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Evolution appears to be especially rapid during speciation, and the genes involved in speciation should be evident in species such as humans that have recently speciated or are presently in the process of speciation. Haldane’s rule is that when one sex is sterile or inviable in interspecific F[sub 1] hybrids, it is usually the heterogametic sex. For mammals, this implicates genes on the X chromosome as those particularly responsible for speciation. A preponderance of sex- and reproduction-related genes on the X chromosome has been shown repeatedly, but also mental retardation genes are more frequent on the X chromosome. We argue that brain, testis, and placenta are those organs most responsible for human speciation. Furthermore, the high degree of complexity of the vertebrate genome demands coordinate evolution of new characters. This coordination is best attained when the same set of genes is redeployed for these new characters in the brain, testis, and placenta. Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Screening of a human brain cDNA library using the C-terminal tail of the melanin-concentrating hormone receptor 1 (MCHR1) as bait in a yeast two-hybrid assay resulted in the identification of the neurite-outgrowth related factor, neurochondrin. This interaction was verified in overlay, pulldown, and co-immunoprecipitation assays. Deletion mapping confined the binding to the C terminus of neurochondrin and to the proximal C terminus of MCHR1, a region known to be involved in G protein binding and signal transduction. This region of the MCHR1 is also able to interact with the actin- and intermediate filament-binding protein, periplakin. Interactions of MCHR1 with neurochondrin and periplakin were competitive, indicating that these two proteins bind to overlapping regions of MCHR1. Although neurochondrin did not interfere with melanin-concentrating hormone-mediated internalization of the receptor, it did inhibit G protein-coupled signal transduction via both Galpha(i/o) and Galpha(q/11) family G proteins as measured by each of melanin-concentrating hormone-induced G protein-activated inwardly rectifying K(+) channel activity of voltage-clamped amphibian oocytes, by calcium mobilization in transfected mammalian cells, and by reduction in the capacity of melanin-concentrating hormone to promote binding of [(35)S]guanosine 5'-3-O-(thio)triphosphate to both Galpha(o1) and Galpha(11). Immunohistochemistry revealed co-expression of neurochondrin and MCHR1 within the rodent brain, suggesting that neurochondrin may be involved in the regulation of MCHR1 signaling and play a role in modulating melanin-concentrating hormone-mediated functions in vivo.

We have recently identified a Melanin-concentrating hormone receptor 1 interacting zinc-finger protein (MIZIP) from a human brain cDNA library. Here, we report the generation of a specific antibody against MIZIP and its distribution in rodent tissues using immunoblotting and immunohistochemical techniques. MIZIP was detected as a 27 kDa protein in brain, liver, and skeletal muscle, and to a lower extend, in lung, testis, and heart. Subcellular fractionation of adult mouse brain revealed the presence of MIZIP and MCHR1 in the cytoplasmic, membrane, and synaptosomal fraction, but not in a postsynaptic density preparation. In cultured rat, embryonic hippocampal neurons MIZIP is somatodendritically localized. In the adult rodent brain, MIZIP is widely distributed. High levels of expression were detected in brain regions involved in olfaction, feeding behavior, sensorimotor integration, and learning and memory, for example, the olfactory bulb, the olfactory tubercle, the caudate putamen, the thalamus and hypothalamus, the nucleus accumbens, the cerebral cortex, the hippocampus formation, and the cerebellum. Co-expression of MIZIP and MCHR1 was observed, for example, in pyramidal neurons of the cerebral cortex and hippocampus, in neurons of the olivary nucleus, lateral hypothalamus, nucleus accumbens, caudate putamen, pontine, and mesencephalic trigeminal nucleus. However, there are also differences in the expression patterns, for example, high expression of MCHR1 was detected in the lateral habenula, but no expression of MIZIP. These data support the notion that MIZIP might interact with MCHR1 in a cell type specific manner in vivo, suggesting a role in the regulation of MCH signalling in distinct regions of the mammalian brain.

MIZIP was originally identified as a highly conserved zinc-finger protein from human brain interacting with the C-terminus of the melanin-concentrating hormone receptor 1. However, the cellular functions of MIZIP are still not known. Here, we focussed on the identification of associated proteins using affinity purification from human cells. This resulted in the identification of alpha- and beta-tubulin. The interaction was confirmed in vitro and in vivo using GST pull-down and immunoprecipitation assays, and was mapped to the MYND zinc-finger of MIZIP and to the N-terminus of tubulin. Immunoprecipitation and immunocytochemistry analyses demonstrate that MIZIP binds to tubulin but not to cellular microtubules in vivo and that ectopic expression of MIZIP does not interfere with the overall structure of the microtubular cytoskeleton. Our results suggest that MIZIP might play an important role in mammalian cells by associating with tubulin and thus might provide a link between MCHR1 and tubulin functions.

In mice genetic ablation of expression of either melanin-concentrating hormone or the melanin-concentrating hormone-1 receptor results in alterations in energy metabolism and a lean phenotype. There is thus great interest in the function and regulation of this receptor. Using the yeast two-hybrid system we identified an interaction of the actin- and intermediate filament-binding protein periplakin with the intracellular C-terminal tail of the melanin-concentrating hormone-1 receptor. Direct association of these proteins was verified in pull-down and coimmunoprecipitation experiments. Truncations and internal deletions delineated the site of interaction to a group of 11 amino acids proximal to transmembrane helix VII, which was distinct from the binding site for the melanin-concentrating hormone-1 receptor-interacting zinc finger protein. Immunohistochemistry demonstrated coexpression of periplakin with melanin-concentrating hormone-1 receptor in specific cells of the piriform cortex, amygdala, and other structures of the adult mouse brain. Coexpression of the melanin-concentrating hormone-1 receptor with periplakin in human embryonic kidney 293 cells did not prevent agonist-mediated internalization of the receptor but did interfere with binding of (35)S-labeled guanosine 5'-3-O-(thio)triphosphate ([(35)S]GTPgammaS) to the G protein Galpha(o1) and the elevation of [Ca(2+)](i). Coexpression of the receptor with the interacting zinc finger protein did not modulate receptor internalization or G protein activation. The interaction of periplakin with receptors was selective. Coexpression of periplakin with the IP prostanoid receptor did not result in coimmunoprecipitation nor interfere with agonist-mediated binding of [(35)S]GTPgammaS to the G protein Galpha(s). Periplakin is the first protein described to modify the capacity of the melanin-concentrating hormone-1 receptor to initiate signal transduction.

The murine homologue of the human glycoprotein (transmembrane) NMB (GPNMB) gene was identified by subtractive cloning from in vitro cultured murine primary osteoblast cells and subsequent RACE-PCR. GPNMB is a highly glycosylated type I transmembrane protein that shares significant sequence homology to several melanosomal proteins. Increasing expression of Gpnmb mRNA was observed during differentiation of murine primary osteoblast cell cultures. To address the potential functions of GPNMB we analysed its mRNA-expression during murine embryonic development. In early development Gpnmb mRNA is detected at high levels in the outer layer of the retina. Later in development expression gets restricted to the retinal pigment epithelium and iris. At the cytoplasmic domain of GPNMB, a conserved di-leucin-based endosomal/melanosomal-sorting signal (ExxPLL) was located, present as well in several known melanosomal proteins. To analyse the subcellular localization we used EGFP-tagged GPNMB transfected in COS7 and HEK293 cells. In both non-pigmented cell lines, the EGFP-GPNMB fusion protein was localized to vesicular, endosomal like structures. Sequence homology to known melanosomal proteins, mRNA expression and subcellular localization are suggestive for GPNMB as an intracellular, endosomal/melanosomal compartment specific protein important for melanin biosynthesis and the development of the retinal pigment epithelium and iris. As the gene coding for human GPNMB was localized to chromosome 7p15, a locus involved in the human inherited disease cystoid macular edema, also known as dominant cystoid macular dystrophy (OMIM 153880) we highly suggest that GPNMB is a candidate gene for this human inherited disease.

Using the yeast-two-hybrid system a novel protein was identified from human brain that interacts with the C-terminus of melanin-concentrating hormone receptor 1 (MCH-R1). This protein, characterized by a Myeloid translocation protein 8, Nervy, DEAF1 proteins (MYND) zinc-finger domain, is termed MCH-R1-interacting zinc-finger protein, MIZIP. It is fully conserved in man, rat, mouse and highly conserved in Xenopus and zebrafish, but not detectable in invertebrates. MIZIP gene organization in human (six exons on chromosome 9q34.3) and mouse is highly conserved, yet in rodents an additional exon is generated giving rise to alternatively spliced mRNAs. MIZIP is expressed in brain, testis and stomach, where expression of MCH and MCH-R1 was previously reported. MIZIP interaction with MCH-R1 was verified by overlay and pull-down assays as well as by co-transfection experiments in human embryonic kidney-293 cells. MIZIP is cytoplasmically localized but gets recruited to the plasma membrane when cells are co-transfected with MCH-R1 supporting the notion that MIZIP is involved in the function of MCH-R1.

We have isolated and characterized a novel differentially spliced gene predominantly expressed in the nervous system, which encodes protein isoforms with significant homology to the alpha-actinin protein superfamily, the Caenorhabditis elegans UNC-53 protein and weak homology to the nuclear membrane protein POM121. Similar to POM121 the primary structures show a hydrophobic region that is likely to form one or more adjacent transmembrane segment(s). Indirect immunofluorescence with antibodies against a synthetic peptide gave staining of the nucleus. Target experiments with EGFP (enhanced green fluorescent protein)-fusion proteins confirmed the nuclear localization. Two further members of this gene family could be isolated. All three pore membrane and/or filament interacting like (POMFIL) genes are differentially expressed in neuronal tumor cell lines. In 40% of tested primary neuroblastomas expression of POMFIL1 is strongly reduced and after brain injury POMFIL1 protein expression is upregulated, indicating that POMFIL1 is involved in the process of neuron growth and regeneration, as well as in neural tumorigenesis.

The BMP2-dependent onset of osteo/chondrogenic differentiation in the acknowledged pluripotent murine mesenchymal stem cell line (C3H10T1/2) is accompanied by the immediate upregulation of Fibroblast Growth Factor Receptor 3 (FGFR3) and a delayed response by FGFR2. Forced expression of FGFR3 in C3H10T1/2 is sufficient for chondrogenic differentiation, indicating an important role for FGF-signaling during the manifestation of the chondrogenic lineage in this cell line. Screening for transcription factors exhibiting a chondrogenic capacity in C3H10T1/2 identified that the T-box containing transcription factor Brachyury is upregulated by FGFR3-mediated signaling. Forced expression of Brachyury in C3H10T1/2 was sufficient for differentiation into the chondrogenic lineage in vitro and in vivo after transplantation into muscle. A dominant-negative variant of Brachyury, consisting of its DNA-binding domain (T-box), interferes with BMP2-mediated cartilage formation. These studies indicate that BMP-initiated FGF-signaling induces a novel type of transcription factor for the onset of chondrogenesis in a mesenchymal stem cell line. A potential role for this T-box factor in skeletogenesis is further delineated from its expression profile in various skeletal elements such as intervertebral disks and the limb bud at late stages (18.5 d.p.c.) of murine embryonic development.

For five members of the family of the small leucine-rich proteoglycans (SLRPs), the expression pattern during fetal development was analyzed. RNA in situ hybridization on whole body sections of mouse embryos was performed for biglycan (Bgn), decorin (Dcn), fibromodulin (Fmod), chondroadherin (Chad), and lumican (Lum). Special attention was given to the question of whether these patterns coincide only with sites of collagen secretion in connective tissue during tissue modeling or if expression can be observed at specific sites of organ differentiation also. In general, Fmod, Lum, and Bgn are expressed at sites of cartilage and bone formation and interstitial tissue deposition; Chad is expressed only at sites of cartilage; and Dcn is expressed only at sites of interstitial tissue deposition. However, there are some distinct developmental stages where no collagen secretion is known to occur. For example, this applies for the expression of Fmod in the forming somites of stage 9.5 postconception (p.c.), for Dcn and Lum in later stage embryos in the pituitary gland and dorsal root ganglia, and for Bgn and Dcn during differentiation in the kidney. These studies provide further evidence for a role of these molecules during connective tissue organization but also for an involvement at specific sites of organ differentiation.

Mutations in the DKC1 gene are responsible for causing X-linked recessive dyskeratosis congenita (DKC) and a more severe allelic variant of the disease, Hoyeraal-Hreidarsson syndrome. Both diseases are characterized by progressive and fatal bone marrow failure. The nucleolar protein dyskerin is the pseudouridine synthase component of the box H+ACA snoRNAs and also interacts with the RNA component (human telomerase, hTR) of the telomerase complex. Dyskerin is therefore thought to function in the processing of pre-rRNA and of the hTR, strengthening the notion that the underlying mechanism of DKC is a premature senescence of cells, especially of the rapidly dividing epithelial and hemopoietic cells. To examine the functions of dyskerin in vivo, it will be necessary to generate mouse models. As a first step, we here provide the genomic structure of the mouse Dkc1 gene and expression analysis of the transcript. Northern hybridizations revealed the tissue-specific expression of an alternative 4.5-kb transcript, in addition to the ubiquitous 2.6-kb transcript. RNA in situ hybridizations on day 10.5-18.5 postconception embryos showed a ubiquitous expression of Dkc1 with a notably higher level of expression confined to the epithelial tissues. In addition, higher level Dkc1 expression was confined to embryonic neural tissues as well as to specific neurons in the cerebellum (Purkinje cells) and the olfactory bulb (mitral cells) of the adult brain. In adult testis, elevated expression was limited to the Leydig cells. The results indicate that some of the pertinent functions of dyskerin may be more tissue-specific than previously thought and are not limited to rapidly dividing cells., (Copyright 2000 Academic Press.)

To identify possible ligands of the orphan somatostatin-like receptor 1 (SLC-1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC-1 and G protein-gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N-terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK-mediated currents but also by the induction of Ca(2+) dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the G(i)- or G(q)-mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior.

A systematic search for expressed sequences in the human Xq28 region resulted in the isolation of 8.5 kb large contigs of human and murine cDNAs with no apparent conserved open reading frames. These cDNAs were found to be derived from the 3"-untranslated region (3"-UTR) of the methyl-CpG-binding protein 2 gene ( MeCP2 ). This long 3"-UTR is part of an alternatively polyadenylated, 10.1 kb MeCP2 transcript which is differentially expressed in human brain and other tissues. RNA in situ hybridization to sections of mouse embryo and adult tissues of an Mecp2 3"-UTR probe showed ubiquitous low level expression in early organogenesis and enhanced expression in the hippocampus during formation of the differentiated brain. Sequence comparison between the human and mouse homologues revealed several blocks of very high conservation separated by less conserved sequences. Additional support for a domain-like conservation pattern of the long 3"-UTR of the MeCP2 gene was obtained by examining conservation in the chimpanzee, orangutan, macaque, hamster, rat and kangaroo. The minimum free energy distribution for the predicted RNA secondary structure was very similar in human and mouse sequences. In particular, the conserved blocks were predicted to be of high minimum free energy, which suggests weak secondary structure with respect to RNA folding. The fact that both the sequence and predicted secondary structure have been highly conserved during evolution suggests that both the primary sequence and the three-dimensional structure of the 3"-UTR may be important for its function in post-transcriptional regulation of MeCP2 expression.

The murine homologue of the human motility-stimulating protein autotaxin (ATX) was identified as a BMP2 upregulated gene by subtractive cloning from mesenchymal progenitors C3H10T1/2 (Bächner, D., Ahrens, M., Betat, N., Schröder, D., Hoffmann. A., Lauber, J., Steinert, P., Flohe, L., Gross, G., 1998. Bmp-2 downstream targets in mesenchymal development identified by subtractive cloning from recombinant mesenchymal progenitors (C3H10T1/2). Dev. Dyn. 213, 398-411). ATX mRNA transcription is induced during BMP2 mediated osteo-/chondrogenic differentiation in vitro several orders of magnitude. To delineate a potential role for ATX in osteo-/chondrogenic development, its expression pattern during murine embryogenesis was examined in comparison with Col1a1 and Col2a1, a marker either of osteoblast, odontoblast and tendon or of chondrocyte development, respectively. Localization of murine ATX was first observed in the floor plate of the neural tube at day 9.5 of mouse embryonic development. Later, enhanced ATX expression levels were observed in proliferating subepithelial mesenchyme, during osteo-/chondrogenic and tooth development, in choroid plexus epithelium, in late kidney development, and in smooth muscles of the ductus deferens and the bladder.

Apolipoprotein E (ApoE) was identified as upregulated by Bmp-2 (bone morphogenetic protein-2) in the murine mesenchymal progenitor cell line C3H10T1/2 by a subtractive cloning strategy. Expression of recombinant Bmps in mesenchymal C3H10T1/2 progenitors results in the differentiation into the osteogenic, the chondrogenic, and the adipogenic lineage. In addition, ApoE is also expressed in primary osteoblasts isolated from murine calvariae late in the in vitro osteoblast developmental sequence. To infer possible roles of ApoE in organogenesis and tissue differentiation, ApoE expression during mouse embryonic development was analyzed in murine midgestation and late embryonic development by in situ hybridization. ApoE is highly expressed at many sites of organ development (liver, brain, heart, eye, lung), probably in a subset of neural crest cells and ectodermal derivatives suggestive for important functions of ApoE during embryonic differentiation and organ development.

ABmp-dependent in vitro model was used to identify cDNAs during the manifestation of mesenchymal lineages. This model involves the recombinant expression of Bmps (Bmp-2, Bmp-4-7) in murine mesenchymal C3H10T1/2 progenitors, which leads to the differentiation into three lineages: the osteogenic, the chondrogenic and the adipogenic lineage, albeit in varying efficiencies. By subtractive cloning, 21 Bmp-2-regulated cDNAs from C3H10T1/2 mesenchymal progenitors were identified; 20 were related to known sequences and 1 was not. During mouse embryonic development, many of these cDNAs are expressed in chondrogenic, osteogenic, and in adipogenic tissues. Novel findings include a G0/G1 switch gene (G0S2), which was demonstrated to be predominantly expressed in adipose tissue during late murine embryonic development. Furthermore, the membrane-standing glycoprotein autotaxin (ATX) is expressed, at precartilage condensations, joint regions, and during tooth development. An as yet undescribed cDNA, 29A, which encodes a putative secreted factor, is expressed in developing osteo-/chondrogenic tissues of vertebrae, ribs, tooth, and the limb bud. C3H10T1/2-progenitors, therefore, may serve as a legitimate model for the investigation of the Bmp-mediated events during mesenchymal differentiation.

Using RNA in situ hybridization we compared the expression patterns of the cell adhesion molecule-like receptor-type protein tyrosine phosphatases LAR, RPTP sigma and RPTP sigma during mouse development. We found that LAR is expressed in basal lamina-associated epithelial tissues of (neuro)ectodermal, neural crest/ectomesenchyme and endodermal origin. RPTP sigma is found in (neuro)ectodermal, neural crest-derived systems and in mesoderm-derived tissues. The expression pattern of RPTP sigma largely parallels that of RPTP sigma, in concordance with their proposed evolutionary history

In vertebrates several proteins containing a covalently bound selenocysteine residue have been identified. Among these, selenoprotein P is the most unusual one: depending on the species, 8-12 selenocysteine residues are cotranslationally integrated into the polypeptide chain. The protein was traced in rat plasma, but its role has not been worked out so far. In order to improve our understanding on selenoprotein P we investigated its tissue-specific expression and its genomic DNA. RNA in situ hybridization analyses confirmed the liver-specific expression in mice. Selenoprotein P was also found to be expressed in testis, brain, gut, and hematopoietic cells. The murine selp gene contains five exons within 10.3 kb with a coding sequence restricted to exons 2 to 5. The complete gene including the selp promoter was sequenced. One TATA motif 38 bp upstream to exon 1 suggests transcription of selp by RNA polymerase II. Within the 1116 bp upstream of exon 1 four hepatic nuclear factor 3beta (HNF3beta) binding motifs were found, which is in line with liver-specific expression of selenoprotein P. The expression in hematopoietic cells might be due to multiple GATA-1 motifs. Two BRN-2 motifs suitable for the binding of brain-specific regulatory factors correlated to the selenoprotein P expression in the cerebellum. Selenoprotein P was also expressed in Leydig cells which could be regulated by binding proteins docking to the SRY motifs present in the promoter region.

Construction of a transcript map in the DXS52 region in Xq28 had previously led to the isolation of a cDNA with a LIM zinc finger domain in the carboxyl terminus. In parallel, the orthologous murine transcript was isolated from the syntenic region. The human and mouse cDNAs have been designated ZNF185 and Zfp185, respectively. By integrating the cDNA sequence with the cosmid-derived genomic sequence the exon-intron structure of the 3' end of the ZNF185 gene was resolved. Comparative sequence analyses of the human genomic sequence with the full-length murine cDNA facilitated prediction of the 5' end of the gene. The selective expression of three transcripts corresponding to the ZNF185 gene and a related gene was shown by Northern and Southern blots. In situ hybridizations revealed a nonubiquitous and stage-specific expression of Zfp185, especially in differentiating connective tissue. Since LIM proteins regulate cellular proliferation and/or differentiation by diverse mechanisms, and some have directly been associated with disease, conceivably ZNF185 may represent a candidate for a disease-causing gene linked to Xq28. Knowledge of the genomic structure will permit detailed mutation analyses.

The complete cDNA for murine Beta-trace protein was isolated by RT-PCR using degenerate primers designed according to amino acid sequences derived from tryptic peptides. It encodes a protein of 165 amino acids (calculated molecular weight 18,472 Da) with a predicted 24-amino-acid leader peptide. In situ analyses during mouse embryonic development and in adult animals revealed a specific temporal expression pattern of Beta-trace. Beta-Trace mRNA was initially detected at 14.5 days postconception in mesenchymal cells destined to become leptomeninges and in the developing testis. Later in development, a lower level of expression was additionally observed in choroid plexus epithelium, in strictly confined regions of the eye (pigment and ciliary epithelium), in the ear (cochlear duct), and within single cells in the brain. Expression was also found in epithelia of the epididymis and the testis Leydig cells of postpubertal animals. The highly specific expression at blood-tissue barriers such as the blood-cerebrospinal fluid, blood-retina blood-aqueous humor, and blood-testis barriers indicates a potential role for this lipocalin in transport and/or in maturation and maintenance of these barriers.

Myotonic dystrophy (DM) is commonly associated with CTG repeat expansions within the gene for DM-protein kinase (DMPK). The effect of altered expression levels of DMPK, which is ubiquitously expressed in all muscle cell lineages during development, was examined by disrupting the endogenous Dmpk gene and overexpressing a normal human DMPK transgene in mice. Nullizygous (-/-) mice showed only inconsistent and minor size changes in head and neck muscle fibres at older age, animals with the highest DMPK transgene expression showed hypertrophic cardiomyopathy and enhanced neonatal mortality. However, both models lack other frequent DM symptoms including the fibre-type dependent atrophy, myotonia, cataract and male-infertility. These results strengthen the contention that simple loss- or gain-of-expression of DMPK is not the only crucial requirement for development of the disease.

A search for genes with sequence homologies to the FMR1 gene resulted in the isolation of mouse and human homologues of the recently described FXR1 gene. The mouse FXR1 gene shares amino acid identity and similarity of 99.1% and 99.6%, respectively, with the human FXR1 gene and amino acid identify and similarity of 67.3% and 79.5% respectively, with the mouse FMR1 gene. The 3' untranslated region of the FXR1 gene is extremely conserved between human and mouse. The gene structure of FXR1 is very similar to that of FMR1 and both genes probably originate from a common ancestral gene. In contrast to the previously published localization, we mapped the transcribed gene to chromosome region 3q28. An intronless form of the FXR1 gene, either processed functional homologue or pseudogene was localized to 12q12. Northern blot analysis of the human FXR1 gene revealed an expression pattern of a housekeeping gene with stronger expression in muscle. RNA in situ hybridization to sections of mouse embryo and adult tissues has shown that during embryonic development the mouse FXR1 mRNA is expressed in different tissues, most prominent in skeletal muscle, the gonads and distinct regions of the central nervous system, and that the expression is restricted to proliferating cells. While FMR1 is highly expressed in proliferating spermatogonia, FXR1 is highly expressed in postmeiotic spermatids.

Protein-tyrosine phosphatases (PTPases) form an important class of cell regulatory proteins. We have isolated overlapping cDNA clones that together comprise an 8 kb transcript encoding a novel murine PTPase which is expressed in various organs. Sequence analysis revealed an open reading frame of 2,460 amino acid residues. The predicted protein, PTP-BL, is a large non-transmembrane PTPase that exhibits 80% homology with PTP-BAS, a recently described human PTPase. PTP-BL shares some intriguing sequence homologies with submembranous proteins. It contains a band 4.1-like motif also present in the tumor suppressors neurofibromatosis 2 and expanded, five 80 amino acid repeats also present in the discs-large tumor suppressor, and a single catalytic phosphatase domain. No obvious homologies to other proteins were found for the N-terminal region of the protein other than human PTP-BAS. RNA in situ hybridization experiments show that the PTP-BL gene is expressed in epithelial cells, predominantly in kidney, lung, and skin. These data suggest a cell cortical localization for PTP-BL in epithelial cells and a possible role in the morphology and motility of epithelial tissues.

Huntington's disease (HD) is associated with an expanded and unstable (CAG) > 35 repeat within a gene of unknown function. We isolated the complete coding region of the rat HD gene (rhd) from cDNA libraries and investigated its expression in different developmental stages of rodent tissues. The rat gene exhibits 90% peptide sequence identity to the human and 96% to the murine sequence. The (CAG)n repeat is markedly reduced in the rat compared to the average human (CAG)n block. Northern blot analysis and in situ hybridizations reveal that in rodents the hd gene is already expressed during embryonal development. As in humans, the rhd gene is expressed in two transcriptional isoforms which result from different polyadenylation signals. In mice, however, a third transcript of intermediate size was found predominantly expressed in brain. This transcript is downregulated in later development. At day 14.5 p.c. the level of rhd expression is similar in the brain and in non-neuronal tissues. In contrast, the expression in non-neuronal tissues is markedly reduced in adult animals and corresponds to the restricted distribution of neuropathologic changes observed in HD patients.

The diverse biological consequences of size-expansion of the unstable (CTG)n repeat in the myotonic dystrophy protein kinase (DM-PK) gene at chromosome region 19q13.3, are still poorly understood. Abnormal (CTG)n length may affect either DM-PK mRNA fate or function, or alternatively, compromise gene transcription by distortion of chromatin configuration. In the latter model involvement of neighbouring genes in DM upon extreme expansion of the repeat cannot be discarded as a possibility and should be studied further. Here we report on the elucidation of the complete genomic structure and expression pattern of the mouse DMR-N9 gene (called 59 gene in humans), which is at 1.1 kbp upstream of the DM-PK gene. This gene contains five exons spanning 7 kbp and codes for a protein of 650 amino acids. Two regions of the predicted protein show significant homology to WD repeats, highly conserved amino acid sequences found in a family of proteins engaged in signal transduction or cell regulatory functions. The start site of transcription has been determined and we have identified putative transcription factor binding sequences in a 400 bp putative promoter area immediately upstream of the transcribed unit. Northern blotting analysis and RNA in situ hybridization revealed ubiquitous low expression in all tissues of the mouse embryo and enhanced expression in adult brain and testis. The onset of transcription is phased early in mouse embryogenesis, before or at day 9.5 of gestation. From day 14.5 onwards DMR-N9 mRNAs were detected in all neural tissues, especially in the telencephalon and mesencephalon. Later, mRNA presence is evident in distinct tubules of the mature testis, restricted to secondary spermatocytes of stages VIII to XII of the spermatogenic proliferation cycle. We conclude that the DMR-N9 gene is a candidate for being involved in the manifestation of mental and testicular symptoms in severe cases of DM.

We have analysed the expression patterns of two human genes coding for two different rab GDIs, rab GDI alpha/XAP-4 and rab GDI beta, proteins involved in the regulation of vesicle-mediated cellular transport. The gene sequences are extremely conserved in evolution, with substantial homology preserved across three eukaryotic kingdoms. Although the sequence homology between the two human rab GDIs studied is very high, their expression patterns are completely different. The Northern blot analysis and in situ hybridization to sections of mouse embryos and postnatal tissues have revealed that the rab GDI alpha/XAP-4 is expressed predominantly in neural and sensory tissues and may thus serve a specific function in neural signal transmission. In contrast to rab GDI alpha/XAP-4, the human rab GDI beta is expressed ubiquitously.

To elucidate the function of the FMR1 gene, we applied RNA in situ hybridization to cryosections of mice from different developmental stages. The murine Fmr-1 was found transcribed in a ubiquitous manner with an expression pattern similar to glyceraldehyd phosphate dehydrogenase, Gapdh, which was used as a control gene. A significant difference in the Fmr-1 expression pattern, however, was markedly enhanced expression specifically confined to the testis and the fetal ovary. In the immature and mature testis an elevated level of Fmr-1 expression is found in type A1 spermatogonia. Expression in the testis is observed in fetal life, reaches the highest level in the immature testis, and declines early in adult life. In the mature ovary no specific Fmr-1 expression signal was found but enhanced levels were seen in the fetal ovary. At this developmental stage proliferation of oogonia takes place. It is suggested that FMR1 serves a special function during germ cell proliferation in males and females. These findings are discussed in the light of the current observation that fragile X patients produce only sperm with a premutation sized allele. Two hypotheses are put forward: (1) In males lack of FMR1 function results in a premeiotic defect preventing spermatogonia with a full mutation to reach meiosis. A fragile X mutation can be passed on to offsprings only as a premutation (selection hypothesis). (2) Transition of a premutation allele to full mutation occurs in a postzygotic stage after separation of the germ line and is restricted to soma cells (restriction hypothesis). Expression of FMR1 in proliferating germ cells is in line with both hypothesis.