Structural characterization of the mouse high growth deletion and discovery of a novel fusion transcript between suppressor of cytokine signaling-2 (Socs-2) and viral encoded semaphorin receptor (Plexin C1)

Abstract

The high growth (HG) mouse mutation is a 460 Kb deletion of chromosome 10 which causes a 30–50% increase in growth in the homozygous animal. We have shotgun sequenced six bacterial artificial chromosomes which span the length of the deletion to an average depth of 13.2× to generate a 649,868 bp sequence. Sequence analysis revealed the presence of three genes, suppressor of cytokine signaling-2 (Socs-2), caspase and RIP adaptor with death domain (Raidd/Cradd), and viral encoded semaphorin receptor (Plexin C1, viral encoded semaphorin receptor). The two deletion breakpoints lie in within the second introns of both Socs-2 and Plexin C1, resulting in the formation of a novel expressed fusion transcript between Socs-2 and Plexin C1 in HG mice. Expression of the fusion transcript, the presence of four splice variants of Raidd/Cradd and the exon structure of Socs-2 were illustrated using polymerase chain reaction. Genomic comparisons of the mouse and human sequence were used to verify the sequence assembly.

Introduction

The study of mutation, either induced or naturally occurring, is one of the oldest tools and a classic paradigm geneticists have exploited to gain insight toward understanding normal biological processes. The high growth (hg) mutation in mice is one such example of a spontaneously occurring mutation which causes a 30–50% increase in the overall body size of homozygous animals. This model provides a unique opportunity to study genes with prominent roles in the regulation of mammalian growth as the hg mutation causes mice to grow proportionately larger without becoming obese (Famula et al., 1988). Endocrine studies examining the levels of growth hormone (GH) and insulin-like growth factor I (IGF-I) in both high growth (HG) and normal mice found HG mice not only lacked the typical pulsatile pattern of GH secretion, but also showed reduced levels of GH and higher levels of IGF-I (Medrano et al., 1991). Previous mapping studies to locate the high growth locus exposed a gross deletion of approximately 500 Kb in chromosome 10 of HG mice (Horvat and Medrano, 1996). Two genes, CASP2 and RIPK1 domain containing adaptor with death domain (Raidd/Cradd) and suppressor of cytokine signaling-2, also known as Cish2 (Socs-2) have been identified within this region through exon trapping and Southern blot experiments (Horvat and Medrano, 1998, Horvat and Medrano, 2001). However, the Raidd/Cradd mouse knockout (T.W. Mak, personal communication) had no reported growth related phenotype, indicating that other genes either within or affected by the deletion were responsible for the high growth phenotype.

The involvement of Socs-2 in creating the high growth phenotype was confirmed with the development of a Socs-2 knockout mouse by Metcalf et al. (2000), which resulted in an animal that was phenotypically similar to the HG mouse. Unlike other genetic mutations such as point mutations or small deletions/insertions, major chromosomal rearrangements such as the high growth deletion may have extensive repercussions to the organism by affecting not only deleted genes, but also those located proximally to the mutation. Since there are some phenotypic differences between the HG and Socs-2^−/− mouse such as increased IGF-I serum levels in the HG mouse as compared to wildtype, it is not known whether the high growth phenotype is due to the lack of a single gene product or a combination of gene products.

To characterize the hg deletion and its flanking sequences, we shotgun sequenced a 650 Kb segment of mouse chromosome 10. Our sequence analysis verified the presence of three known genes, Socs-2, Raidd/Cradd, and viral encoded semaphorin receptor (Plexin C1, also known as Vespr or viral encoded semaphorin receptor), each of which we examined in both HG and wildtype mice in order to determine if or how their transcriptional expression patterns were affected by the deletion. We also describe our search for novel genes not previously mapped to this region, comparatively analyze the human and mouse genomic sequences of the high growth region, and discuss possible mechanisms for the creation of this spontaneous mutation. Here we have not only verified the complete absence of Raidd/Cradd gene expression and aberrant expression of Socs-2 and Plexin C1 in HG mice, but also significantly narrowed the possibility that additional, unknown genes within the deletion also contribute to the overall high growth phenotype. This highlights the potential utility of the HG mouse not only as a natural model for overgrowth by deregulation of cytokine signal transduction, but also as a model for understanding Raidd/Cradd's role in apoptosis, Plexin C1 in the immune system, and possible interactions among these adjacent genes.