Structure and chromosomal localization of the mouse oncomodulin gene

The rat gene encoding oncomodulin (OM), a small calcium-binding protein, is under the control of a solo LTR derived from an endogenous intracisternal A-particle. The latter sequence is the only OM promoter analyzed so far. In order to study cell-type-specific OM expression in a species lacking LTR sequences in the OM locus, we initially synthesized an OM cDNA from mouse placenta. By sequencing, we found a 137-bp-long 5 leader region that differed markedly from its rat counterpart but had high similarity to several mouse genomic sequences. Primers specific to this sequence in addition with primers specific for an exon 2/intron 2 sequence were used to screen a mouse ES cell line genomic P1 library. One positive clone contained the whole OM gene, including intron 1 of 25 kb and a 5 flanking region of 27 kb lacking an LTR. The region upstream of exon 1 contains no TATA or CCAAT boxes but has a homopurine/homopyrimidine stretch of 102 bp as well as a (CA)₂₂ repeat. The latter sequence is polymorphic and was therefore, used to map the OM gene to the distal end of the long arm of mouse Chromosome (Chr) 5 by interspecific backcross analysis. Additonally we localized the OM gene by in situ hybridization to the region G1-3 on Chr 5, confirming the genetic linkage results. Finally, the OM gene was found to be structurally conserved and to exist in a single copy in mammals.     

Structure and chromosomal localization of the human thrombospondin gene

Thrombospondin (THBS1) is a large modular glycoprotein component of the extracellular matrix and contains a variety of distinct domains, including three repeating subunits (types I, II, and III) that share homology to an assortment of other proteins. Determination of THBS1 gene structure has revealed that the type I repeat modules are encoded by symmetrical exons and that the heparin-binding domain is encoded by a single exon. To further elucidate the higher level organization of THBS1, the gene was localized to the q11-qter region of chromosome 15.     

Structure and chromosomal localization of the mouse bombesin receptor subtype 3 gene

Bombesin (BN)-like peptides/neurotransmitters mediate a broad range of physiological funtions in the gastrointestinal tract and the central nervous system through binding to their specific, high-affinity mammalian bombesin receptors. This family of heptahelical, G protein-coupled receptors includes the gastrin-releasing peptide receptor (GRP-R, or bb2), neuromedin B receptor (NMB-R, or bb1), and the bombesin receptor subtype 3 (BRS-3, or bb3). The tissue distribution of BRS-3 is quite dissimilar compared to the other two BN receptors, GRP-R and NMB-R, and a natural ligand for BRS-3 is currently unknown. Nothing is known about mechanisms regulating BRS-3 gene expression and possible association with disease. To gain insight into the underlying structure and chromosomal localization of the BRS-3 genes, bacteriophage P1 genomic clones, harboring the genes for the human and mouse BRS-3, respectively, were isolated and their structure and chromosomal localizations determined. The protein-coding region of both genes is divided into three exons and spans approximately 5 kb. The loci of the BRS-3 genes were mapped to a syntenic region of the human (Xq25) and mouse (XA7.1–7.2) X-chromosome, respectively. The structural data of the BRS-3 genes derived from this study will permit future investigations of the mechanisms regulating their expression.     

Structure, expression and chromosomal localization of human p80-coilin gene.

Coiled bodies (CBs) are non-capsular nuclear bodies with a diameter of 0.3-1 micron and appear to be composed of coiled fibrils. Human autoantibodies to CBs recognize an 80-kD nuclear protein highly enriched in CBs, and this protein has been named p80-coilin. CBs are known to assemble and disassemble during the cell cycle, with the highest number of CBs occurring at mid to late G1 where p80-coilin is assembled into several small nuclear body-like structures. In S and G2 phases, CBs become larger and their number decreases and often they are undetectable during mitosis. Using a human autoantibody as a probe for expression cloning, we initially isolated a partial cDNA encoding p80-coilin. In this report, the 5' end of the complete cDNA for p80-coilin was obtained using the 5'-RACE (rapid amplification of cDNA ends) methodology. The size of the reconstructed full-length cDNA corresponds to the 2.7-kb mRNA detected in Northern blot analysis. The complete p80-coilin protein consists of 576 amino acids with a predicted molecular mass of 62,608. A putative p80-coilin pseudogene was also detected during the rescreening of p80-coilin cDNA. To confirm the validity of the cDNA sequence, three overlapping genomic DNA clones representing the human p80-coilin gene were selected for further analysis. The complete gene for p80-coilin contains 7 exons spanning approximately 25kb. Sequence analysis of exons 1 and 2 in genomic DNA clones confirmed the accuracy of the 5' cDNA sequence derived from the 5'-RACE procedure. Furthermore, the human p80-coilin gene was localized to chromosome 17q22-23 by fluorescence in situ hybridization.     

Genomic organization and chromosomal localization of the mouse IKBKAP gene.

The autosomal recessive disorder familial dysautonomia (FD) has recently been demonstrated to be caused by mutations in the IKBKAP gene, so named because an initial report suggested that it encoded an IkappaB kinase complex associated protein (IKAP). Two mutations in IKBKAP have been reported to cause FD. The major mutation is a T-->C transition in the donor splice site of intron 20 and the minor mutation is a missense mutation in exon 19 that disrupts a consensus serine/threonine kinase phosphorylation site. We have characterized the cDNA sequences of the mouse, rat and rabbit IKBKAP-encoded mRNAs and determined the genomic organization and chromosomal location of mouse IKBKAP. There is significant homology in the amino acid sequence of IKAP across species and the serine/threonine kinase phosphorylation site altered in the minor FD mutation of IKAP is conserved. The mouse and human IKBKAP genes exhibit significant conservation of their genomic organization and the intron 20 donor splice site sequence, altered in the major FD mutation, is conserved in the human and mouse genes. Mouse IKBKAP is located on the central portion of chromosome 4 and maps to a region in which there is conserved linkage homology between the human and mouse genomes. The homologies observed in the human and mouse sequences should allow, through the process of homologous recombination, for the generation of mice that bear the IKBKAP mutations present in individuals with FD. The characterization of such mice should provide significant information regarding the pathophysiology of FD.     

Structure and chromosomal localization of the human glycogenin-2 gene GYG2

Glycogenin-2 is one of two self-glucosylating proteins involved in the initiation phase of the synthesis of the storage polysaccharide glycogen. Cloning of the human glycogenin-2 gene, GYG2, has revealed the presence of 11 exons and a gene of more than 46 kb in size. The structure of the gene explains much of the observed diversity in glycogenin-2 cDNA sequences as being due to alternate exon usage. In some cases, there is variation in the splice junctions used. Over regions of protein sequence similarity, the GYG2 gene structure is similar to that of the other glycogenin gene, GYG. A genomic GYG2 clone was used to localize the gene to Xp22.3 by fluorescence in-situ hybridization. Localization close to the telomere of the short arm of the X chromosome is consistent with mapping information obtained from glycogenin-2 STS sequences. Glycogenin-2 maps between the microsatellite anchor markers AFM319te9 (DXS7100) and AFM205tf2 (DXS1060), and its 3' end is 34.5 kb from the 3' end of the arylsulphatase gene ARSD. GYG2 is outside the pseudoautosomal region PAR1 but still in a region of X-Y shared genes. As is true for several other genes in this location, an inactive remnant of GYG2, consisting of exons 1-3, may be present on the Y chromosome.     

Genomic structure and chromosomal localization of the mouse gene Punc

The mouse gene Punc encodes a member of the immunoglobulin superfamily of cell surface proteins. It is highly expressed in the developing embryo in nervous system and limb buds. At mid-gestation, however, expression levels of Punc decrease sharply. To allow investigation of such a regulatory mechanism, the genomic locus encompassing the Punc gene was cloned, characterized, and mapped. Fluorescent in situ hybridization was used to determine the chromosomal location of the Punc gene of mouse and human. Mouse Punc maps to Chromosome (Chr) 9 in the region D-E1, whereas the human PUNC gene is localized to Chr 15 at 15q22.3-23, a region known to be syntenic to mouse 9D-E1. The human PUNC gene therefore maps close to a genetic locus that is linked to Bardet-Biedl Syndrome, an autosomal recessive human disorder. Confirmation for the location of human PUNC was obtained through sequence relationships between mouse Punc cDNA, human PUNC cDNA, genomic sequence upstream of the murine Punc gene, and human STS markers that had been previously mapped on Chr 15. The STS sequence WI-14920 is in fact derived from the 3′-untranslated region of the human PUNC gene. WI-14920 had been placed at 228cR from the top of the Chr 15 linkage group, which provided positional information for the human PUNC gene at high resolution. Thus, this study identifies PUNC as the gene corresponding to a previously anonymous marker and serves as a basis to investigate its role in genetic disorders. Received: 8 July 1998 / Accepted: 14 October 1998     

Genomic structure and chromosomal localization of the mouse persyn gene

Synucleins are a family of small intracellular proteins expressed mainly in the nervous system. The involvement of synucleins in neurodegeneration and malignancy has been demonstrated, but the physiological functions of these proteins remain elusive. Further studies including generation of animals with modified persyn expression are necessary to clarify the functions of these proteins and the mechanisms of their involvement in human diseases. We cloned and determined the organization and chromosomal localization of the mouse gene coding for persyn, a member of the synuclein family. The gene is composed of five exons, and its general structure is very similar to that of the human persyn gene. Using fluorescence in situ hybridization, we assigned the persyn gene to the boundary of bands B and C on mouse chromosome 14. We found a fragment of the gene that directs expression of the persyn protein in sensory neurons and could be used for generation of transgenic animals.