Representational difference analysis in a lupus-prone mouse strain results in the identification of an unstable region of the genome on chromosome 11

Abstract

BXSB mice develop a lupus-like autoimmune syndrome. We have previously identified several intervals that were linked to disease and, in an attempt to characterise lupus susceptibility genes within these intervals, we have sought to isolate differentially expressed genes. Representational difference analysis was used to compare gene expression between BXSB and C57BL/10 mice using spleen and thymus as a source of mRNA. The majority of differentially expressed sequences identified were immunoglobulin and gp70-related sequences, overexpression of these being characteristic of the disease. Among other isolated sequences were a sialyltransferase gene, a mouse tumour virus superantigen gene (Mtv-3), and the virus-related sequence, hitchhiker. In BXSB the sialyltransferase gene not only overexpressed spliced transcripts, but also produced high levels of unspliced mRNA. Further analysis demonstrated that the copy number of the three linked sequences: sialyltransferase, Mtv-3 and hitchhiker, was amplified in BXSB and that the structural organisation of this locus varies in different mouse strains. This locus consists of three parts, Mtv-3-hitchhiker-sialyltransferase, hitchhiker-sialyltransferase, and sialyltransferase alone. Different combinations of the regions are present in different mouse strains. Linkage analysis demonstrated that this region at the distal end of chromosome 11 is weakly linked to phenotypic markers of disease.

Figure 1

Semi-quantitative RT–PCR analysis of the hitchhiker sequence and sialyltransferase gene. (A) RT–PCR using hitchhiker primers 12 and 13 together with control primers were performed at 20, 25 and 30 cycles, using an equal amount of BXSB and B10 thymus cDNA as template. (B) Sialyltransferase gene primers 7 and 8 were used in semi-quantitative RT–PCR analysis, with an equal amount of BXSB and B10 thymus cDNA (top) and spleen cDNA (bottom) as templates. Bands for unspliced and spliced transcripts together with the control products are indicated.

Figure 2

PCR analysis of genomic DNA for the presence of the normal and variant sialyltransferase sequences. PCR was performed using primers 1 and 2, specific for the normal sialyltransferase gene (A), and using primers 17 and 5 specific for the variant sialyltransferase gene with the hitchhiker insertion (B), in additon to control primers.

Figure 3

RT–PCR analysis of the sialyltransferase gene. (A) RT–PCR analysis of variant and normal forms of the sialyltransferase gene. The RT–PCR reactions contain three primers: primer 2 which is common to both forms, primer 1 which is specific to normal form, and primer 3 which is specific to variant form. (B) RT–PCR analysis of splicing patterns of variant form of sialyltransferase gene. PCR using primers 6 and 16 which span two introns was performed. Spliced and unspliced bands are indicated.

Figure 4

PCR analysis for the presence of Mtv-3 (region 3). PCR was performed using primers 4 and 5, together with control primers, indicating the presence or absence of region 3, respectively.

Figure 5

Schematic representation of the genomic organisation of the locus containing the sialyltransferase gene. The sialyltransferase gene, hitchhiker sequence and Mtv-3 are represented by thick bars, and their transcripts by arrowed thick bars. Exon–intron organisation is illustrated for the sialyltransferase and hitchiker loci. Primers and their orientation are indicated by numbered arrowheads (Table 1). The differences between the 3′ UTRs of the normal and variant form of the sialyltransferase gene are indicated by shading of the arrowheads.

Figure 6

Southern blot analysis of DNA from different mouse strains. (A) Genomic DNA from BXSB and B10 was digested with BamHI, transferred to nylon membrane and hybridised with a radiolabelled hitchhiker gene probe generated by PCR using primers 14 and 15. (B) The same Southern blot shown in (A) was rehybridised with a sialyltransferase gene probe generated from a PCR product amplified using primers 7 and 8. The upper bands represent the normal form of the gene while the lower bands are the variant form of the gene. (C) The same probe used in (B) was hybridised to a blot of KpnI, SacI, BglII, PstI, BamHI and HindIII digested BALB/c DNA, and HindIII digested NZB, MRL, NZW, B10, BXSB and 129 DNA. A single band was detected in BALB/c DNA regardless of the enzyme used, indicating the presence of a single copy of the sialyltransferase gene. The upper bands in other strains, representing the variant form of the gene, were analysed by densitometry to obtain copy numbers (indicated on the figure). The lower band was used as a single copy control.