Close linkage of the olfactory marker protein gene to the mouse deafness mutation shaker-1.

One thousand sixty-six progeny have been generated from a backcross segregating for the mouse deafness mutation, shaker-1 (sh-1). One thousand fifty-two mice were analyzed for a protein polymorphism segregating for the distal flanking marker, beta-globin (Hbb), and 13 recombinants between Hbb and sh-1 were identified. One thousand eight mice were analyzed for a restriction fragment length polymorphism segregating for the proximal flanking marker, tyrosinase (c), and 54 recombinants between c and sh-1 were identified, completing a panel of 67 recombinant mice from the backcross in the vicinity of the sh-1 mutation. This panel allows the identification of markers closely linked to the sh-1 mutation that may act as start points for a chromosomal walk to the gene. One such marker, the olfactory marker protein gene (Omp), is recombinant with sh-1 in only one mouse from the recombinant panel. Thus, the Omp gene lies 0.1 cM from sh-1, on average, a distance of 200 kb. Haplotype analysis indicates that Omp lies proximal to sh-1.     

Sequence and characterization of twoHSP70 genes in the colonial protochordateBotryllus schlosseri

Two genes belonging to the heat shock protein 70 gene family have been cloned from the colonial protochordateBotryllus schlosseri. The two intronless genes(HSP70.1 andHSP70.2) exhibit 93.6% sequence identity within the predicted coding region, and 83.3% and 81.7% sequence identity in the 5′ and 3′ flanking regions, respectively. The predicted amino acid sequences are 95% identical and contain several signatures characteristic of cytoplasmic eukaryoticHSP70 genes (Gupta et al. 1994; Rensing and Maier 1994). Northern blotting and sequence analysis suggest that both genes are heat-inducible merebees of theHSP70 gene family. Given these characteristics,HSP70.1 andHSP70.2 appear to be good candidates for protochordate homologues of the major histocompatibility complex-linkedHSP70 genes of human, mouse, and rat (Milner and Campbell 1990; Walter et al. 1994). Further experiments to determine whether there is functional evidence for such similarity are in progress. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers US 1901 (HSP70.1) and US 1902 (HSP70.2)     

Structural Organization of the Mouse and Human GALR1 Galanin Receptor Genes (GalnrandGALNR) and Chromosomal Localization of the Mouse Gene

The neuropeptide galanin elicits a range of biological effects by interaction with specific G-protein-coupled receptors. Human and rat GALR1 galanin receptor cDNA clones have previously been isolated using expression cloning. We have used the human GALR1 cDNA in hybridization screening to isolate the gene encoding GALR1 in both human (GALNR) and mouse (Galnr). The gene spans approximately 15–20 kb in both species; its structural organization is conserved and is unique among G-protein-coupled receptors. The coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop, while exon 3 encodes the remainder of the receptor, from transmembrane domain 6 to the C-terminus of the receptor protein. The mouse and human GALR1 receptor proteins are 348 and 349 amino acids long, respectively, and display 93% identity at the amino acid level. The mouseGalnrgene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the humanGALNRgene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein.     

The Structure and Expression of the Murine Wildtype p53-Induced Phosphatase 1 (Wip1) Gene

The human wildtype p53-induced phosphatase 1 (Wip1; GenBank symbol Ppm1d) gene encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner. We have cloned and sequenced the mouse Wip1 gene and its encoded mRNA. The mouse Wip1 gene is composed of six exons and spans over 36 kb of DNA. The mouse cDNA sequence predicts a 598-amino-acid protein with a molecular mass of roughly 66 kDa. Comparison of human and mouse Wip1 sequences revealed 83% overall identity at the amino acid level. The 5′-flanking region of exon 1 had promoter elements characteristic of a housekeeping gene. The Wip1 coding sequences share conserved functional regions with other PP2Cs from a diverse array of species. Expression of Wip1 mRNA was detected ubiquitously in adult and embryonic tissues, though expression in the testis was much higher than in other tissues. Wip1 has been mapped near the p53 gene on mouse chromosome 11.     

Periplasmic production of human pancreatic prokallikrein in Escherichia coli

Summary The human pancreatic prokallikrein gene has been fused to the DNA sequence coding for the signal peptide of the Escherichia coli major outer membrane protein F (OmpF) and expressed under the control of tac promoter in E. coli. By induction with isopropyl--d-thiogalactopyranoside, the cells produced prokallikrein very efficiently. The fused OmpF signal peptide was verified as being processed correctly at the cleavage site of the OmpF signal peptide, and the N-terminal amino acid sequence of the product was found to be identical to that of native human prokallikrein. However, the prokallikrein produced by E. coli formed insoluble aggregates and was always collected in the insoluble fraction. An electron micrograph of prokallikrein-producing cells indicated that the prokallikrein was secreted into the periplasmic space and formed insoluble inclusion bodies there. By treating the insoluble inclusion bodies with oxidized and reduced glutathione in 1 M guanidine-HCl solution, a portion of them could be solubilized in water and showed kallikrein activity of 8 units (approx. 264 g kallikrein) per litre of culture by trypsin activation.     

Isolation of the Mouse Homologue of BRCA1 and Genetic Mapping to Mouse Chromosome 11

The BRCA1 gene is in large part responsible for hereditary human breast and ovarian cancer. Here we report the isolation of the murineBrca1homologue cDNA clones. In addition, we identified genomic P1 clones that contain most, if not all, of the mouseBrca1locus. DNA sequence analysis revealed that the mouse and human coding regions are 75% identical at the nucleotide level while the predicted amino acid identity is only 58%. A DNA sequence variant in theBrca1locus was identified and used to map this gene on a (Mus m. musculusCzech II × C57BL/KsJ)F1 × C57BL/KsJ intersubspecific backcross to distal mouse chromosome 11. The mapping of this gene to a region highly syntenic with human chromosome 17, coupled with Southern and Northern analyses, confirms that we isolated the murineBrca1homologue rather than a related RING finger gene. The isolation of the mouseBrca1homologue will facilitate the creation of mouse models for germline BRCA1 defects.     

Isolation and characterization of gmsti,a stress-inducible gene from soybean (Glycine max) coding for a protein belonging to the TPR (tetratricopeptide repeats) family

In close vicinity of two fus nuclear genes (chloroplast-specific translation elongation factor cEF-G) of soybean (Glycine max) we localized a split nuclear gene coding for a protein with tetratricopeptide repeats (TPR). A full-length cDNA was sequenced (1871 nucleotides). It encodes a protein (569 amino acids) with high sequence identity to the yeast STI1 stress-inducible and the human transformation-sensitive IEF SSP 3521 protein which both carry TPR elements. The soybean gene is heat-inducible. This is the first evidence for the existence of plant genes coding for proteins which belong to the TPR family. We call the gene gmsti and the protein GMSTI in analogy to the yeast counterpart.     

A rat homeobox gene, rNKx-2.5, is a homologue of the tinman gene in Drosophila and is mainly expressed during heart development

 We report the cloning of a rat homeobox-containing gene, rNkx-2.5, and investigation of its expression in adult tissues and during embryonic development. The rNkx-2.5 gene is a homologue of the tinman gene in Drosophila. Both genes share an identical TN domain (tinman-like amino-terminal decapeptide) and about 66.7% sequence identity within their homeodomain sequences. In vertebrates, the rNKx-2.5 gene is most closely related to the mouse NKx-2.5 (mNKx-2.5) gene. Coding sequences for both NKx-2.5 genes have 94.1% sequence identity, including three identical conserved domains, the TN, homeo and NK-2 domains (NK-2 specific domain, carboxy-terminal to the homeodomain in vertebrate tinman homologues). The rat NKx-2.5 gene is encoded by a 1.4-kb mRNA and in adult tissues is mainly expressed in heart, with weaker expression in testis, spleen and lung. During embryonic development, expression of rNKx-2.5 is strongly observed in developing heart, specifically in the myocardium of both atrial and ventricular chambers. In addition, rNKx-2.5 expression marks other developing tissues, including tongue, thyroid, stomach, spleen and pulmonary veins. These data show that rNKx-2.5 is expressed in a pattern similar but not identical to that previously observed for mNKx-2.5. Received: 24 February 1997 / Accepted: 23 June 1997     

Molecular cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal chromosome 1

 Human membrane cofactor protein (MCP, CD46) is widely distributed and is one of the plasma membrane complement inhibitors. We isolated cDNA clones encoding genetic homologues of human MCP from a rat testis cDNA library. Northern blot analysis indicated that rat MCP is preferentially expressed in testis, similar to what is found with guinea pig MCP. We identified several different cDNAs, which were presumably generated by alternative splicing from a single-copy gene. The most prevalent isoform corresponded to the Ser/Thr/Pro-rich C type of human MCP. Mouse MCP cDNA was cloned by polymerase chain reaction based on the nucleotide sequence of rat MCP. The deduced amino acid sequence showed 77.8% identity to rat MCP. Mouse MCP was also preferentially expressed in testis. Unique expression in testis in rat and mouse as well as guinea pig suggests that MCPs in these species not only act as complement regulatory proteins but may also have more specialized functions in fertilization or reproduction. Genetic mapping by linkage analysis indicated that the mouse Mcp gene is located on distal chromosome 1, closely linked to the complement receptor 2 (Cr2) gene. Received: 24 February 1998 / Revised: 11 May 1998     

Nucleotide sequence of a mouse Tcp-1 pseudogene: A nucleotide record for a t complex gene carried by an ancestor of the mouse

We have isolated clones of a processed pseudogene of mouse t complex polypeptide 1 (Tcp-1) and determined the nucleotide sequence of the pseudogene. The pseudogene was 1363 bp long and had no intron. The Tcp-1 pseudogene had 88.4% or 88.3% nucleotide identity to the mouse Tcp-1 cDNA of wild-type (Tcp-1)^bor t haplotype (Tcp-1)^a, and 87.5% identity to the rat Tcp-1 cDNA. On 12 nucleotide positions where the open reading frames (ORFs) of mouse Tcp-1 ^band Tcp-1 ^acDNAs have bp substitutions, the Tcp-1 pseudogene had 6 bp identical to Tcp-1 ^b, 5 bp identical to Tcp-1 ^aand 1 bp not identical to neither. On ten amino acid positions where TCP-1B and TCP-1A polypeptides have substitutions, deduced amino acids of the Tcp-1 pseudogene had four amino acids identical to TCP-1B, five amino acids identical to TCP-1A and one amino acid identical to neither. These results suggest that the ancestral mouse Tcp-1 gene would have had no significant difference between the resemblance to Tcp-1 ^band that to Tcp-1 ^abefore they were diverged and that amino acids of TCP-1B and TCP-1A would have been substituted in similar high rates.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number D00851.