Combining QTL data for HDL cholesterol levels from two different species leads to smaller confidence intervals.

Quantitative trait locus (QTL) analysis detects regions of a genome that are linked to a complex trait. Once a QTL is detected, the region is narrowed by positional cloning in the hope of determining the underlying candidate gene-methods used include creating congenic strains, comparative genomics and gene expression analysis. Combined cross analysis may also be used for species such as the mouse, if the QTL is detected in multiple crosses. This process involves the recoding of QTL data on a per-chromosome basis, with the genotype recoded on the basis of high- and low-allele status. The data are then combined and analyzed; a successful analysis results in a narrowed and more significant QTL. Using parallel methods, we show that it is possible to narrow a QTL by combining data from two different species, the rat and the mouse. We combined standardized high-density lipoprotein phenotype values and genotype data for the rat and mouse using information from one rat cross and two mouse crosses. We successfully combined data within homologous regions from rat Chr 6 onto mouse Chr 12, and from rat Chr 10 onto mouse Chr 11. The combinations and analyses resulted in QTL with smaller confidence intervals and increased logarithm of the odds ratio scores. The numbers of candidate genes encompassed by the QTL on mouse Chr 11 and 12 were reduced from 1343 to 761 genes and from 613 to 304 genes, respectively. This is the first time that QTL data from different species were successfully combined; this method promises to be a useful tool for narrowing QTL intervals.

Candidate genes for obesity revealed from a C57BL/6J x 129S1/SvImJ intercross.

OBJECTIVE: To identify the genes controlling body fat, we carried out a quantitative trait locus (QTL) analysis using C57BL/6J (B6) and 129S1/SvImJ (129) mice, which differ in obesity susceptibility after consuming an atherogenic diet. METHODS: Mice were fed chow until 8 weeks and an atherogenic diet from 8 to 16 weeks; body fatness was measured by X-ray absorptiometry in 528 (B6 x 129) F(2) at 8 and 16 weeks. A high-density genome scan was performed using 508 polymorphic markers. After identifying the genetic loci, we narrowed the QTL using comparative genomics and bioinformatics. RESULTS: The percentage of body fat was significantly linked to loci on chromosomes (Chr) 1 (22, 68 and 173 Mb), 4 (74 Mb), 5 (73 Mb), 7 (88 Mb), 8 (43 and 80 Mb), 9 (55 Mb), 11 (115 Mb) and 12 (32 Mb); three suggestive loci on Chrs 6 (76 Mb), 9 (30 Mb) and 16 (26 Mb) and two pairs of interacting loci (Chr 2 at 99.8 Mb with Chr 7; Chr 1 at 68 Mb with Chr 11). Comparative genomics narrowed the QTL intervals by 20-57% depending on the chromosome; in most cases, haplotype analysis further narrowed them by about 90%. CONCLUSIONS: Our analysis identified 15 QTL for percentage of body fat. We narrowed the QTL using comparative genomics and haplotype analysis and suggest several candidate genes: Apcs on Chr 1, Ppargc1a on Chr 5, Ucp1 on Chr 8, Angptl6 on Chr 9 and Lpin1 on Chr 12.

The emerging role of ACE2 in physiology and disease.

The renin-angiotensin-aldosterone system (RAAS) is a key regulator of systemic blood pressure and renal function and a key player in renal and cardiovascular disease. However, its (patho)physiological roles and its architecture are more complex than initially anticipated. Novel RAAS components that may add to our understanding have been discovered in recent years. In particular, the human homologue of ACE (ACE2) has added a higher level of complexity to the RAAS. In a short period of time, ACE2 has been cloned, purified, knocked-out, knocked-in; inhibitors have been developed; its 3D structure determined; and new functions have been identified. ACE2 is now implicated in cardiovascular and renal (patho)physiology, diabetes, pregnancy, lung disease and, remarkably, ACE2 serves as a receptor for SARS and NL63 coronaviruses. This review covers available information on the genetic, structural and functional properties of ACE2. Its role in a variety of (patho)physiological conditions and therapeutic options of modulation are discussed.

Mapping of rabbit microsatellite markers using chromosome-specific libraries.

Recently, rabbit microsatellite markers were developed from a chromosome 1-specific library, and seven new markers were incorporated into the genetic map of the rabbit. We have now developed microsatellite markers from chromosomes 3-, 5-, 6-, 7-, 12-, and 19-specific libraries. Linkage analysis was performed with use of these new markers, five recently physically mapped markers (PMP2, TCRB, ALOX15, MT1, and Sol33), microsatellite markers located in the HBA gene cluster, the MHC region and FABP6 gene, and seven biochemical markers (Es-1, Es-3, Est-2, Est-4, Est-6, Est-X, and HP). This analysis enabled us to verify the specificity of the libraries and to determine the position and orientation of the linkage groups on the chromosomes.

Fourteen chromosomal localizations and an update of the cytogenetic map of the rabbit.

In order to improve the informativeness of the cytogenetic map of the rabbit genome, fourteen markers were regionally mapped to individual chromosomes. The localizations comprise eleven gene loci (PRLR, GHR, HK1, ACE, TF, 18S+28S rDNA, CYP2C4, PMP2, TCRB, ALOX15 and MT1) and three microsatellite loci (Sat13, Sol33 and D1Utr6). Five of the genes contain known microsatellite sequences. To achieve these localizations, homologous and heterologous small insert clones, and clones from a rabbit Bacterial Artificial Chromosome (BAC) library were used as probes for fluorescence in situ hybridization experiments. Results indicate that especially BAC clones are a valuable tool for cytogenetic mapping. Some of the genes were selected for mapping on the basis of human- rabbit comparative painting data, to achieve localizations on gene-poor rabbit chromosomes. Our data are, in general, in agreement with the human-rabbit comparative painting data. By mapping microsatellite sequences that have also been used in linkage studies, links are provided between the genetic and physical maps of the rabbit genome. Linkage groups I, VI and XI could be assigned to chromosomes 1, 5 and 3 respectively. Moreover, in this paper we give an overview of the current status of the rabbit cytogenetic map. This map now comprises 62 physically mapped genes, which are scattered over all autosomes, except chromosome 2, and the X chromosome.

Mapping of rabbit chromosome 1 markers generated from a microsatellite-enriched chromosome-specific library.

A genomic DNA library was produced from flow-sorted rabbit chromosome 1 and enriched for fragments containing CA-repeats. Clones containing CA-repeats were identified and primers for amplification of the microsatellite were developed after sequencing the clone. The degree of polymorphism was tested in rabbits from different breeds. This approach identified 12 microsatellite markers which could be used for studying linkage relationships in the progeny of an F(2)-intercross: (AX/JUxIIIVO/JU) F(2), and two backcrosses: (OS/JxX/J)X/J and (WH/JxX/J)X/J. Seven of these markers were mapped on chromosome 1.

Genetic analysis and mapping of biochemical markers in an F2 intercross of two inbred strains of the rabbit (Oryctolagus cuniculus).

A total of 40 biochemical and four immunological markers found to be polymorphic in the rabbit in previous studies were screened in the AX/JU and IIIVO/JU inbred strains. Although the strains are considered unrelated, only eight (biochemical) markers werefound to be polymorphic between the two strains. These eight markers were analyzed in an F2 intercross population. Linkage was found for Est-5 and C on chromosome 1 and for Es-1, Est-2, Est-4, Est-6 and HP on linkage group VI. Two polymorphic markers, Es-3 and Mhr-1 could not be linked to any of the other markers.

Mapping of microsatellite loci and association of aorta atherosclerosis with LG VI markers in the rabbit.

Twenty-three rabbit microsatellites were extracted from the EMBL nucleotide database. Nine of these markers, together with nine earlier published microsatellite markers, were found to be polymorphic between the AX/JU and IIIVO/JU inbred strains. By using an F(2) intercross we could integrate five markers into the rabbit linkage map. One anonymous microsatellite marker could be assigned to chromosome 1, and one microsatellite marker, located within the metallothionein-1 gene, could be added to linkage group VI (LG VI). Three microsatellite markers (one anonymous, one located within the PMP2 gene, and one located within the FABP6 gene) constitute a new linkage group (LG XI). We also measured the degree of dietary cholesterol-induced aorta atherosclerosis in the F(2) animals. A significant cosegregation was found between the degree of aorta atherosclerosis and the allelic variation of the biochemical marker Est-2 on LG VI in male rabbits. This association was not found in female rabbits.

Isolation and characterization of KIUBP2, a ubiquitin hydrolase gene of Kluyveromyces lactis that can suppress a ts-mutation in CBF2, a gene encoding a centromeric protein of Saccharomyces cerevisiae.

The Kluyveromyces lactis UBP2 gene was isolated as a suppressor of a temperature-sensitive mutation in CBF2, a gene coding for a centromere-binding protein of Saccharomyces cerevisiae. The UBP genes are hydrolases than can cleave a ubiquitin moiety from a protein substrate. KlUBP2 is not essential for growth since a disruption of the KlUBP2 gene had little effect, except for a slight decrease in the growth rate. The stability of centromere-containing plasmids was not influenced either. In addition to KlUBP2, five S. cerevisiae genes involved in the ubiquitination pathway could suppress the ts-mutation in the CBF2 gene, namely UBA1, UBA2, UBP1, UBP2 and YUH1, although YUH1 was the only one that could do this like KlUBP2 from a single-copy plasmid. Surprisingly, these genes encode proteins with antagonistic activity as two, UBA1 and UBA2, are ubiquitin-activating enzymes whereas the other three are de-ubiquitinating hydrolases.

Rabbit calcium-sensing receptor (CASR) gene: chromosome location and evidence for related genes.

Diverse cellular functions are regulated by the calcium-sensing receptor, encoded by the CASR gene, which plays an important role in calcium homeostasis. Here we provide the sequence for exon VII of the rabbit CASR gene and show that it is 91% identical to the human gene at the nucleotide level, and 95% identical at the amino acid level. The gene was mapped by fluorescence in situ hybridization, using a cosmid isolated from a genomic library, to chromosome 14q11 and this was confirmed independently by PCR amplification of flow sorted chromosomes. In addition, the cosmid detected sites with lower frequencies on four other chromosomes: 3q, 5p, 8p, and 13p. Two of these sites (5p and 13p) were also detected by a related but unidentical cosmid, and map to regions that are homologous to the mouse calcium-sensing receptor related sequences (Casr-rs); suggesting that they may represent CASR-related sequences in the rabbit. The data support the presence of a family of genes related to the calcium-sensing receptor in the G-protein coupled receptor (GPCR) superfamily, as well as extend the existing knowledge of homology for several human and rabbit chromosomes.

Complete homology maps of the rabbit (Oryctolagus cuniculus) and human by reciprocal chromosome painting.

Fluorescence in situ hybridization (FISH) was used to construct a homology map to analyse the extent of evolutionary conservation of chromosome segments between human and rabbit (Oryctolagus cuniculus, 2n = 44). Chromosome-specific probes were established by bivariate fluorescence activated flow sorting followed by degenerate oligonucleotide-primed PCR (DOP-PCR). Painting of rabbit probes to human chromosomes and vice versa allowed a detailed analysis of the homology between these species. All rabbit chromosome paints, except for the Y paint, hybridized to human chromosomes. All human chromosome paints, except for the Y paint, hybridized to rabbit chromosomes. The results obtained revealed extensive genome conservation between the two species. Rabbit chromosomes 12, 19 and X were found to be completely homologous to human chromosomes 6, 17 and X, respectively. All other human chromosomes were homologous to two or sometimes three rabbit chromosomes. Many conserved chromosome segments found previously in other mammals (e.g. cat, pig, cattle, Indian muntjac) were also found to be conserved in rabbit chromosomes.