| First Author | Paterson T | Year | 1997 |
| Journal | Mouse Genome | Volume | 95 |
| Issue | 4 | Pages | 898-90 |
| Mgi Jnum | J:45476 | Mgi Id | MGI:1195497 |
| Citation | Paterson T, et al. (1997) Physical map of four tandemly repeated genes encoding murine alpha1-protease inhibitor. Mouse Genome 95(4):898-90 |
| abstractText | Full text of Mouse Genome contribution: PHYSICAL MAP OF FOUR TANDEMLY REPEATED GENES ENCODING MURINE alpha1-PROTEASE INHIBITOR. T. Paterson1 and L. Mullins2. 1Corresponding Author: National Science Laboratory, Scottish National Blood Transfusion Service, 12 Bristo Place, Edinburgh, EH1 1EZ, Scotland. Tel: +44 131 2252875. Fax: +44 131 2201893. email: tpaterso@hgmp.mrc.ac.uk. 2Centre for Genome Research, Edinburgh University, West Mains Road, Edinburgh, EH9 3JQ, Scotland. Introduction Five alpha1-protease inhibitor (alphal-PI) gene products are transcribed in M. musculus C57BL/6 adult liver1 from a multigene family on chromosome 12(2, 3). alpha1-PI inhibits the activity of a number of serine proteases: elastase and trypsin in man4 (where it is referred to as alphal- antitrypsin) and elastase and chymotrypsin in mouse5. However, its primary physiological role is in the regulation of neutrophil elastase, and in humans alpha1-PI deficiency leads to abnormal degradation of connective tissue, manifesting as emphysema and other conditions4. Multigene alpha1-PI and alphal-antichymotrypsin families seem to have arisen in a number of genera, but not man. In mouse members of the alpha1-PI family possess antichymotrypsin activity, whilst members of the neighbouring 'antichymotrypsin' (contrapsin) family are active against trypsin5. The five murine alpha1-PI cDNA sequences are highly related (greater than 98% sequence similarity in exons, introns and flanking sequences)1, and the majority of amino acid differences between the proteins reside within the reactive-site loop which is important in determining serpin substrate specificity (coded by exon 5 of the cDNAs). Studies have shown that only some of the murine alpha1-PI proteins are efficient inhibitors of neutrophil elastase (6, 7). A mouse deficient in alpha1-PI could provide a useful model for antitrypsin deficiency in man, but production of a suitable 'knockout' mouse mutant is complicated by the number of copies of highly related neighbouring genes which encode protease inhibitors with overlapping but subtly differing specificities. Results and Discussion Initial attempts to isolate clones spanning the alpha1-PI gene cluster from Lambda and cosmid libraries suggested that rearrangements were occurring, and indeed YAC clones spanning this region (obtained from K. Krauter, Colorado) showed frequent spontaneous deletions when analysed by Field Inversion Gel Electrophoresis (FIGE, Hoeffer). PCR primers specific for the conserved exon 2 of all five cDNAs were used to screen a phage Pl-packaged artificial chromosome library of 129/Ola mouse DNA containing 40,000 clones of 80-100kb (single genome coverage) cloned using the vector pAd10sacBll8 (L. Mullins, unpublished). Four stable clones containing exon 2 of alpha1-PI were identified. Two of these also contained exon 3 and exon 5 sequences (again identified by PCR) and were shown by ÔalleleÕ-specific PCR of exon 5, and later by sequencing, to contain genes Pl3 (clone J84) and both Pl1 and Pl2 (clone B12). An Spel/Xhol restriction enzyme map was generated of these overlapping clones using FIGE, Southern blotting and hybridization with a large number of different probes (see Figure 1, B12: map co-ordinates 45-122kb; J84: 118-192 kb). Seven BAC clones containing exon 2 (identified by hybridization) were obtained from the Research Genetics (Alabama) 129SV-derived BAC library. The specific genes present were identified by dot-blot hybridization using exon 5 specific oligonucleotides. One clone (391C22) carried four genes, P11-4, but was unstable and rearranged spontaneously on growth, and was unsuitable for mapping. Clone 241P6 carried Pl1 and Pl2 (co-ordinates 77-210kb) and confirmed the PAC derived map. Clone 144O20 carried Pl3 and Pl4 on a 72kb Sa/l fragment (0-72kb) and a further 50kb upstream of the cluster (unmapped). One clone (391C22) could not be resolved with the map presented in Figure 1 and could represent a rearranged plasmid. No clones containing the Pl5 exon 5 sequences were located in any of the libraries screened, suggesting the Pl5 gene may not be present in the 129 mouse strain. The positions of exon containing fragments were mapped by hybridization, and at a gross level agreed with the gene structure reported by Krauter et al2. The presence of three Spel sites within all four copies of intron 1 confirms the conservation of gene structure. Hybridization studies with a variety of probes showed that several kilobases extending 5' and 3' to each gene were conserved, and probably reflect a large unit of duplication in the evolution of the cluster. It may therefore be necessary to extend quite far out from the cluster to obtain unique flanking markers. Acknowledgements PAC and Lambda libraries of 129 mouse were generously provided by The Centre for Genome Research, Edinburgh (A. Smith and L. Mullins). YAC clones spanning the region were made available by K. Montgomery (New York) and K. Krauter (Colorado). L, Mullins was funded by the BBSRC. References 1. Borriello, F. and Krauter, K.S. (1991) Proc. Natl. Acad. Sci. USA 88, 9417-9421. 2. Krauter, K.S., Citron, B.A., Hsu, M.-T., Powell, D., and Darnell, J.E. (1986) DNA 5, 29-36. 3. Hill, R.E., Shaw, P.H., Barth, R.K., and Hastie, N.D. (1985) Mol. Cell. Biol. 5, 2114-2122. 4. Travis, J. and Salvesen, G.S. (1983) Ann. Rev. Biochem. 52, 655-709. 5. Takahara, H. and Sinohara, H. (1983) J. Biochem. 93, 1411-1419. 6. Paterson, T. and Moore, S. (1996) Bioch. Biophys. Res. Comm. 219, 64-69. 7. Dubin, A., Mak, P. and Travis, J. (1996) Acta. Biochem. Polonica 43, 475-480. 8. Pierce, J.C. and Sternberg, N.L. (1992) Methods in Enzymology 216, 549-574. Figure 1. (Legend). Restriction map of the murine alpha1 Protease Inhibitor gene cluster. The five exons (I-V) of the four genes (PI1-4) are shown boxed above the map, scaled in kilobases. S = SpeI; X = XhoI. Only two Sal1 sites are shown. |
