The Sp6 locus uses several promoters and generates sense and antisense transcripts.

The SP/KLF transcription factor family contains over 25 members sharing a DNA-binding domain composed of three zinc fingers of the C(2)H(2) type. We previously identified the sixth member of the SP subfamily (Sp6). The 5' end of the Sp6 transcript was not cloned and was predicted bioinformatically. A mouse molar tooth cDNA was then isolated differing from the Sp6 sequence by its 5' end, and was named epiprofin. Sp6 and epiprofin are currently used as synonyms. Here, we show that the Sp6 transcript possesses a first exon distinct from the epiprofin one: the Sp6 gene thus uses two promoters, generating two transcript variants which differ in their first exon. Furthermore, we identified an Sp6 opposite strand transcript (Sp6os) and examined, by quantitative RT-PCR experiments, the presence and the abundance of these two transcripts in mouse tissues. We also mapped the mouse locus by FISH to chromosome 11D.

Exposure to oestrogen prenatally does not interfere with the normal female-typical development of odour preferences.

The neural mechanisms controlling mate recognition and heterosexual partner preference are sexually differentiated by perinatal actions of sex steroid hormones. We previously showed that the most important action of oestrogen during prenatal development is to defeminise and, to some extent, masculinise brain and behaviour in mice. Female mice deficient in alpha-foetoprotein (AFP) due to a targeted mutation in the Afp gene (AFP-KO) do not show any female sexual behaviour when paired with an active male because they lack the protective action of AFP against maternal oestrogens. In the present study, we investigated whether odour preferences, another sexually differentiated trait in mice, are also defeminised and/or masculinised in AFP-KO females due to their prenatal exposure to oestrogens. AFP-KO females of two background strains (CD1 and C57Bl/6j) preferred to investigate male over female odours when given the choice between these two odour stimuli in a Y-maze, and thus remained very female-like in this regard. Thus, the absence of lordosis behaviour in these females cannot be explained by a reduced motivation of AFP-KO females to investigate male-derived odours. Furthermore, the presence of a strong male-directed odour preference in AFP-KO females suggests a postnatal contribution of oestrogens to the development of preferences to investigate opposite-sex odours.

Rat gene mapping in the post-genome sequencing era: the continued utility of cell hybrids to localize rat genes (Cks2, Ephb4, Fabp5, Il13ra1, Rpl10, Ssr4).

Finding the position of a gene is now easily done when the genome sequence is available: the gene position is generally found by a simple query of genomic databases such as those available at the Ensembl browser or the NCBI. We were interested in determining the position of 125 cancer-related rat genes and we found that the position of most of these genes (110) could indeed be identified in this manner. However, in 15 cases, the gene position was not available in these databases, or the results were ambiguous. We then explored a more specialized database, namely the Rat Genome Database, and experimentally mapped these genes using standard and radiation cell hybrids. The 15 genes in question could be localized unambiguously. In four cases, the radiation cell hybrids were indispensable: the sequence of these four genes could not be found in the rat genome sequence. On the basis of the sample we examined, it thus appears that a classical gene mapping method is still required to localize about 3% of the rat genes, as if 3% of the rat gene sequences were lacking in the current rat genome sequence.

Regulation of the alpha-fetoprotein promoter: Ku binding and DNA spatial conformation.

This work shows that the proximal promoter of the mouse Afp gene contains a Ku binding site and that Ku binding is associated with down-regulation of the transcriptional activity of the Afp promoter. The Ku binding site is located in a segment able to adopt a peculiar structured form, probably a hairpin structure. Interestingly, the structured form eliminates the binding sites of the positive transcription factor HNF1. Furthermore, a DNAse hypersensitive site is detected in footprinting experiments done with extracts of AFP non-expressing hepatoma cells. These observations suggest that the structured form is stabilised by Ku and is associated with extinction of the gene in AFP non-expressing hepatic cells.

Physiological role of the putative ammonium transporter RhCG in the mouse.

Ammonium excretion into urine is a major process essential to the regulation of acid-base homeostasis. We have shown that Rh-type proteins, including renal RhCG, belong to the Mep/Amt family of ammonium transporters and promote bi-directional ammonium transport upon heterologous expression in yeast. To study the physiological role of RhCG and to test a potential function in ammonium excretion, we have generated mice bearing an invalidation of the corresponding gene.

Genetic analysis of susceptibility to endometrial adenocarcinoma in the BDII rat model.

Most cancers are genetically complex and heterogeneous, a serious obstacle to identifying specific genes underlying the disease. If inbred animal models are used, then both the genetic constitution and environmental influences can be carefully controlled. Females of the BDII inbred rat strain are genetically predisposed to endometrial cancer; more than 90% of virgin BDII females will develop endometrial adenocarcinoma (EAC) during their life span. BDII females were crossed to males from inbred strains with low EAC incidence (SPRD or BN). When F(1) males were backcrossed to BDII females to generate N(1) populations of offspring, about one fourth of the female progeny developed EAC. With transmission disequilibrium test analysis, significant association was detected in three chromosomal regions (on RNO1, RNO11, and RNO17) in the SPRD crosses and in the short arm of RNO20 in the BN crosses. It appears that several susceptibility genes with minor but cooperating effects are responsible for the susceptibility. Furthermore, it seems clear from the interstrain crosses not only that the onset of tumors depends on the presence of susceptibility alleles from the EAC-prone BDII strain, but also that tumor development is affected by the contribution of a genetic component derived from the nonsusceptible strains.

Chromosome evolution of MMU16 and RNO11: conserved synteny associated with gene order rearrangements explicable by intrachromosomal recombinations and neocentromere emergence.

Comparative mapping between the rat and mouse genomes has shown that some chromosomes are entirely or almost entirely conserved with respect to gene content. Such is the case of rat chromosome 11 (RNO11) and mouse chromosome 16 (MMU16). We determined to what extent such an extensive conservation of synteny is associated with a conserved gene order. Therefore, we regionally localized several genes on RNO11. The comparison of the gene map of RNO11 and MMU16 unambiguously shows that the gene order has not been conserved in the Murinae lineage, thereby implying the occurrence of intrachromosomal evolutionary rearrangements. The transition from one chromosome configuration to the other one can be explained either by two intrachromosomal recombinations or by a single intrachromosomal recombination accompanied by neocentromere emergence.

Characterization, expression pattern and chromosomal localization of the spermatogenesis associated 6 gene (Spata6).

We report the cloning and characterization of the spermatogenesis associated 6 gene (Spata6) encoding a predicted protein of 488 amino acids. It exhibits similarity with the motor domain of kinesin related proteins and with the Caenorhabditis elegans neural calcium sensor protein (NCS-2). The gene encodes three mRNAs of approximately 2.6, approximately 1.8 and approximately 1.2 kb. The expression of the 2.6 kb mRNA is detected at low levels in testis, ovary, thymus and placenta, while the 1.8 and 1.2 kb transcripts are exclusively expressed in testis. The 1.8 and 1.2 kb transcripts are specifically expressed in haploid germ cells. Data from in situ hybridization experiments suggested that mRNA expression of Spata6 in spermatids is higher than in spermatocytes and spermatogonia. RT-PCR analysis and whole mount in situ hybridization demonstrate that the Spata6 transcript is expressed during embryonic development and is localized in neural tube, somites and limb buds of mouse embryo. The Spata6 gene consists of 15 exons ranging in size between 40 and 596 bp. The 2.6 and 1.8 kb transcripts have different 5' untranslated sequences but have the same translational initiation site and therefore may encode the same protein with a predicted molecular weight of 49.7 kDa. The 1.2 kb transcript is derived from a proximal promoter between exons 7 and 8, and contains a translation initiation codon AUG, which is in frame with initiator AUG codon of the 2.6 and 1.8 kb transcripts. Therefore, the 1.2 kb transcript may code for a truncated protein of 32 kDa. Western blot analysis with the antiserum raised against a synthetic peptide from the C-terminal of the deduced Spata6 protein detects only a single protein of 53 kDa in all tissues studied. The Spata6 gene was localized to chromosome 5, region q34-35 in the rat and to chromosome 1, region p32-35 in the human. In an effort to determine the function of Spata6, we inactivated the mouse gene in embryonic stem cells through homologous recombination. Although the heterozygous mutant cells were able to generate low coat colour chimeric mice, all chimeras did not transmit the targeted allele to their progeny suggesting that a high contribution of Spata6(+/-) cells lead to the lethality of the chimeric embryos.

Genomic organisation and chromosomal assignment of ODF2 (outer dense fiber 2), encoding the main component of sperm tail outer dense fibers and a centrosomal scaffold protein.

ODF2 (outer dense fiber 2) was first described as the main protein component of the sperm tail cytoskeleton, the outer dense fibers, but was shown recently to be a component of the centrosomal scaffold in chicken. In mouse two related ODF2 cDNA clones were isolated which have been suggested to be most likely the result of alternative splicing. We show here the exon/intron organisation of mouse ODF2 and demonstrate that alternative splicing results in related cDNA sequences and most likely explains, at least partially, the highly complex protein pattern detected on Western blots. ODF2 was mapped to rat chromosome 3 and more specifically by FISH analysis at bands 3q11-->3q12. In addition, we demonstrate that ODF2 is indeed a component of the centrosome and the mitotic spindle poles in mammals.

Genetic identification of multiple susceptibility genes involved in the development of endometrial carcinoma in a rat model.

There are clear indications that inheritance plays an essential role in certain cases of human endometrial cancer, and there are at least 2 forms of early-onset heritable endometrial adenocarcinomas (EACs). Females of the BDII inbred rat strain are known to be genetically predisposed to endometrial carcinoma, and we have performed a genetic analysis of susceptibility to endometrial cancer in this strain. F(2) populations were generated by crossing BDII females with males from 2 different strains with a low incidence of EAC, and the occurrence of endometrial cancer was studied. Three chromosome regions associated to EAC susceptibility were identified, and the susceptibility genes in these regions were designated Ecs1, Ecs2 and Ecs3. Our results indicate that the genes affecting susceptibility to EAC are different in the 2 crosses, suggesting that the genes behind the susceptibility in BDII animals may interact with different genes in different genetic backgrounds.

Independent amplification of two gene clusters on chromosome 4 in rat endometrial cancer: identification and molecular characterization.

The BDII rat is genetically predisposed to hormone-dependent endometrial adenocarcinoma and was used to model human cancer. Tumors arising spontaneously in strain crosses involving BDII rats were analyzed by means of comparative genome hybridization. The most common aberration was amplification of the proximal region of rat chromosome 4, centered around bands q12-q22. The copy numbers of 15 cancer-related genes from the region were examined in tissue cultures of 11 endometrial carcinomas (10 endometrial adenocarcinomas and 1 endometrial squamous cell carcinoma) and one peritoneal mesothelioma. Amplification in rat chromosome 4 was detected in six tumors (50%), five of which carried two separate amplified regions, situated at 4q12-q13 and 4q21-q22, interrupted by a nonamplified segment at 4q13-q21.1. The genes Cdk6 (cyclin-dependent kinase 6) and Met (hepatocyte growth factor receptor) were located in the core of each amplified region and were amplified most recurrently and at the highest levels among the genes tested. Using fluorescence in situ hybridization on tumor metaphases, it was observed that the amplified Cdk6 and Met sequences were situated on typical homogeneously staining regions (HSRs). In three tumors, both genes were amplified in the same HSRs, whereas in two tumors, the amplified sequences of each gene were situated in separate HSRs. In addition, Cdk6 and Met amplification was consistently associated with a corresponding increase in gene expression, suggesting that the two genes might represent the targets for the amplifications. In the sixth tumor, which carried amplified sequences of Met but not of Cdk6, coexpression of Met and the normally silent hepatocyte growth factor gene (Hgf; the ligand of Met) was observed. This finding suggests that an autocrine signaling circuit might be operating in this particular tumor. Taken together, our findings suggest that up-regulation of Cdk6 and/or Met may contribute to the development of endometrial cancers in the BDII rat.

Cloning of the rat IL-5Ralpha gene: analysis of 5'-upstream region and expression by B cells.

Although rats are widely used for the analysis of allergic reactions and parasitic infections where IL-5 is involved, nothing is currently known of the expression of IL-5 receptor in this species. In this study, the cDNA sequence, genomic structure and the transcriptional regulation of the rat IL-5Ralpha were analyzed. The rat IL-5Ralpha gene, which we localized to chromosome 4q34-q41, spans more than 25 kb and consists of 12 exons. Promoter activity was seen in different cell lines and analysis by deletion experiments allowed to identify two negative regulatory regions which did not differ when tested either with IL-5Ralpha-negative or positive cells. Finally, the investigation of the expression of IL-5Ralpha showed that it is expressed in lung, spleen, liver, and purified rat B cells from normal rat. This can provide an explanation for the role of rat IL-5 as B-cell growth factor and a relevant model in order to better understand the activity of IL-5 on human B cells.

Mapping of the rat glypican genes.

The glypicans compose a family of glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycans that play a role in the control of cell division and growth regulation. So far, six members (GPC1-6) of this family are known in vertebrates. The rat glypican gene 3 (Gpc3) was previously assigned to chromosome Xq36 (Shen et al., 1997). Using standard and radiation cell hybrids, we localized the five other rat glypican genes.

Amplification of Mycn, Ddx1, Rrm2, and Odc1 in rat uterine endometrial carcinomas.

The BDII rat is genetically predisposed to estrogen-dependent endometrial adenocarcinoma and represents a valuable model for this type of tumor. Tumors arising in strain crosses involving the BDII rats had previously been screened for DNA copy number changes using comparative genome hybridization (CGH). It was found that extra copies of the proximal region of rat chromosome (RNO) 6 commonly could be detected in these tumors. Based on RH-mapping data and comparative mapping with mouse and human, seven cancer-related genes were predicted to be situated in RNO6q14-q16. Rat PACs were isolated for the N-myc proto-oncogene (Mycn), apolipoprotein B (Apob), the DEAD box gene 1 (Ddx1), ornithine decarboxylase 1 (Odc1), proopiomelanocortin (Pomc1), ribonucleotide reductase, M2 polypeptide (Rrm2), and syndecan 1 (Sdc1). The localization of the genes to the region was verified by FISH (fluorescence in situ hybridization) mapping, and the detailed order among them was determined by dual-color FISH. By Southern blot analysis, it was found that the Mycn locus was highly amplified in two out of 10 cell cultures derived from the tumors. In one of them (designated RUT30), the amplification level of Mycn was estimated at 140x. Two other genes were coamplified (Ddx1 and Rrm2) at much lower levels. Similarly, in another culture (designated RUT2), Mycn was amplified more than 40x, whereas three of the other genes (Ddx1, Rrm2, and Odc1) were coamplified at lower levels. Using FISH on metaphase chromosomes from the cell cultures analyzed, the amplified sequences were shown to be located in typical HSRs. With competitive RT-PCR, distinct overexpression of Mycn and Ddx1 could be demonstrated in both RUT2 and RUT30. In addition, Mycn was overexpressed in two other tumors not exhibiting Mycn amplification. Taken together, our results suggest that overexpression of Mycn plays an important role in the development of endometrial cancer in the BDII rat. In humans, Mycn amplification has been reported mainly from tumors of neuronal origin. To our knowledge, this is the first report of Mycn amplification and overexpression in hormone-dependent tumors.

Identification of the gene encoding Brain Cell Membrane Protein 1 (BCMP1), a putative four-transmembrane protein distantly related to the Peripheral Myelin Protein 22 / Epithelial Membrane Proteins and the Claudins.

BACKGROUND: A partial cDNA clone from dog thyroid presenting a very significant similarity with an uncharacterized mouse EST sequence was isolated fortuitously. We report here the identification of the complete mRNA and of the gene, the product of which was termed "brain cell membrane protein 1" (BCMP1). RESULTS: The 4 kb-long mRNA sequence exhibited an open-reading frame of only 543 b followed by a 3.2 kb-long 3' untranslated region containing several AUUUA instability motifs. Analysis of the encoded protein sequence identified the presence of four putative transmembrane domains. Similarity searches in protein domain databases identified partial sequence conservations with peripheral myelin protein 22 (PMP22)/ epithelial membrane proteins (EMPs) and Claudins, defining the encoded protein as representative of the existence of a novel subclass in this protein family.Northern-blot analysis of the expression of the corresponding mRNA in adult dog tissues revealed the presence of a huge amount of the 4 kb transcript in the brain. An EGFP-BCMP1 fusion protein expressed in transfected COS-7 cells exhibited a membranous localization as expected. The sequences encoding BCMP1 were assigned to chromosome X in dog, man and rat using radiation hybrid panels and were partly localized in the currently available human genome sequence. CONCLUSIONS: We have identified the existence in several mammalian species of a gene encoding a putative four-transmembrane protein, BCMP1, wich defines a novel subclass in this family of proteins. In dog at least, the corresponding mRNA is highly present in brain cells. The chromosomal localization of the gene in man makes of it a likely candidate gene for X-linked mental retardation.

Analysis of genetic changes in rat endometrial carcinomas by means of comparative genomic hybridization.

Animals of the BDII inbred rat strain are known to be genetically predisposed to endometrial adenocarcinoma (EAC). Using them as models of human EACs, we studied tumors arising in F1 and F2 progeny from BDII animals crossed to animals from two other inbred strains, in which EACs were quite rare. In order to identify chromosomal regions exhibiting DNA copy number changes, comparative genomic hybridization (CGH) was applied in a series corresponding to 27 different solid tumors, most of which were classified as EACs, from these animals. The main findings from the study were that, although many different chromosomes were involved in copy number variation, some of the changes detected were recurrent and quite specific. Among specific changes found were gains in rat chromosome (RNO) regions 4q12 approximately q22, 6q14 approximately q16, and whole chromosome arms in some of the small metacentric chromosomes (e.g., RNO14, 16, and 18). RNO10 was involved in gain in the terminal and proximal regions. Each of these regions contains previously identified cancer-related genes representing possible candidates to be involved in the development of EAC. Furthermore, it was observed that there were clear differences in the pattern of copy number changes between tumors occurring in the two different crosses, and also between solid tumors and cell cultures. Endometrial cancer is the most common human gynecological cancer, but not much is known about specific genetic changes influencing this disease. Two genetic alterations that have been reported from human endometrial cancer are amplification of the ERBB2 gene and mutations in the 12 codon of the KRAS gene. One case of Erbb2 amplification was found but there were no Kras mutations in the rat material studied. We conclude that molecular genetic analysis of the rat BDII model will provide important new information about EAC in mammals.

Rat-mouse and rat-human comparative maps based on gene homology and high-resolution zoo-FISH.

The laboratory rat, Rattus norvegicus, and the laboratory mouse, Mus musculus, are key animal models in biomedical research. A deeper understanding of the genetic interrelationsships between Homo sapiens and these two rodent species is desirable for extending the usefulness of the animal models. We present comprehensive rat-human and rat-mouse comparative maps, based on 1090 gene homology assignments available for rat genes. Radiation hybrid, FISH, and zoo-FISH mapping data have been integrated to produce comparative maps that are estimated to comprise 83-100% of the conserved regions between rat and mouse and 66-82% of the conserved regions between rat and human. The rat-mouse zoo-FISH analysis, supported by data for individual genes, revealed nine previously undetected conserved regions compared to earlier reports. Since there is almost complete genome coverage in the rat-mouse comparative map, we conclude that it is feasible to make accurate predictions of gene positions in the rat based on gene locations in the mouse.

Rat adrenoleukodystrophy-related (ALDR) gene: full-length cDNA sequence and new insight in expression.

X-linked adrenoleukodystrophy (X-ALD) is an inherited demyelinating disorder due to mutations in the ALD gene, which encodes a peroxisomal ABC half-transporter (ALDP). It has been suggested that ALDP assembles with ALDRP (adrenoleukodystrophy-related protein), a close homologous half-transporter, to form a functional heterodimer. For the first time full-length ALDRP cDNA (5.5 kb) was cloned, and 5' and 3' RACE analysis revealed that alternative usage of polyadenylation sites generates the two transcripts of 3.0 and 5.5 kb observed in the rat in Northern blot analysis. Southern blotting and chromosomal mapping demonstrated one ALDR locus in the rat genome. Characterisation of the 3' flanking region suggested that an ID sequence might be responsible for high expression of the 5.5 kb ALDRP transcript in rat brain. ALDR gene expression was found to be high in the liver of rats before weaning and very low in adult rats; the reverse developmental regulation was observed in the brain. Fenofibrate, which is a potent inducer of the ALDR gene in the liver of adult rats, could not induce the ALDR gene in suckling rats. The exact significance of this result with regard to development of an efficient pharmacological gene therapy for X-ALD is discussed.