Comparative and genetic analysis of the porcine glucocerebrosidase (GBA) gene

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Abstract

The genomic sequence of the porcine (Sus scrofa) glucocerebrosidase (GBA) gene (∼5.7 kb), encoding glucocerebrosidase (glucosylceramidase; acid beta-glucosidase; EC 3.2.1.45), was determined and compared with human (Homo sapiens) GBA and GBAP (pseudogene). The porcine gene harbours 11 exons and 10 introns, and the genomic organization is identical with human GBA. The exon sequences, coding for signal peptide and mature protein, show 81% and 90% sequence identity, respectively, with the corresponding human GBA sequences. Short interspersed elements, SINEs (PREs), are present in introns 2, 4 and 7. There is no evidence of a pseudogene in pig. The deduced protein sequence of GBA consists of 39 amino acids of signal peptide (long form) and 497 amino acids of the mature protein; the latter shows 90% sequence identity with the human protein. Four polymorphisms were observed within the porcine gene: insertion/deletion of one of the two SINEs (PREs) in intron 2 (locus PREA); deletion of a 37- to 39-bp stretch in intron 4 (one direct repeat and 5′ end of PRE); deletion of a 47-bp stretch in the middle part of PRE in intron 4 (locus PREB); and single-base transition (C–T) in intron 6 (locus HaeIII–RFLP). GBA was assigned to chromosome 4q21 by FISH and was localized to the same region by linkage analysis and RH mapping, i.e., to the chromosome 4 segment where quantitative trait loci for growth and some carcass traits are located.

Introduction

Glucocerebrosidase (GBA; glucosylceramidase; acid beta-glucosidase; EC 3.2.1.45), a lysosomal membrane protein that cleaves the O-beta-d-glucosidic linkage of glucosylceramide and aryl-beta-glucosides, is encoded by the GBA gene which is located on chromosome 1q21 in humans Shafit-Zagardo et al., 1981, Barneveld et al., 1983, Ginns et al., 1985. The human (Homo sapiens) glucocerebrosidase cDNA was first cloned and sequenced by Sorge et al. (1985) and Tsuji et al. (1986), and the genomic sequence as well as genomic organization of the gene (GBA) and pseudogene (GBAP) was determined by Horowitz et al. (1989). The presence of the pseudogene, which shows 96% sequence identity with the functional gene and is located approximately 16 kb downstream from this, complicates the study of GBA in humans. Both the gene and the pseudogene contain 11 exons and 10 introns, but they differ in length (GBA is ∼7.6-kb long, while GBAP is ∼5.7-kb long). In exon 9 of GBAP, a 55-bp deletion occurs (Horowitz et al., 1989) and exactly the same deletion is found in GBA of some Gaucher's disease patients (Beutler and Gelbart, 1993). The pseudogene is consistently transcribed, and in some cases, the level of transcription is equal to that of the functional gene (Sorge et al., 1990). However, the pseudogene harbours many point mutations and deletions that preclude it from directing synthesis of active protein (Beutler, 1993).

In the human gene, numerous mutations have been described that lead to an inherited glucocerebrosidase deficiency resulting in the accumulation of glucocerebroside in various tissues, a disorder known as Gaucher's disease (Beutler, 1993).

Specific precautions are needed to avoid incidental coinvestigation of the pseudogene Tayebi et al., 1996, Finckh et al., 1998 not only in human but also in animal species. In fact, it would be practically impossible to conclude whether the gene or pseudogene was approached without the prior knowledge of the sequence.

A human GBA cDNA probe was used to search for RFLPs in porcine (Sus scrofa) GBA, and the gene was mapped to the chromosome 4 linkage group ATP1B1GBAEAL Marklund et al., 1993, Archibald et al., 1995. GBA is located in a chromosome region where QTLs for growth and some carcass traits have been found Andersson et al., 1994, Andersson-Eklund et al., 1998, Walling et al., 2000, De Koning et al., 2001, Malek et al., 2001, Geldermann et al., 2003. However, no sequence information on porcine GBA was available prior to the present study.

In humans, at least six other genes and two pseudogenes are located in the 75-kb region containing GBA (Winfield et al., 1997). The knowledge of genes in the homologous region in pigs, their structures, chromosomal and linkage positions, is important for comparative genomics and should facilitate the search for candidate or causative genes for growth and carcass traits. The aim of the present study was to sequence and study the organization of the porcine GBA gene and to compare it with the human GBA and GBAP, to search for polymorphisms by using polymerase chain reaction (PCR) and PCR–RFLP and to study chromosomal assignment and genetic mapping.

Section snippets

Polymerase chain reaction (PCR) amplification of the GBA gene

The porcine (Sus scrofa) GBA gene was amplified using three pairs of PCR primers (1F-1R, 2F-2R and 3F-3R) that were designed based on the human GBA sequence (Horowitz et al., 1989, Winfield et al., 1997; EMBL accession numbers J03059; AF023268; NCBI NM_000157) as well as from the porcine sequence that we determined (EMBL AJ575649). Another pair of primers (4F-4R) was used to identify a BAC clone (Rogel-Gaillard et al., 1999) containing GBA, and two other pairs of primers (5F-2R and 6F-6R) were

PCR amplification, subcloning and sequence analysis of GBA gene

By using PCR primers 1F-1R, 2F-2R and 3F-3R in PCR on porcine genomic DNA, we were able to amplify overlapping fragments of GBA, from exon 2 to the 3′UTR in exon 11. We did not succeed in designing gene-specific primers to amplify the part of the gene covering exons 1 and 2 and intron 1; therefore, a fragment with this sequence was obtained from the BAC clone no. 280D6. The PCR fragments and the BAC fragment were subcloned and the complete sequence of 5687 bp was determined. The sequence

Acknowledgements

We greatly appreciate Drs. Martine Yerle and Denis Milan (INRA, Castanet-Tolosan, France) for providing the IMpRH panel and Dr Patrick Chardon (INRA, Jouy-en-Josas, France) for providing the BAC library. Dr. Michal Kopečný is thanked for useful discussions. We acknowledge the expert technical assistance of Marie Datlová, Marc Mattheeuws and Dominique Vander Donckt. This research was supported by the Grant Agency of the Czech Republic (Grant no. 523/00/0669) and Grant Agency of the Ministry of

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