The Genomic Structure of an Insertional Mutation in the Dystonia Musculorum Locus

We have previously identified a line of transgenic mice, Tg4, in which an hsp68-lacZ hybrid gene has inserted into the dystonia musculorum (dt) locus on chromosome 1. We have confirmed the localization of the Tg4 integration site to the proximal region of mouse chromosome 1 by interspecific backcross analysis. One end of the integration complex has been cloned and we have used single-copy probes from the flanking region to screen a mouse genomic library. Several overlapping lambda phage clones have been isolated and arranged into a contig spanning 75 kb of genomic DNA. Probes from the genomic contig have enabled us to characterize the wildtype and Tg4 loci. We report that the integration of the transgene was accompanied by a deletion of 45 kb of host genomic sequences with no other detectable rearrangement in the Tg4 genome.     

The Mouse Region Syntenic for Human Spinal Muscular Atrophy Lies within theLgn1Critical Interval and Contains Multiple Copies ofNaipExon 5

Spinal muscular atrophy (SMA) is a relatively common, autosomal recessively inherited neurodegenerative disorder that maps to human chromosome 5q13. This region of the human genome has an intricate genomic structure that has complicated the evaluation of SMA candidate genes. We have chosen to study the mouse region syntenic for human SMA in the hope that the homologous mouse interval would contain the same genes as human 5q13 on a simpler genomic background. Here, we report the mapping of such a region to mouse chromosome 13 and to the critical interval forLgn1,a mouse locus responsible for modulating the intracellular replication and pathogenicity of the bacteriumLegionella pneumophila.We have generated a mouse YAC contig across theLgn1/Smainterval and have mapped the two flanking gene markers for the human SMA locus, MAP1B and CCNB1, onto this contig. In addition, we have localized the two SMA candidate genes, SMN and NAIP, to theLgn1critical region, making these two genes candidates for theLgn1phenotype. Upon subcloning of the YAC contig into P1s and BACs, we have detected a large, low copy number repeat that contains at least one copy ofNaipexon 5. Identification of theLgn1gene will either provide a novel function for SMN or NAIP or reveal the existence of another, yet uncharacterized gene in the SMA critical region. Mutations in such a gene might help to explain some of the phenotypic variability among the human SMAs.     

Comparative analysis of the PCOLCE region in Fugu rubripes using a new automated annotation tool

The Japanese pufferfish Fugu rubripes with a genome of about 400 Mb is becoming increasingly recognized as a vertebrate model organism for comparative gene analysis (see Elgar 1996 for review). We have isolated and sequenced two Fugu cosmids spanning a genomic region of 66 kb containing the Fugu homolog to the human PCOLCE-I (Gl?ckner et al. 1998). We then examined if RUMMAGE-DP, a newly developed analysis tool for gene discovery which was designed for human and mouse genomic DNA, can be used for automatic annotation of Fugu genomic sequence. The exon prediction programs contained in RUMMAGE-DP performed better overall for the human sequence than for the Fugu contig. The GENSCAN program was the only exon prediction programme that performed equally well for both organisms. We show that RUMMAGE-DP is very useful in automatic analysis of Fugu sequences. Comparative analysis of the genomic structure of the PCOLCE-I genes in Fugu and human reveals that the exon/intron structure throughout the protein coding region is almost identical. We defined an additional domain based on the high degree of similarity of 26 aa between mammals and Fugu. The PCOLCE-I protein in both organisms contains two highly conserved CUB domains. Exons 6 and 7 are the only coding exons that differ in length between the two species. We assume that these exons do not code for any catalytic domain of the protein. Analysis of the remaining five Fugu genes within the 66 kb interval revealed no conserved synteny with the corresponding human 7q22 region. Received: 13 October 1998 / Accepted: 25 July 1999     

Cloning and expression of hbves, a novel and highly conserved mRNA expressed in the developing and adult heart and skeletal muscle in the human

bves is a novel mRNA expressed in the developing heart in chick and mouse. Here we describe hbves, the human homolog of chick and mouse bves. Northern and dot blot analyses reveal restricted expression in the heart and skeletal muscle in the embryo and adult. BLAST searches of the NCBI databases confirm that hbves is novel. Portions of the sequence are an exact match with genomic PAC 52202, which localizes to Chromosome (Chr) 6q21. Presumably, these matches and intervening sequences match the intron-exon borders of the gene. Computer conformation analysis of the derived amino acid predicts three transmembrane helices with an extracellular C-terminus that is conserved in chick, mouse, and human. bves is highly conserved among all three species at the amino acid level with 75% identity and 92% similarity. Received: 15 February 1999 / Accepted: 29 April 1999     

Cloning, expression and chromosomal location of NKX6B to 10q26, a region frequently deleted in brain tumors

Nkx6-2 (former Gtx) is a murine-homeobox-containing gene localized distally on Chromosome (Chr) 7. Analysis of the expression pattern, together with DNA binding assays, suggests that this gene product might be important for differentiated oligodendrocyte function and in the regulation of myelin gene expression. We now report on the cloning and characterization of the human homolog (NKX6B). DNA sequence analysis of an 11-kb genomic fragment revealed that the complete human gene spans 1.2 kb and is composed of three exons. NKX6B is predicted to encode a polypeptide of 277 amino acids with 97% identity to mouse Nkx6-2. Northern blot experiments showed that NKX6B expression is tightly controlled in a tissue-specific fashion with the highest site of expression being the brain. Finally, using STS content mapping and RH analyis, we demonstrated that NKX6B maps to the 10q26, a region where frequent loss of heterozygosity has been observed in various malignant brain tumors. These results may implicate NKX6B as a candidate tumor suppressor gene for brain tumors, particularly for oligodendrogliomas. Received: 14 June 2000 / Accepted: 21 September 2000     

Genomic organization and mapping of the human and mouse neuronal β2-nicotinic acetylcholine receptor genes

As a first step in determining whether there are polymorphisms in the nicotinic acetylcholine receptor (nAChR) genes that are associated with nicotine addiction, we isolated genomic clones of the β2-nAChR genes from human and mouse BAC libraries. Although cDNA sequences were available for the human gene, only the promoter sequence had been reported for the mouse gene. We determined the genomic structures by sequencing 12 kb of the human gene and over 7 kb of the mouse gene. While the sizes of exons in the mouse and human genes are the same, the introns differ in size. Both promoters have a high GC content (60–80%) proximal to the AUG and share a neural-restrictive silencer element (NRSE), but overall sequence identity is only 72%. Using a 6-Mb YAC contig of Chr 1, we mapped the human β2-nAChR gene, CHRNB2, to 1q21.3 with the order of markers cen, FLG, IVL, LOR, CHRNB2, tel. The mouse gene, Acrb2, had previously been mapped to Chr 3 in a region orthologous to human Chr 1. We refined mapping of the mouse gene and other markers on a radiation hybrid panel of Chr 3 and found the order cen, Acrb2, Lor, Iv1, Flg, tel. Our results indicate that this cluster of markers on human Chr 1 is inverted with respect to its orientation on the chromosome compared with markers in the orthologous region of mouse Chr 3. Received: 26 January 1999 / Accepted: 10 May 1999     

Cloning, Structure, and Chromosome Localization of the Mouse Glutaryl-CoA Dehydrogenase Gene

Glutaryl-CoA dehydrogenase (GCDH) is a nuclear-encoded, mitochondrial matrix enzyme. In humans, deficiency of GCDH leads to glutaric acidemia type I, an inherited disorder of amino acid metabolism characterized by a progressive neurodegenerative disease. In this report we describe the cloning and structure of the mouse GCDH (Gcdh) gene and cDNA and its chromosomal localization. The mouse Gcdh cDNA is 1.75 kb long and contains an open reading frame of 438 amino acids. The amino acid sequences of mouse, human, and pig GCDH are highly conserved. The mouse Gcdh gene contains 11 exons and spans 7 kb of genomic DNA. Gcdh was mapped by backcross analysis to mouse chromosome 8 within a region that is homologous to a region of human chromosome 19, where the human gene was previously mapped.     

Sequence-ready physical map of the mouse Chromosome 16 region with conserved synteny to the human Velocardiofacial syndrome region on 22q11.2

Proximal mouse Chromosome (Chr) 16 shows conserved synteny with human Chrs 16, 8, 22, and 3. The mouse Chr 16/human Chr 22 conserved synteny region includes the DiGeorge/Velocardiofacial syndrome region of human Chr 22q11.2. A physical map of the entire mouse Chr 16/human Chr 22 region of conserved synteny has been constructed to provide a substrate for gene discovery, genomic sequencing, and animal model development. A YAC contig was constructed that extends ca. 5.4 Mb from a region of conserved synteny with human Chr 8 at Prkdc through the region conserved with human Chr 3 at DVL3. Sixty-one markers including 37 genes are mapped with average marker spacing of 90 kb. Physical distance was determined across the 2.6-Mb region from D16Mit74 to Hira with YAC fragmentation. The central region from D16Jhu28 to Igl-C1 was converted into BAC and PAC clones, further refining the physical map and providing sequence-ready template. The gene content and borders of three blocks of conserved linkage between human Chr 22q11.2 mouse Chr 16 are refined. Received: 4 November 1998 / Accepted: 21 December 1998     

小麦尿卟啉原Ⅲ合成酶基因克隆及序列分析

根据水稻已公布的尿卟啉原Ⅲ合成酶(UROS)基因和小麦EST的保守序列,设计特异性引物对小麦尿卟啉原Ⅲ合成酶基因的部分片段进行克隆,得到了364 bp的cDNA(命名为UROS1)。以UROS1作为种子进行电子克隆,得到一段长为1210 bp的cDNA序列,并设计特异性引物克隆到1个1077 bp cDNA序列。对该片段分析结果表明,克隆得到的小麦UROS基因包含了信号肽区和全长的成熟肽区。小麦UROS基因与水稻UROS基因的同源性为86%左右,其推导氨基酸序列与水稻和拟南芥蛋白序列同源性分别约91%和79%。动物、植物以及微生物间核酸序列的保守性较低,氨基酸序列保守性也不高,但都存在UROS保守结构域(Hem D)。进化分析显示,该酶在不同物种间的进化速度差异较大。     

Characterization of the murine polycystic kidney disease (Pkd2) gene

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most frequent genetically transmitted disorders among Europeans with an attributed frequency of 0.1%. The two most common genetic determinants for ADPKD are the PKD1 and PKD2 genes. In this study we report the genomic structure and pattern of expression of the Pkd2 gene, the murine homolog of the human PKD2 gene. Pkd2 is localized on mouse Chromosome (Chr) 5 proximal to anchor marker D5Mit175, spans at least 35 kb of the mouse genome, and consists of 15 exons. Its translation product consists of 966 amino acids, and the peptide shows a 95% homology to human polycystin2. Functional domains are particularly well conserved in the mouse homolog. The expression of mouse polycystin2 in the developing embryo at day 12.5 post conception is localized in mesenchymally derived structures. In the adult mouse, the protein is mostly expressed in kidney, which suggests its functional relevance for this organ. Received: 13 March 1998 / Accepted: 11 May 1998     

Colinearity of novel genes in the class II regions of the MHC in mouse and human

To examine the degree of conservation of gene organization in and around the class II regions of the major histocompatibility complexes of mouse and human, we have established the positions of sequences homologous to five human non-class II genes (RING1-5) in mouse, and the positions of sequences homologous to three mouse non-class II genes (KE3-5) in human. The resulting comparative map reveals that the organization of genes in the entire proximal region of the MHCs of mouse and human is remarkably conserved, apart from the H-2K gene pair in mouse, which can be accounted for by a 60 kilobase (kb) insertion. The characterization of the novel human gene RING5 is also presented. This gene, which is widely expressed, maps 85 kb proximal to the DPB2 gene. Partial nucleotide sequencing of a RING5 cDNA clone reveals that it is the human homolog of the mouse KE4 gene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M58660.     

Fine mapping of Hyplip1 and the human homolog, a potential locus for FCHL

Familial combined hyperlipidemia (FCHL) is a common genetic dyslipidemia predisposing to premature coronary heart disease (CHD). We previously identified a locus for FCHL on human Chromosome (Chr) 1q21-q23 in 31 Finnish FCHL families. We also mapped a gene for combined hyperlipidemia (Hyplip1) to a potentially orthologous region of mouse Chr 3 in the HcB-19/Dem mouse model of FCHL. The human FCHL locus was, however, originally mapped about 5 Mb telomeric to the synteny border, the centromeric part of which is homologous to mouse Chr 3 and the telomeric part to mouse Chr 1. To further localize the human Hyplip1 homolog and estimate its distance from the peak linkage markers, we fine-mapped the Hyplip1 locus and defined the borders of the region of conserved synteny between human and mouse. This involved establishing a physical map of a bacterial artificial chromosome (BAC) contig across the Hyplip1 locus and hybridizing a set of BACs to both human and mouse chromosomes by fluorescence in situ hybridization (FISH). We narrowed the location of the mouse Hyplip1 gene to a 1.5-cM region that is homologous only with human 1q21 and within approximately 5–10 Mb of the peak marker for linkage to FCHL. FCHL is a complex disorder and this distance may, thus, reflect the well-known problems hampering the mapping of complex disorders. Further studies identifying and sequencing the Hyplip1 gene will show whether the same gene predisposes to hyperlipidemia in human and mouse. Received: 9 September 2000 / Accepted: 30 October 2000     

Rapid evolution of human pseudoautosomal genes and their mouse homologs

Comparative studies of genes in the pseudoautosomal region (PAR) of human and mouse sex chromosomes have thus far been very limited. The only comparisons that can presently be made indicate that the PARs of humans and mice are not identical in terms of gene content. Here we describe additional comparative studies of human pseudoautosomal genes and their mouse homologs. Using a somatic cell hybrid mapping panel, we have assigned the mouse homolog of the human pseudoautosomal interleukin 3 receptor alpha subunit (IL3RA) gene to mouse Chromosome (Chr) 14. Attempts to clone the mouse homolog of the human pseudoautosomal adenine nucleotide translocase-3 (ANT3) gene resulted in the isolation of the murine homologs of the human ANT1 and ANT2 genes. The mouse Ant1 and Ant2 genes are very similar in sequence to their human homologs, and we have mapped them to mouse Chromosomes (Chrs) (8 and X respectively) that exhibit conserved synteny with the chromosomes on which the human genes are located. In contrast, the homolog of ANT3 appears to be either very divergent or absent from the mouse genome. Southern blot analysis of DNA from a variety of mammalian species shows restricted conservation of human pseudoautosomal genes, a trend that also applies to the two cloned mouse homologs of these genes and to neighboring human genes in distal Xp22.3. Our observations combined with those of other workers lead us to propose a model for the evolution of the PAR that includes both rapid sequence evolution and the incremental reduction in size of the region during mammalian evolution. Received: 4 May 1995 / Accepted: 21 August 1995     

Structure of the mouse 3-Methyladenine DNA Glycosylase gene and exact localization upstream of the α-globin gene cluster on Chromosome 11

In this paper we describe the genomic organization of the mouse 3-Methyladenine DNA Glycosylase (MPG) gene and localize three putative regulatory elements around this gene. The MPG gene plays a key role in the excision repair of methylated adenine residues and has been localized upstream of the -globin gene cluster in human and mouse. The human MPG gene has been fully characterized, whereas up to now only the cDNA sequence of the mouse MPG gene had been published. Here, we describe a detailed restriction map, the intron/exon structure, the CpG-rich putative promoter sequence, and the exact localization of the mouse MPG gene with respect to the murine -globin gene cluster. Our analysis reveals a remarkable different exon/intron structure of the mouse MPG gene compared with its human homolog. Two prominent DNase hypersensitive sites (HSS) were found 0.1 and 1.5 kb upstream of the coding sequence. In addition to these elements, an erythroid prominent HSS was mapped at the intron/exon boundary of the last exon. The characterization and localization of the MPG gene in mouse makes it now possible to carry out transgenic and gene targeting experiments and are essential to understand the control of gene expression of the MPG gene in particular and of the whole region in general.The nucleotide sequence data reported in this paper will be submitted to Genbank.     

Isolation and characterization of a cosmid contig for the GCPS gene region

The zinc finger gene GLI3 has been shown to be involved in the embryonal development of the limbs and skull. Mutations in GLI3 lead to the development of the human Greig cephalopolysyndactyly syndrome (GCPS) and the mouse mutations extra toes (Xt) and anterior digit deformity (add). The GCPS locus on human chromosome 7p13 has recently been isolated in a yeast artifical chromosome (YAC) contig. Here, we describe the establishment of a cosmid contig that was derived from two of the YAC clones, that spans 550 kb of human DNA, and that includes the GLI3 gene. In this contig, three GCPS translocation breakpoints have been mapped to distinct EcoRI fragments in the 3 half of the gene. In addition, exon-carrying fragments have been identified and the size of the GLI3 gene could be determined as at least 280 kb. The gene is flanked by a CpG island that lies on the 5 side and that is in close proximity to the first exon detected by the cloned GLI3 cDNA. Further upstream, five segments were found that have been conserved between man and mouse. In the mouse, this region has been characterized as the transgene integration site resulting in the add phenotype. Both the CpG island and the conserved regions are probable candidates for a search for GLI3 promoter and control elements.