Fadel Tissir

Gene-based anchoring of the rat genetic linkage and cytogenetic maps: new regional localizations, orientation of the linkage groups, and insights into mammalian chromosome evolution.

Abstract. In order to generate anchor points connecting the rat cytogenetic and genetic maps, the cytogenetic position of 62 rat markers (including 55 genes) already localized genetically was determined by fluorescence in situ hybridization. Whenever possible, markers located near one end of the linkage groups were included. These new localizations allowed us to unambiguously orient the 20 autosomal and the X chromosome linkage groups. The position of the centromere in the linkage map could also be determined in the case of several metacentric chromosomes. In addition, the regional localization of 15 other rat genes was determined. These new data bring useful information with respect to comparative mapping with the mouse and the human and to mammalian evolution. They illustrate, for instance, that groups of genes can remain syntenic during mammalian evolution while being subjected to intrachromosomal rearrangements in some lineages (synteny is conserved while gene order is not). This analysis also disclosed cases of synteny conservation in one the two rodent species and the human, while the synteny is split in the other rodent species: such configurations are likely examples of lineage-specific interchromosomal rearrangements associated with speciation.

The rat genes encoding the pancreatitis-associated proteins I, II and III (Pap1, Pap2, Pap3), and the lithostathin/pancreatic stone protein/regeneration protein (Reg) colocalize at 4q33-->q34.

Using fluorescence in situ hybridization, we determined that the three rat PAP genes, and the related REG gene map in the same chromosomes region, namely 4q33-->q34. This rat chromosome region is thus homologous to the human 2p12 region, which also contains the PAP gene, the REG1A gene, and a REG-related gene (REGL).

Localization of the genes encoding the three rat angiotensin II receptors, Agtr1a, Agtr1b, Agtr2, and the human AGTR2 receptor respectively to rat chromosomes 17q12, 2q24 and Xq34, and the human Xq22

Using fluorescence in situ hybridization, we determined the regional localization of the 3 rat genes encoding angiotensin II receptors at 17q12 (Agtr1a), 2q24 (Agtr1b) and Xq34 (Agtr2). In parallel, we showed that the type 2 human gene, AGTR2, also maps on the X chromosome, at band Xq22.

Assignment of rat Jun family genes to chromosome 19 (Junb), chromosome 5q31-33 (Jun), and chromosome 16 (Jund)

By means of somatic cell hybrids segregating rat chromosomes, we determined the chromosome localization of three rat genes of the Jun family: Jumb (Chr 19), Jun (=c-Jun) (Chr 5) and Jund (Chr 16). The Jun gene was also localized to the 5q31–33 region by fluorescence in situ hybridization. These rat gene assignments reveal two new homologies with mouse and human chromosomes, and provide a new example of synteny conserved in the human and a rodent species (the mouse), but split between the two rodent species.

Rat chromosome 1 : Regional localization of seven genes (Slc9a3, Srd5a1, Esr, Tcp1, Grik5, Tnnt3, Jak2) and anchoring of the genetic linkage map to the cytogenetic map

Seven genes were regionally localized on rat Chromosome (Chr) 1, from 1p11 to 1q42, and two of these genes were also included in a linkage map. This mapping work integrates the genetic linkage map and the cytogenetic map, and allows us to orient the linkage map with respect to the centromere, and to deduce the approximate position of the centromere in the linkage map. These mapping data also indicate that the Slc9a3 gene, encoding the Na+/H+ exchanger 3, is an unlikely candidate for the blood pressure loci assigned to rat Chr 1. These new localizations expand comparative mapping between rat Chr 1 and mouse or human chromosomes.

Mapping of the rat ribosomal protein S18 gene (Rps 18) to chromosome 20p12

Using fluorescent in situ hybridization, we mapped the rat ribosomal protein S18 to chromosome 20p12

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

Abstract. 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.

Regional localization of the rat genes encoding the cAMP-specific phosphodiesterases 3 (Pde4d) and 4 (Pde4b) and the tyrosinase-related protein 1 (Tyrp1)

We regionally mapped rat genes encoding the cAMP-specific phosphodiesterases 3 (Pde4d) and 4 (Pde4b) and the tyrosinase-related protein 1 (Tyrp1)using fluorescent in situ hybridization (FISH)

Assignment of the gene encoding the serotonin 5HT1B receptor to rat Chromosome 8q31 by fluorescence in situ hybridization

Species:Mouse Locus name: methionine synthase or 5-methyltetrahydrofolatehomocysteine methyltransferase Locus symbol:Mtr Map position: proximal–D13Mit1–1.06 cM ± 1.06 SE– Mtr, D13Bir4, D13Bir6–1.06 ± 1.06–D13Abb1e–2.13 ± 1.49–D13Bir7–distal Method of mapping:Mtr was localized by RFLP analysis of 96 animals from an interspecific backcross panel ((C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei) provided by The Jackson Laboratory, Bar Harbor, Me. (BSS panel) [1]. Database deposit information: The data are available from the Mouse Genome Database, accession number MGD-JNUM-39061. Molecular reagents:A 1095-bp mouse cDNA was obtained by reverse transcription/PCR of mouse liver RNA, with degenerate oligonucleotides based on regions of homology within the methionine synthase sequences of lower organisms. The two primers (D1730 and D1733), as described by Leclerc et al. [2], were successful in amplifying both human and mouse cDNAs. The PCR products from both species were subcloned and sequenced; they showed 89% identity. The mouse cDNA was labeled by random priming and hybridized to Southern blots of EcoRI-digested mouse genomic DNA. Allele detection:Allele detection was performed by RFLP analysis of an EcoRI polymorphism. The C57BL/6J strain has alleles of approximately 13 kb, while theMus spretusstrain has alleles of approximately 9 kb and 4 kb. A constant band of approximately 0.5 kb was seen in both strains. Previously identified homologs: Human MTR has been mapped to chromosomal band 1q43 by fluorescence in situ hybridization [2–4]. Discussion: Methionine synthase (EC 2.1.1.13, 5-methyltetrahydrofolate-homocysteine methyltransferase) catalyzes homocysteine remethylation to methionine, with 5-methyltetrahydrofolate as the methyl donor and methylcobalamin as a cofactor. Nutritional deficiencies and genetic defects in homocysteine metabolism result in varying degrees of hyperhomocysteinemia. Dramatic elevations in plasma and urinary homocysteine levels are associated with the inborn error of metabolism, homocystinuria. Consequent to the recent isolation of the human cDNA for methionine synthase [2–4], two groups of investigators have identified mutations in methionine synthase in homocystinuric patients [2, 5]. Mild elevations in plasma homocysteine are thought to be a risk factor for both vascular disease and neural tube defects [6–8]. A genetic variant in methylenetetrahydrofolate reductase (MTHFR), the enzyme that synthesizes 5-methyltetrahydrofolate for the methioninesynthase reaction, is the most common genetic determinant of hyperhomocysteinemia identified thus far [9]. Mild defects in the methionine synthase reaction are also potential candidates for hyperhomocysteinemia and the associated multifactorial diseases. A common variant has been reported for the human methionine synthase gene, but its physiologic consequences have not yet been determined [2, 4]. The mapping of the human MTR gene to 1q43 and of the mouse gene to proximal Chromosome (Chr) 13 is consistent with previous findings of human/mouse homologies between these 2 chromosomal regions; the human and mouse nidogen genes have been mapped to 1q43 and proximal Chr 13, respectively [10]. Several genes have already been implicated in neural tube defects in mice [11]. Studies involving the mouse methionine synthase gene will be useful in assessing the role of this important enzyme in the development of birth defects and/or vascular disease.

The rat genetic and cytogenetic maps

Summary The rat map was improved by determining the regional chromosome localization of 82 genes, thereby orienting each linkage group. New anonymous markers were also generated on rat chromosome 2.