Sint1, a common integration site in SL3-3-induced T-cell lymphomas, harbors a putative proto-oncogene with homology to the septin gene family

Abstract

The murine retrovirus SL3-3 is a potent inducer of T-cell lymphomas when inoculated into susceptible newborn mice. Previously, DNAs from twenty SL3-3-induced tumors were screened by PCR for provirus integration sites. Two out of 20 tumors demonstrated clonal provirus insertion into a common region. This region has now been isolated and characterized. The region, named SL3-3 integration site 1 (Sint1), maps to the distal end of mouse chromosome 11, corresponding to human chromosome 17q25, and may be identical to a mouse mammary tumor virus integration site in a T-cell lymphoma, Pad3. Two overlapping genomic lambda clones spanning about 35 kb were isolated and used as a starting point for a search for genes in the neighborhood of the virus integration sites. A genomic fragment was used as a hybridization probe to isolate a 3-kb cDNA clone, the expression of which was upregulated in one of two tumors harboring a provirus in Sint1. The cDNA clone is predicted to encode a protein which shows 97.0% identity to a human septin-like protein encoded by a gene which has been found as a fusion partner gene of MLL in an acute myeloid leukemia with a t(11;17)(q23;q25). Together these findings raise the possibility that a proto-oncogene belonging to the septin family, and located about 15 kb upstream of the provirus integration sites, is involved in murine leukemia virus-induced T-cell lymphomagenesis.

FIG. 1

Primer positions for RT-PCR analyses. The 3.0-kb Sint1 cDNA is indicated as a line with the identified exon included as a shaded box. Arrows indicate positions and numbers of primers. The expected (brackets) and observed sizes of the PCR fragments are indicated. The PCR analyses were performed in two rounds (see Materials and Methods), and the products from the second PCR were analyzed by gel electrophoresis (not shown).

FIG. 2

(A) Relative locations and orientations of the integrated proviruses in two independent tumors (15 and 16). Box, preintegration site PCR probe A; arrows 1 and 2, primers used to amplify probe A; arrows a and b, provirus-specific primers; H, HindIII restriction site. (B) Restriction map of the cloned Sint1 locus. Upper lines indicate the two overlapping genomic λ-phage clones, while the bottom line shows the resulting map. SacI (S), XhoI (X), PstI (P), HindIII (H), and EcoRI (E) sites are indicated. Underlined are the CpG indicator enzymes (see text). Dotted lines, regions only partly analyzed by restriction enzyme mapping and sequencing; boxes A and B, hybridization probes; triangles, provirus integration sites.

FIG. 3

Sint1 maps in the distal region of mouse chromosome 11. Sint1 was placed on mouse chromosome 11 by interspecific backcross analysis. The segregation patterns of Sint1 and flanking genes in 108 backcross animals that were typed for all loci are shown at the top. For individual pairs of loci, more than 108 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J × M. spretus)F₁ parent. Black, boxes, presence of a C57BL/6J allele; white boxes, presence of an M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 11 linkage map showing the location of Sint1 in relation to linked genes is shown at the bottom. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in this study can be obtained from the Genome Data Base, a computerized database of human linkage information maintained by the William H. Welch Medical Library of Johns Hopkins University (Baltimore, Md.).

FIG. 4

Northern blot analyses of Sint1 expression. All three panels are identical Northern blots hybridized with the probes indicated at the left. The blots contain about 2 μg of poly(A)⁺ RNA per lane from eight different mouse tissues (left) and from mouse embryos at different time points (right). Size markers (in kilobases) are at the left of each autoradiogram.

FIG. 5

Correlation between Sint1 genomic region and Sint1 cDNA. (Top) Part of the genomic region; (middle) genomic 3.5-kb HindIII/NotI subclone; (bottom) corresponding cDNA clone. Triangles, provirus integration sites. Restriction enzyme site abbreviations are as defined for Fig. 1. Hybridization probes are shown as boxes A, B, and C. The grey box containing an arrow indicates the location and orientation of the exon in the genomic clone as well as in the cDNA clone. The 5′ and 3′ splicing signal (ss) sequences are shown with the conserved intron dinucleotides underlined.

FIG. 6

Sint1 cDNA and predicted amino acid sequences. In the 3′-UTR, the AT degradation motifs are underlined and the polyadenylation site consensus sequence is boxed.

FIG. 7

Comparison of the predicted amino acid sequences encoded by Sint1 cDNA and a human brain cDNA (KIAA0991; accession no., AB023208) or the human MSF cDNA (accession no., AF123052). The KIAA0991 and MSF sequences are 100% identical in the region shown. Boxed are the GTP-binding motifs. The sequences show 97.0% identity and 97.9% similarity (it should be noted, however, that the KIAA0991 and MSF sequences are 88 and 234 aa residues longer, respectively, than the Sint1 sequence). Alignment was performed with the Genetics Computer Group program BESTFIT. Vertical lines, identical residues; colons, well-conserved replacements that scored better than 0.5 in the PAM-250 matrix; dots, replacements scoring better than 0.1. The lengths of the Sint1, KIAA0991, and MSF sequences are 334, 422, and 568 aa, respectively.

FIG. 8

RNase protection assay. (Top) RNase protection of RNA from seven different SL3-3 MLV-induced tumors assayed with Sint1 and mouse β-actin RNA probes. Lane M, molecular DNA size markers (200, 300, 400, and 500 bp). Arrows indicate the protected fragments, the sizes of which are 325 bp for Sint1 and 210 bp for mouse β-actin. (Bottom) Localization of the Sint1 RNA probe as well as the Sint1/β-actin values after quantification of radioactive protected fragments in two experiments (experiment 2 is the one shown at the top).