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.

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.

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.

Improved radiation hybrid map of rat chromosome 2: colocalization of the genes encoding corticotropin-releasing hormone and IL6-receptor with quantitative trait loci regulating the inflammatory response.

We established a radiation hybrid (RH) map of several genes and anonymous markers in the lower half of rat chromosome 2, a chromosome region that contains quantitative trait loci (QTLs) for blood pressure, diabetes and inflammatory response. Two of the newly localized genes (Crh and Il6r) encode proteins involved in the regulation of inflammatory and immune events. Our data show that they reside within regions that were genetically defined as QTLs controlling the inflammatory response. These genes are thus both functional and positional candidates.

Analysis of candidate genes included in the mammary cancer susceptibility 1 (Mcs1) region.

The rat strain COP is resistant to spontaneous and carcinogen-induced mammary cancer, whereas the strain WF is susceptible. Using genetic linkage analysis of (WF x COP) F1 x WF backcrosses, LC Hsu, LA Shepel and co-workers showed that a region at the centromeric end of Chromosome (Chr) 2 (2q1) segregates with the sensitivity to mammary cancer development. The responsible locus was named Mcs1 (for mammary cancer susceptibility 1). We have developed the chromosome map of the 2q1 region by localizing 18 genes, 4 ESTs, and several anonymous markers, using radiation hybrids and fluorescence in situ hybridization. The region containing Mcs1 was delineated to 2q12-q14. Five of the genes (Mef2c, Map1b, Ccnh, Rasa, Rasgrf2) were assigned to this region and were shown to be expressed in the rat mammary glands, while three possible functional candidate genes, Pi3kr1, Rad17, and Naip, were excluded from the critical region. Since cyclin H, encoded by Ccnh, plays an important role in the control of the cell cycle and since the proteins encoded by Rasa and Rasgrf2 control the activity of the RAS oncoprotein, the corresponding genes appeared as both functional and positional Mcs1 candidates. RT-PCR experiments on RNA extracted from mammary glands of the two rat strains (COP, WF) was done. No amino acid sequence difference was found between the two strains. These results argue against the hypothesis that any of these three genes is Mcs1.

Identification of KLF13 and KLF14 (SP6), novel members of the SP/XKLF transcription factor family.

Using the sequence of the SP1 zinc-finger DNA-binding domain as a probe to screen a mouse EST database, we identified two novel members of the SP/XKLF transcription factor family, KLF13 and KLF14. The mouse Klf13 cDNA (1310 bp in length) contains a single open reading frame of 288 amino acids with a DNA-binding domain closely related to that of the human RFLAT-1 protein and a putative transactivator N-terminal domain rich in proline and alanine residues. The mouse Klf13 gene seems to be the homologue of the human RFLAT1 gene. The mouse Klf14 sequence is homologous to a human genomic sequence from chromosome 17 that is believed to code for a protein with three zinc fingers at the end of its C-terminal domain. Using reverse transcription-polymerase chain reaction, we showed ubiquitous expression of Klf13 and Klf14 in adult mice. A third member of this family was also identified in a human EST database; this sequence was found to be identical to KLF11 (TIEG2), recently identified by Cook et al. (1998, J. Biol. Chem. 273: 25929-25936). The corresponding mouse cDNA was isolated and sequenced. The three genes were localized in the human and the rat: chromosomes 15 (human KLF13), 17q21.3-q22 (human KLF14; HGMW-approved symbol SP6), and 2p25 (human KLF11) and chromosomes 1q31-q32 (rat Klf13), 10q31-q32.1 (rat Klf14) (SP6), and 6q16-q21 (rat Klf11).

Localization of 54 rat genes, and definition of new synteny groups conserved in the human and the rat.

In order to improve the rat gene map and comparative mapping with the human and the mouse, we determined the chromosome localization of 54 rat genes. Most genes encode transcription factors or other regulatory proteins of cancer relevance. The human homologs of four genes were also assigned to their respective chromosome. These data generated anchor points between the recently established radiation hybrid maps and the genetic and cytogenetic maps. They improve comparative mapping between the rat, the mouse, and the human gene maps, and in particular they disclose four new synteny groups conserved in the rat and the human. These new localizations should also be useful for the identification of genes involved in the control of quantitative traits such as cancer susceptibility or diabetes.

Localization of new, microdissection- generated, anonymous markers and of the genes Pcsk1, Dhfr, Ndub13, and Ccnb1 to rat chromosome region 2q1.

The centromeric region of rat chromosome 2 (2q1) harbors unidentified quantitative trait loci of genes that control tumor growth or development. To improve the mapping of this chromosome region, we microdissected it and generated 10 new microsatellite markers, which we included in the linkage map and/or radiation hybrid map of 2q1, together with other known markers, including four genes: Pcsk1 (protein convertase 1), Dhfr (dihydrofolate reductase), Ndub13 (NADH ubiquinone oxidoreductase subunit b13), and Ccnb1 (cyclin B1). To generate anchor points between the different maps, the gene Ndub13 and the microsatellite markers D2Ulb25 and D2Mit1 were also localized cytogenetically. The radiation map generated in region 2q1 extends its centromeric end of about 150 cR.

Genes encoding atrial and brain natriuretic peptides as candidates for sensitivity to brain ischemia in stroke-prone hypertensive rats.

-Previous studies suggested that atrial natriuretic peptide gene (Anp) and brain natriuretic peptide gene (Bnp) are plausible candidate genes for susceptibility to stroke and for sensitivity to brain ischemia in the stroke-prone spontaneously hypertensive rat (SHRSP). We performed structural and functional analyses of these 2 genes in SHRSP from Glasgow colonies (SHRSPGla) and Wistar-Kyoto rats from Glasgow colonies (WKYGla) and developed a radiation hybrid map of the relevant region of rat chromosome 5. Sequencing of the coding regions of the Anp and Bnp genes revealed no difference between the 2 strains. Expression studies in brain tissue showed no differences at baseline and at 24 hours after middle cerebral artery occlusion. Plasma concentrations of atrial natriuretic peptide (ANP) did not differ between the SHRSPGla and WKYGla, whereas concentrations of brain natriuretic peptide were significantly higher in the SHRSPGla as compared with the WKYGla (n=11 to 14; 163+/-21 pg/mL and 78+/-14 pg/mL; 95% confidence interval 31 to 138, P=0.003). We did not detect any attenuation of endothelium-dependent relaxations to bradykinin or ANP in middle cerebral arteries from the SHRSPGla; indeed the sensitivity to ANP was significantly increased in arteries harvested from this strain (WKYGla: n=8; pD2=7. 3+/-0.2 and SHRSPGla: n=8; pD2=8.2+/-0.15; P<0.01). Moreover, radiation hybrid mapping and fluorescence in situ hybridization allowed us to map the Anf marker in the telomeric position of rat chromosome 5 in close proximity to D5Rat48, D5Rat47, D5Mgh15, and D5Mgh16. These results exclude Anp and Bnp as candidate genes for the sensitivity to brain ischemia and pave the way to further congenic and physical mapping strategies.

Rat Chromosome 2: assignment of the genes encoding cyclin B1, interleukin 6 signal transducer, and proprotein convertase 1 to the Mcs1-containing region and identification of new microsatellite markers.

The rat Chromosome (Chr) 2 harbors several genes controlling tumor growth or development, blood pressure, and non-insulin-dependent diabetes mellitus. We report that the region (2q1) containing the mammary susceptibility cancer gene Mcs1 also harbors the genes encoding cyclin B1, interleukin 6 signal transducer (gp130), and proprotein convertase 1. We also generated 13 new anonymous microsatellite markers from Chr 2-sorted DNA. These markers, as well as a microsatellite marker in the cyclin B1 gene, were genetically mapped in combination with known markers. A cyclin B1-related gene was also cytogenetically assigned to rat Chr 11q22-q23.