcnf ) is highly expressed in the developing embryonic nervous system and the labyrinthine layer of the placenta [ 12 , 13 ] . in the adult ,
high transcript levels are restricted to the developing germ cells [ 5 , 14 , 15 , 16 ] .
northern analysis reveals a transcript of 7.5 kilobases ( kb ) in somatic cells and an additional message of approximately 2.4 kb in male germ cells .
this size difference is at least partially due to different polyadenylation sites , and it is therefore assumed that both transcripts code for identical proteins of 495 amino acids .
the protein sequence is encoded by 11 exons . when differentiation of p19 embryonal carcinoma cells is triggered by retinoic acid , the transcript and the protein are temporarily upregulated and then downregulated .
isolation of a human cdna coding for a protein ( hgcnf ) with an identity to the mouse protein of 98.7% , similar regulation in mouse p19 cells and in the human embryonal carcinoma cell line nt2/d1 , together with the presence of two mrnas of approximately 7.5 and 2.2 kb in human testis , suggested similar functions for mouse and human gcnf [ 18 , 19 , 20 ] .
the cloning of human cdnas that give rise to different hgcnf isoforms , however , suggests a higher complexity in humans . currently , four different hgcnf cdnas have been isolated that code for isoforms ranging in size from 454 to 480 amino acids .
we have investigated the genomic structure of mammalian gcnf to determine how the different gcnf isoforms are generated .
here we compare the exon / intron structure of the previously characterized mouse gene with the human ortholog .
our study shows that alternative splicing generates at least three of the different gcnf isoforms .
to understand how the different human gcnf mrna isoforms are generated , we have identified all human protein - coding exons .
the alignment of the full - length human gcnf cdna ( genbank accession number s83009 ) with the genome sequencing data at the ncbi localized the first protein - coding exon on chromosome-9-derived working draft sequence element nt_008491 .
the genomic sequence was aligned with the previously identified mouse exon 1 containing the putative translational start site .
the comparison was extended up to position -100 with respect to the mouse cdna reaching farthest in the furthest 5 ' direction .
in addition , 100 base - pairs ( bp ) of the first identified intron were included ( figure 1 ) . while the transcriptional start sites of gcnf are still elusive , the 5 ' ends of the first protein - coding exons can not be defined .
with respect to the sequence in figure 1 , the furthest 5'-reaching human cdna ( s83309 ) starts with nucleotide 171 .
the putative translational start codons are in positions 346 and 350 of the mouse and human sequences , respectively .
these start codons are present in all full - length mammalian gcnf cdnas characterized so far , suggesting a common amino terminus for the different gcnf isoforms .
the comparison of the mouse 5'-untranslated sequence with the human genomic dna reveals high conservation with identical sequence elements of up to 50 nucleotides .
the presence of 18 cg dinucleotides conserved between human and mouse is suggestive of a regulatory function of the untranslated sequence .
five different human cdnas with alternative 5 ' ends have , however , been reported to genbank ( s83309 , u80802 , af004291 , nm001489/u64876 , x99975 ) . a comparison with the genomic sequence ( nt_008491 ) shows the sequence variation ( figure 2 ) .
single - nucleotide polymorphism among cdnas isolated from different human libraries may reflect variants in the human population .
two cdnas ( u64876/nm001489 , x99975 ) differ in their untranslated region with respect to the genomic sequence .
therefore , it can not be ruled out that these cdna ends may have been generated during the cloning process .
in addition , one of the cloned cdnas , coding for hgcnf-3 ( af004291 ) , has a deletion in the coding region of the first exon , giving rise to an open reading frame of 454 amino acids .
the 5 ' part of hgcnf-3 has been isolated by the polymerase chain reaction , suggesting that this deletion may have been generated during the synthesis .
the isolation of additional cdnas may give a clue as to which variants are true gcnf isoforms .
the functional significance of the different isoforms is , at present , unknown but may lead to different transcriptional properties of gcnf isoforms .
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 .
two short exons of 42 bp and 45 bp , respectively , follow the first protein - coding exon in the mouse .
short exons are relatively rare in mammalian genomes . the structure of the second protein - coding exon is conserved ( figure 3a ) .
interestingly , on the basis of the genomic cdna , the third exon is highly conserved as well ( figure 3b ) .
the human splicing apparatus preferentially , or exclusively , skips this putative exon , however .
as splicing is highly regulated , a splice enhancer present in the mouse genome may not be present in the human genome .
consequently , all known human gcnf isoforms lack the amino acids encoded by the putative third exon . of the 243 bp exon 4 that encodes the core of the dna - binding domain
one of the reported sequences ( u64876/nm_001489 ) has a c to a transversion , however , which changes a codon for asparagine to one for lysine .
splicing of exon 2 to exon 4 at the position characterized in the mouse results in isoform hgcnf-2 .
in addition to this splice acceptor position , a splice acceptor site located 12 nucleotides further downstream is used to generate hgcnf-1 .
two hgcnf-2 variants , hgcnf-2a and hgcnf-2b , which differ by a single amino acid , have been isolated . as speculated , alternative splicing generates the isoform 2b with a deletion of a serine residue . splicing to an acceptor site of exon 7 located three nucleotides
the sequence and structure of exons 8 to 11 are also highly conserved ( figure 3g , h , i , j ) .
the comparison of the 11th exon was extended up to the end of the human cdna sequence of s88309 .
highly conserved sequence elements of up to 91 identical nucleotides indicate a regulatory function of the 3'-untranslated sequence following the translational stop codon .
the aceview analysis at the ncbi based on the draft sequence and a blast search with s83309 of the celera genomics freely accessible whole - genome sequence data gave mostly similar intron sizes .
both analyses revealed a large first intron of 37,652 bp in the public sequence data , and 37,157 bp in the private data .
the size of the second intron separating exon 2 and exon 4 was only available in the ncbi database ( 14,869 bp ) . according to the ncbi and celera databases , introns 3 to 9 have sizes of 10,486 ( ncbi ) ( 10,471 , celera ) bp , 3629 ( 3615 ) bp , 190,321 ( 1708 ) bp , 1963 ( 1960 ) bp , 2716 ( 9019 ) bp , 1905 ( 1912 ) bp , and 1927 ( 1928 ) bp , respectively .
the comparison of both analyses shows that the deduced sizes of two of the human introns differs greatly .
it seems likely that these inconsistencies will be corrected in the final assembly of the human genome .
to understand how the different human gcnf mrna isoforms are generated , we have identified all human protein - coding exons .
the alignment of the full - length human gcnf cdna ( genbank accession number s83009 ) with the genome sequencing data at the ncbi localized the first protein - coding exon on chromosome-9-derived working draft sequence element nt_008491 .
the genomic sequence was aligned with the previously identified mouse exon 1 containing the putative translational start site .
the comparison was extended up to position -100 with respect to the mouse cdna reaching farthest in the furthest 5 ' direction .
in addition , 100 base - pairs ( bp ) of the first identified intron were included ( figure 1 ) . while the transcriptional start sites of gcnf are still elusive , the 5 ' ends of the first protein - coding exons can not be defined .
with respect to the sequence in figure 1 , the furthest 5'-reaching human cdna ( s83309 ) starts with nucleotide 171 .
the putative translational start codons are in positions 346 and 350 of the mouse and human sequences , respectively .
these start codons are present in all full - length mammalian gcnf cdnas characterized so far , suggesting a common amino terminus for the different gcnf isoforms .
the comparison of the mouse 5'-untranslated sequence with the human genomic dna reveals high conservation with identical sequence elements of up to 50 nucleotides .
the presence of 18 cg dinucleotides conserved between human and mouse is suggestive of a regulatory function of the untranslated sequence .
five different human cdnas with alternative 5 ' ends have , however , been reported to genbank ( s83309 , u80802 , af004291 , nm001489/u64876 , x99975 ) . a comparison with the genomic sequence ( nt_008491 ) shows the sequence variation ( figure 2 ) .
single - nucleotide polymorphism among cdnas isolated from different human libraries may reflect variants in the human population .
two cdnas ( u64876/nm001489 , x99975 ) differ in their untranslated region with respect to the genomic sequence .
therefore , it can not be ruled out that these cdna ends may have been generated during the cloning process .
in addition , one of the cloned cdnas , coding for hgcnf-3 ( af004291 ) , has a deletion in the coding region of the first exon , giving rise to an open reading frame of 454 amino acids .
the 5 ' part of hgcnf-3 has been isolated by the polymerase chain reaction , suggesting that this deletion may have been generated during the synthesis .
the isolation of additional cdnas may give a clue as to which variants are true gcnf isoforms .
the functional significance of the different isoforms is , at present , unknown but may lead to different transcriptional properties of gcnf isoforms .
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 .
two short exons of 42 bp and 45 bp , respectively , follow the first protein - coding exon in the mouse .
short exons are relatively rare in mammalian genomes . the structure of the second protein - coding exon is conserved ( figure 3a ) .
interestingly , on the basis of the genomic cdna , the third exon is highly conserved as well ( figure 3b ) .
the human splicing apparatus preferentially , or exclusively , skips this putative exon , however .
as splicing is highly regulated , a splice enhancer present in the mouse genome may not be present in the human genome .
consequently , all known human gcnf isoforms lack the amino acids encoded by the putative third exon .
of the 243 bp exon 4 that encodes the core of the dna - binding domain , 225 bp are identical in both species ( figure 3c ) .
one of the reported sequences ( u64876/nm_001489 ) has a c to a transversion , however , which changes a codon for asparagine to one for lysine .
splicing of exon 2 to exon 4 at the position characterized in the mouse results in isoform hgcnf-2 .
in addition to this splice acceptor position , a splice acceptor site located 12 nucleotides further downstream is used to generate hgcnf-1 .
two hgcnf-2 variants , hgcnf-2a and hgcnf-2b , which differ by a single amino acid , have been isolated . as speculated , alternative splicing generates the isoform 2b with a deletion of a serine residue . splicing to an acceptor site of exon 7 located three nucleotides
the sequence and structure of exons 8 to 11 are also highly conserved ( figure 3g , h , i , j ) .
the comparison of the 11th exon was extended up to the end of the human cdna sequence of s88309 .
highly conserved sequence elements of up to 91 identical nucleotides indicate a regulatory function of the 3'-untranslated sequence following the translational stop codon .
the aceview analysis at the ncbi based on the draft sequence and a blast search with s83309 of the celera genomics freely accessible whole - genome sequence data gave mostly similar intron sizes .
both analyses revealed a large first intron of 37,652 bp in the public sequence data , and 37,157 bp in the private data .
the size of the second intron separating exon 2 and exon 4 was only available in the ncbi database ( 14,869 bp ) . according to the ncbi and celera databases , introns 3 to 9 have sizes of 10,486 ( ncbi ) ( 10,471 , celera )
bp , 3629 ( 3615 ) bp , 190,321 ( 1708 ) bp , 1963 ( 1960 ) bp , 2716 ( 9019 ) bp , 1905 ( 1912 ) bp , and 1927 ( 1928 ) bp , respectively .
the comparison of both analyses shows that the deduced sizes of two of the human introns differs greatly .
it seems likely that these inconsistencies will be corrected in the final assembly of the human genome .
in summary , our analysis reveals a conserved structure for gcnf , allows the verification and systematic analysis of splice variants , and may be the basis of a better understanding of gcnf .
the conservation of the intron - exon boundaries is consistent with the extremely high degree of amino - acid conservation between the human and the mouse proteins .
the generation of the proteins hgcnf-1 , hgcnf-2a and hgcnf-2b can be explained by alternative splicing of the rna .
the sequence of the third coding mouse exon , including the splice sites , is highly conserved ; however , at present no human cdna has been isolated containing this putative exon .
alternative splicing provides a plausible means for generating diversity and may contribute to a higher instructive complexity in human gcnf .
exons of gcnf were identified by a blast   search with the human gcnf cdna sequence ( s83009 ) in the " unfinished high throughput genomic sequences " and in the homo sapiens genomic contig sequences at the ncbi [ 24 , 25 ] .
intron sizes given by the aceview analysis   were compared with the numbers obtained by a blast search of celera 's assembled sequence of the human genome .
the putative human gcnf exon 3 was identified by a blast search with the sequence of the third mouse exon .
sequences were aligned using the wisconsin package version 10.0 of the genetics computer group ( gcg ) , madison , wisconsin .
exons of gcnf were identified by a blast   search with the human gcnf cdna sequence ( s83009 ) in the " unfinished high throughput genomic sequences " and in the homo sapiens genomic contig sequences at the ncbi [ 24 , 25 ] .
intron sizes given by the aceview analysis   were compared with the numbers obtained by a blast search of celera 's assembled sequence of the human genome .
the putative human gcnf exon 3 was identified by a blast search with the sequence of the third mouse exon .
sequences were aligned using the wisconsin package version 10.0 of the genetics computer group ( gcg ) , madison , wisconsin .
the mouse - derived ( m ) dna sequence ( upper line , genbank accession number af254575 ) of the first protein - coding exon and flanking sequences are compared with the human - derived ( h ) sequence ( lower line , s83309 for the coding , and nt_008491 for the flanking sequences ) .
identical nucleotides are highlighted in the human sequence by bold letters , gaps in the alignment are shown as dots .
the deduced amino - acid sequence of the human protein is shown in the single - letter code .
the bold p ( proline ) marks the position of an arginine in the mouse protein .
sequence comparison of the 5 ' ends of human gcnf cdnas coding for a full - length protein with the genomic dna sequence .
human cdna sequences have been aligned omitting the cloning sites at the 5 ' ends .
one cytosine and five adenosines in the genomic sequence not found in any of the cdnas are underlined .
the deduced amino - acid sequences ( aab50876 , aac52054 ) are given in the single - letter code .
the upper line of nucleotide sequence shows the murine protein - coding exons and their flanking sequences ( af254815-af254821 ) .
mgcnf indicates the deduced mouse protein sequence ; hgcnf indicates the deduced human protein sequences ; gcnf indicates identical protein sequences .
forty - one out of 42 nucleotides are identical and the flanking splicing signals are conserved .
a homologous sequence coding for identical amino acids was found in the human genomic sequence .
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 .
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 -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 .
 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 . 
Background
Results and discussion
Structure of the first coding exon
Conserved structure of exons 2 to 11
Conclusion
Materials and methods
Database search
Sequence analysis
Figures and Tables