*Institut de Pharmacologie Moléculaire et Cellullaire, UPR 411 Centre National de la Recherche Scientifique, Valbonne, France;
and
Anatomie Pathologique, Faculté de Médecine Laënnec, Lyon, France
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Abstract |
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Introduction |
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Several examples of chimeric genes that have evolved according to the gene fusion model are now documented in insects (Long and Langley 1993
) or vertebrates (Chen, Devries, and Cheng 1997a, 1997b
). Recently, we provided evidence for the emergence of a chimeric gene in Primates (Viale et al. 1998
). This gene was named the variant melanin-concentrating hormone (MCH) gene based on partial homology with the authentic MCH gene (Breton et al. 1993
; Breton, Schorpp, and Nahon 1993
). The authentic MCH gene was mapped on human chromosome 12q23 (Viale et al. 1997
) and encodes a cyclic peptide which is a likely leptin target (Qu et al. 1996
; Huang et al. 1999
). MCH has also been established as a major regulator of food intake behavior (Presse et al. 1996; Qu et al. 1996
; Rossi et al. 1997
; Shimada et al. 1998
).
The variant MCH gene arose by duplication from the authentic MCH gene and was found at two loci on chromosome 5, 5p14 and 5q13 (Pedeutour, Szpirer, and Nahon 1994
). We recently revealed that the first event of transposition from the ancestral chromosome 12 to chromosome 5p occurred in the Catarrhini before divergence of the Cercopithecidae and that the last duplication on both arms of chromosome 5 took place in the Hominidea lineage (Viale et al. 1998
). Another striking feature of the variant MCH genes is the expression of at least one form in brain areas of adult humans where the authentic MCH gene is silent (Viale et al. 1998
). Therefore, the MCH gene family provides a unique model with which to investigate the switch in brain-specific expression of genes that diverged during primate evolution.
Sequences of the region surrounding the authentic MCH gene have recently been determined (Viale et al. 1997
). Tissue-specific expression of the human MCH mRNA and processing of the MCH precursor have also been explored (Viale et al. 1997, 1999
). In contrast, few studies are available regarding the genomic organization, tissue-specific expression, and protein characterization of the variant MCH genes (Breton, Schorpp, and Nahon 1993
; Miller, Burmeister, and Thompson 1998
; Miller, Thompson, and Burmeister 1998
; Viale et al. 1998
). Here, we established the structure of the 5'- and 3'-end flanking regions of one variant MCH gene, and we refined the mapping of PMCHL1 and PMCHL2 loci on chromosome 5. Variant MCH gene transcripts were identified and characterized by RT-PCR and RACE-PCR, and the specific distribution of these RNAs was investigated using fetal, newborn, and adult human brain. Finally, we tested whether the longest of the open reading frames (ORFs) found in the putative variant MCH mRNA, referred to previously as ORF1 (Viale et al. 1998
), may be translated in vitro and in a cellular model.
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Materials and Methods |
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YAC Contig Analysis
YAC contigs spanning the SMA region (Melki et al. 1994
; Lefebvre et al. 1995
) were generously provided by Drs. J. Melki (Institut Necker, Paris, France) and D. Le Paslier (Centre d'Etude du Polymorphisme Humain, Paris, France). The YAC clones were analyzed in detail by PCR.
Subcloning and Sequencing of Variant MCH Gene Fragments
Six genomic clones, named hMCH41 and
hMCH104 (from a
EMBL3 library) and
hMCH1,
hMCH2,
hMCH3, and
hMCH 4 (from two
GEM11 libraries) had previously been isolated (Breton, Schorpp, and Nahon 1993
). All of these clones carried a common BglII fragment of about 10 kb bearing exon IIv and exon IIIv of the variant gene (Breton, Schorpp, and Nahon 1993
; unpublished data). This fragment was subsequently isolated from
hMCH41 and subcloned in Bluescript SK vector to generate the phMCH-L37 clone (Viale et al. 1998
). Several overlapping fragments were generated after exon III nuclease digestion (Sambrook, Fritsch, and Maniatis 1989
) of phMCH-L37, and the resulting independent DNAs were sequenced by automated fluorescent DNA sequencing (Applied Biosystems model 373A sequence facilities at the Institut de Pharmacologie Moléculaire et Cellulaire [IPMC]). In some cases, parts of the PMCHL genes were amplified from genomic DNA fragments isolated from hybrid YAC or phage clones through a "genome-walk" method and directly sequenced without further subcloning. Briefly, YAC and phage clones were digested separately with five different restriction enzymes generating blunt-ended termini (DraI, EcoRV, StuI, AluI, and PvuII), and the DNA restriction fragments were ligated to a derived-splinkerette adapter (Devon, Porteous, and Brookes 1995
). The genomic libraries thus obtained were then used as templates in nested PCR reactions with gene-specific primers (Inv21L and Inv21L2; j and k, respectively, in fig. 1 ) and the adapter primers Splink1 (5'-CGA ATC GTA ACC GTT CGT ACG AGA A-3') and Splink2 (5'-TCG TAC GAG AAT CGC TGT CCT CTC C-3'). DNA sequences were analyzed using Bisance service software (Dessen et al. 1990
).
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Southern Blotting
Human genomic DNA from CEPH control families (kindly provided by Dr A. Gal, Institut für Humangenetik, Lübeck, Germany) and cell lines was digested with BglII (or six other endonucleases), electrophoresed onto a 1% agarose slab gel, transferred to a nylon membrane, and hybridized with either the P1/HMCH2 PCR fragment probe, which recognized both the authentic and the variant MCH genes (Viale et al. 1998
), or a specific variant probe named hMCHL1 (see fig. 1
and table 2
).
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Radioactive Detection
Ten picomoles of one oligonucleotide primer was end-labeled using T4 polynucleotide kinase (Nahon et al. 1989
) and amplified. Templates were 20 ng of DNA from cell lines. 32P-labeled PCR products were electrophoresed on standard denaturing 6% polyacrylamide DNA sequencing gels and exposed to Bio-max film (Kodak).
Variant MCH Gene Transcript Analysis
RNA Isolation
Whole-cell RNA was extracted by a modification of the acid guanidium thiocyanate phenol chloroform method (Chomczynski and Sacchi 1987
). The final RNA samples were resuspended in DEPC-treated water and kept at -80°C.
Reverse-Transcription PCR Experiments
Total RNA from the different brain areas was reverse-transcribed into single-strand cDNA with the moloney murine leukemia virus reverse transcriptase (GIBCO BRL, France) using either poly-d(T) or variant MCH genespecific primers (UBI3 for sense transcripts and V1L for antisense transcripts; cf. table 2
). This served as a template for PCR using Taq DNA polymerase (Appligene, France), as previously described (Presse et al. 1992
). The RT-PCR products were detected either by Southern blot analysis with the P1/HMCH2 probe or by nested-PCR reaction with primers corresponding to the authentic MCH gene (HPCR4 and HPCR3) or the variant MCH genes (V1L/P4 and V1L/P7 or V1L/P4 and Inv19L/P7; cf. table 2 and fig. 1
). To reveal genomic DNA contamination, reverse transcription of RNA was performed in the absence of the enzyme, and PCR was carried out under standard conditions. The level of hybridization of variant MCH and GAPDH mRNA was quantified by densitometric scanning and computerized image analysis (NIH Image, version 1.51). The ratio of variant MCH mRNA/GAPDH mRNA was calculated, and the values of this ratio for each RNA sample were compared with the maximal value of the ratio of the control group (hypothalamic RNA sample corresponding to newborn 2) using a four-point scale (-, +, ++, and +++).
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For immunofluorescence microscopy, the transfected COS cells were grown on glass coverslips for 24 h. They were fixed for 15 min with 4% (v/v) paraformaldehyde in 1 x PBS. After rinsing twice with 1 x PBS, cells were permeabilized by incubation for 10 min with 0.1% Triton X-100/2% BSA/1/20 normal goat serum (vector 51000). The anti-FLAG M2 antibody (Kodak, SIGMA; dil. 1/400) and the anti-NEI antibody (dil. 1/500) (Bittencourt et al. 1992
) were incubated with cells for 1 h at room temperature. After three washings with 1 x PBS, fixed cells were incubated with either fluorescein isothiocyanate (FITC)labeled goat anti-serum anti-rabbit antibody (to detect NEI) or rhodamine-labeled goat anti-mouse antibody (to detect FLAG/M2 epitopes) at 1:200 dilution. The cells were rinsed three times in 1 x PBS and twice with 10 mM Tris-HCl [pH 7.4] buffer and mounted with 23 µl of Vectra shield medicine (Vector Lab.) on a glass slide and finally observed under fluorescence.
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Results |
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Two CA/TA repeats were found in the 5'-flanking region. Analysis of the 3'-flanking sequences revealed the insertion of an Alu-Sq element into a sequence 80% identical to part of the intron Aexon II junction of the authentic MCH gene (fig. 1
). This sequence, named Inv-MCHv, is oriented in the opposite direction of the MCHv sequence of the variant MCH gene and likely represents the remains of truncation/insertion events of the MCH gene that occurred during primate evolution (Viale et al. 1998
).
High-Resolution Mapping of the Two Variant MCH Genes
We first tried to detect a restriction fragment length difference after cleavage with six different restriction enzymes (including BglII; see fig. 2A
) that may distinguish copies of the variant MCH gene on 5p and 5q. We failed to reveal such a variation by Southern blot analysis using either a panel of 20 human genomic DNAs (5 are illustrated in fig. 2A
) or the six genomic MCH clones derived from different phage libraries (see Materials and Methods; data not shown).
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To determine the precise location of the PMCHL1 gene on 5p14, a PCR screening strategy was used. DNAs isolated from somatic cell hybrids containing deletions of human chromosome 5p (Overhauser et al. 1994
; fig. 3
, top) were amplified with 32P-labeled HV-GM1/unlabeled HV-GM2 primers which detected both PMCHL1 and PMCHL2. A representative example of the hybridization pattern obtained is shown in figure 2C.
As expected, the 240-bp PCR product was found in every DNA sample. In contrast, the 270-bp PCR product was present or absent, depending on the length of the deleted region of chromosome 5. Based on these data, the PMCHL1 locus was mapped at the distal end of 5p14.3 (fig. 3
, top).
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Sequence Analysis Revealed Several Point Mutations Between PMCHL1 and PMCHL2 Genes
Inasmuch as different CA/TA repeat lengths could be selectively associated with one of the two variant MCH genes, it was now possible to ascribe the variant MCH gene sequences to either of the chromosome 5 loci. Since the 270-bp PCR product was identified on all our phage clones and on phMCH-L37 (fig. 2B,
lane L37), those clones encompassed the PMCHL1 gene.
To characterize PMCHL2, different PCR products were generated from either YAC clones (742E10 and 751E3; fig. 3 ) or cell hybrids carrying deleted regions of chromosome 5p (JH132 and HHW740; fig. 3 ) using primers that bracketed the putative coding part of the variant MCH gene. The sequences were identical among these DNAs but exhibited several reliable differences when compared with the phMCH-L37. As shown in figure 4 , 22 mutations were distributed along the sequence between HMCH379 and HMCH7 primers and f', respectively, in fig. 1 ). Some of them were associated with restriction cleavage sites, such as the MaeI, AvaII, and PvuII sites at positions 2664, 2828, and 3010, respectively, that allowed discrimination between the two variant MCH genes (GenBank accession numbers: PMCHL1, AF238382; PMCHL2, AF238383).
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Thereafter, reverse transcription and PCR amplification were used to reveal the expression of variant MCH gene transcripts in different areas of fetal, newborn, and adult human brains (table 3 ). Polyadenylated forms of the variant MCH RNAs were first analyzed using oligo d(T) for reverse transcription and HMCH-P1 and HMCH2 primers (see table 2 ) for PCR amplification. A single product of 570 bp was identified in different RNA samples extracted from hippocampus and cerebellum of adults (fig. 5B ). Direct sequencing of this fragment showed that it was colinear with the genomic sequence of PMCHL1, indicating that it corresponded to unspliced transcripts of that gene (not shown). Contamination with genomic DNA appeared unlikely due to the absence of amplification in the nonreverse-transcribed (NRT) samples using specific-variant MCH gene primers or specific-GAPDH gene primers (fig. 5B ). The 570-bp PCR product was found in the hippocampus (or cortex), cerebellum, and hypothalamus at all stages of human development that we analyzed (table 3 ). In contrast, this product was lacking in the cerebellum and spleen of adult 2348 (fig. 5B ), as well as in all peripheral fetus and newborn tissues (not shown).
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As summarized in table 3
, sense variant MCH RNAs were found in at least one cortex and cerebellum at all stages of human brain development, whereas these transcripts were absent in the hypothalami of two fetuses, one neonate (our present data), and all adults we tested previously (Viale et al. 1998
). The antisense variant MCH RNAs were clearly identified in the cortex, hippocampus, and hypothalamus at all stages of development we analyzed. However, these antisense RNAs were not strongly expressed in cerebellar extracts from fetuses and adults.
Expression of a Putative Translational Product from the Variant MCH Gene In Vitro and in Transfected COS Cells
The longest ORF deduced from the RACE-PCR and RT-PCR DNA sequences corresponded to ORF1. It initiated from an ATG codon (methionine, boxed in fig. 6
) in a reasonable Kozak context (Kozak 1991
) and had a purine A at the -3 position. This sequence potentially encodes a short protein of 72 amino acids (8 kDa), designated here variant (V)-MCH-protein (p)8 (VMCH-p8). The VMCH-p8 sequence exhibits strong homology (89% identity at the protein level) to the part of pro-MCH encoded in exon II of the MCH gene (fig. 6 ). Sequence conservation was marked in the region of the NEI peptide which was located upstream of the MCH sequence in the same precursor of rats, mice, and humans (Nahon et al. 1989
; Presse et al. 1990
) (amino acids 131144 of pro-MCH, amino acids 5265 of VMCH-p8; boxed in fig. 6
). Several nucleotide substitutions were noted, but many of these changes resulted in amino acid conservations (Asn90, Phe114, Gly119, Thre123). Sequence differences were confined to the N-terminal part (amino acids 18 of VMCH-p8) and the C-terminus (amino acids 7172 of VMCH-p8).
Predicted hydrophobicity and secondary structure plots of VMCH-p8 (not shown) indicated that this protein lacked an initial stretch of hydrophobic residues that could act as a signal peptide. Therefore, VMCH-p8 is unlikely to be a secreted protein or a neuropeptide precursor. Sequence analysis using PSORT II programs predicted that VMCH-p8 could be a nuclear protein (reliability 52.2% with the k-NN prediction test). A stretch of basic residues is located at amino acids 59 of VMCH-p8 (underlined in fig. 6 ). This sequence corresponds to a classical nuclear localization signal (NLS) according to the PSORT II subprogram.
To assess the translational potential of the RNA encoding VMCH-p8, a PCR fragment encompassing ORF1 was produced and introduced at the BamH1-EcoR1 sites in pKS or pGEM vectors (see fig. 7A ). These constructs were added to a coupled transcription/translation system (TNT-T7 coupled reticulocyte lysate kit). We failed to reveal a variant MCH protein using these constructs. Using the pEXO-VAR construct, which carries ß-globin mRNA-stabilizing sequences, a single protein of the expected molecular mass was identified, its synthesis depending on the amount of pEXO-VAR construct added to the TNT-T7 translation system (fig. 7B ).
A pCMV-FLAG/ORF1 construct was transfected to COS-7 cells under two different conditions of transfection (experiments A and B, Materials and Methods), and indirect immunofluorescence microscopy was used with either anti-FLAG M2 antiserum or anti-NEI antiserum. A similar punctate pattern of fluorescence was observed with the M2 antiserum (fig. 7C, upper panel) and the anti-NEI antiserum (fig. 7C, bottom panel). The majority of the tagged protein accumulated in the perinuclear space of the endoplasmic reticulum (arrowhead in fig. 7C ), while a small fraction was associated with the nucleus (arrow in fig. 7C, bottom panel). Cells transfected with the pCMV vector alone displayed only fluorescent background (not shown).
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Discussion |
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First, we extended the sequence analysis of the human variant MCH gene to the 3' and 5' regions flanking the exon-intron structure previously described (Breton et al. 1993
). Surprisingly, a sequence corresponding to the intron Aexon II part of the authentic MCH gene was found located in an inverse orientation at the 3' end, close to the MCHv region of the variant MCH gene. An Alu-Sq sequence was also identified inserted into the so-called Inv-MCHv region bracketed by short direct repeats, as expected at a site of mobile element insertion. Because the retrotransposition of the Alu-Sq subfamily occurred about 44 MYA (Mighell, Markham, and Robinson 1997
), this Alu repeat could be involved in the truncation process that led to the creation of the variant MCH gene in the equivalent of chromosome 5p14 in the Catarrhini ancestor (Viale et al. 1998
). Alternatively, it was recently transposed during primate evolution. Sequencing of the 3'-flanking region of the variant MCH gene in Cercopithecinae species should clarify this issue.
Two CA/TA repeat elements were mapped close to the exon IIvintron Bexon IIIv region of the variant MCH gene. One of these repeats displayed different lengths, depending on whether it came from the 5p or the 5q copy of the variant MCH gene. We established therefore that the phMCH-L37 carried the sequence of the 5p locus and that multiple point mutations clearly differentiate the PMCHL1 and PMCHL2 genes (22 positions in a 1.1-kb fragment encompassing the exon IIvintron Bvexon IIv region). These results confirmed and extended previous data obtained from PCR-SSCP to a radiation hybrid panel (Miller, Thompson, and Burmeister 1998
). In particular, the site for the PvuII restriction enzyme is lacking in the PMCHL1 sequence, and it may be conveniently used to discriminate the two variant MCH genes and transcripts. We used this technique extensively to characterize the transcripts revealed in the human brain (see below).
Using the size difference of the CA/TA repeat, we precisely located the PMCHL1 and PMCHL2 genes on human chromosome 5, refining the map proposed by Miller, Thompson, and Burmeister (1998)
. PMCHL1 was assigned to a 1-Mb region in chromosome 5p14.3. Deletions covering this region do not contribute to the cri-du-chat syndrome or mental retardation (Overhauser et al. 1994
). However, PMCHL1 may be a candidate gene for multiple sclerosis (Ebers et al. 1996
; Kuokkanen et al. 1996
) and craniometaphyseal dyplasia (Nurnberg et al. 1997
). On chromosome 5q13.3, the variant PMCHL2 gene was found in YAC clones 798B3, 742E10, and 751E3, close to the SMA locus. However, the PMCHL2 gene does not lie within the critical region where deletions, gene conversions, or mutations have been associated with clinical expression of the disease (Melki et al. 1994
; Lefebvre et al. 1995
; Roy et al. 1995
). Thus, it is unlikely that PMCHL2 may be directly involved in the SMA phenotype. However, PMCHL2 falls in a region of susceptibility for the Wagner disease and erosive vitreoretinopathy (Brown et al. 1995
) (see fig. 3
). Nevertheless, these results do not provide direct evidence that either of the variant MCH genes is associated with these diseases. In this context, a broad region of chromosome 5 was linked to obesity in French families (Hager et al. 1998
), but its association with PMCHL1 and PMCHL2 loci is largely hindered by the inability to pinpoint the locus.
An important aspect of the work presented here is the demonstration that both sense and antisense transcripts from the PMCHL1 gene are present in different areas of the developing human brain. We also demonstrate that PMCHL2 is totally silent during development of the human brain. The divergent expression patterns of PMCHL1 and PMCHL2 are likely the result of a different genomic environment in the flanking region, but this remains to be established.
Miller, Burmeister, and Thompson (1998)
have previously identified antisense unspliced RNAs from PMCHL1 in the hypothalamus of one adult human. We confirmed these data and showed also that antisense RNAs overlapping the MCHv region were widely produced in fetal, newborn, and adult brains. No ORF of significant length existed in the antisense orientation of the MCHv region (fig. 1 ). This raises the possibility that antisense PMCHL1 transcripts function as untranslated RNAs. Direct interaction with the complementary sense transcripts originating from either the variant MCH gene or the authentic MCH gene may contribute to control splicing, stability, and/or translational efficiency (reviewed by Dolnick 1997
; Vanhée-Brossollet and Vaquero 1998
). Considering the expression patterns of the sense and antisense PMCHL1 gene transcripts in the human brain, they fulfill the criteria for the "determinator-inhibitor pair" model proposed by McCarrey and Riggs (1986)
. Alternatively, there are a growing number of examples of RNAs lacking extensive ORFs which regulate important functions without direct interaction with complementary transcripts (reviewed in Erdmann et al. 1999). Some of these untranslated RNAs play a role in gene regulation during normal development, such as those transcribed from the Xist gene, involved in the X-chromosome inactivation process (Brown et al. 1992
). Others act as tumor suppressors and modulate cell growth, such as the noncoding RNAs synthesized from the H19 gene (Brannan et al. 1990
; Hao et al. 1993
).
Sense unspliced transcripts from PMCHL1 were identified at all stages of human development in the cortex and cerebellum but not in the fetal and adult hypothalami (see table 3
; Viale et al. 1998
). In addition, no expression of these sense RNAs was found in one fetus and one newborn. Although the basis for this difference is unknown, it may be related to the postmortem interval (PMI) and the agonal state (events prior to death) (Barton et al. 1993
). Indeed, the lack of expression of the sense transcripts correlated with the longest PMI in fetal tissues (see tables 1 and 3
). In addition, hypoxia during the premorterm period in SIDS neonates may also influence the stability of mRNA as found in animal models (White and Lawson 1997
). Thus, quantitative assessment of PMCHL1 gene expression during human brain development is difficult to evaluate. In a similar respect, unspliced sense transcripts are absent in the hypothalamus at all stages of development, except for one newborn (case 2). Again, the proximal cause of death may have selectively influenced PMCHL1 gene expression in this particular case.
The sense PMCHL1 gene transcripts identified in the human brain contain a coding sequence (ORF1) of 72 amino acids which corresponds to an 8-kDa protein named VMCH-p8. Because of the lack of a signal peptide (originally present in exon I of the MCH gene), this ORF has probably lost its original function. In addition, the absence in the human brain of PMCHL1 transcripts where intron Bv is spliced indicates that the production of a putative variant MCH peptide is unlikely. Indeed, by using RP-HPLC coupled with a specific RIA, we failed to identify this variant MCH peptide in various brain regions of fetus, newborn, or adult individuals (unpublished data). However, an intriguing feature of the exon IIv sequence is the high degree of conservation between the MCH and PMCHL1 genes at the DNA and protein sequences both in humans (fig. 6
; Breton et al. 1993
) and in the variant orthologs in Primates (Viale et al. 1998
). This likely reflects a constraint in the ORF1 sequence. Arguments for the translational capacity of the ORF1-containing RNAs come from in vitro translation and COS cell transfection experiments shown here. An NLS motif was identified in the N-terminal part of VMCH-p8 (see fig. 6
) which could contribute to directing this protein to the nucleus, as found for other nuclear proteins (Nigg 1997
). This hypothesis is currently being tested with constructs deleted or mutated in the putative NLS sequence and transfected into different cell types.
In conclusion, the variant PMCHL1 gene represents a unique example of the assembly of unrelated sequences through a complex rearrangment process that conveyed the conversion of a neuropeptide-encoding gene (i.e., a secreted protein) to a gene encoding a putative nucleus-targeted protein (i.e., an intracellular protein) during primate evolution. All of the requirements for PMCHL1 being a new expressed gene, or a gene "in search of a function" (Marshall, Raff, and Raff 1994
), are present here. It is transcriptionally active, albeit at a low level. This means that insertion of the transposed ancestor of this gene occurred in a favorable environment of cis-regulatory elements driving the striking tissue-specific expression of the variant PMCHL1 gene. This is at odds with the other copy of the variant MCH gene (PMCHL2), which appeared not to be transcribed at all in the human brain. Most interestingly, the active PMCHL1 gene may code for a protein, VMCH-p8, but proof of expression of this protein in vivo is presently lacking. The next challenge is to characterize VMCH-p8, as well as other putative variant MCH proteins in developing human brain, and to elucidate their function(s).
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Acknowledgements |
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Footnotes |
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1 Present address: Laboratory of Molecular Genetics, Rockefeller University, New York.
2 Present address: Centre National de la Recherche Scientifique INSERM de Pharmacologie-Endocrinologie, Montpellier, France.
3 Keywords: variant MCH gene
antisense RNA
human brain
human chromosome 5
expressed pseudogene
primate evolution
4 Address for correspondence and reprints: NAHON Jean-Louis, Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique UPR 411, Sophia-Antipolis, 06560 Valbonne, France. E-mail: nahonjl{at}ipmc.cnrs.fr
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