1. by Jean-Michel Cayuela, Betty Gardie, and François Sigaux
Disruption of the Multiple Tumor Suppressor Gene MTS1/p16INK4a/CDKN2 by Illegitimate V(D)J Recombinase Activity in T-Cell Acute Lymphoblastic Leukemias
by Jean-Michel Cayuela, Betty Gardie, and François Sigaux
Blood
Volume 90(9):
November 1, 1997
©1997 by American Society of Hematology

2. Restriction map of the MTS locus and schematic representation of its configuration in T-ALL samples.
Restriction map of the MTS locus and schematic representation of its configuration in T-ALL samples. (A) An updated MTS locus map is shown. The probes used and the 2 breakpoint clusters MTS1 bcrα and MTS1 bcrβ are indicated. C5.3, 2.7, and 2.3, sequence tagged sites (STSs); E, exon; *, polymorphic BamHI site; +, the 16 breakpoints include that of the T84 sample, for which the MTS1 configuration was not fully characterized. (B) Schematic representation of the 2 chromosome 9s in cases with rearrangements occurring in the locus (22 T-ALL samples and the MOLT4 cell line).21 In 1 sample, T106, 2 breakpoints occurring on each chromosome 9 were detected. In T39, breakpoints were different at presentation (T39p) and relapse (T39r). Dashed lines indicate deletions; vertical arrows indicate breakpoint-containing regions. The T84 configuration is not shown. All rearrangements within MTS1 bcrβ suppress the possibility to express p16INK4a (encoded by exon 1α, exon 2, and exon 3) and p19ARF (encoded by exon 1β, exon 2, and exon 3). In rearrangements within MTS1 bcrα, MTS1 exon 1β is deleted and p16INK4a encoding exons remain unchanged. Transcripts initiated from the promoter located 5′ to MTS1 exon 1α and the p16INK4a protein are expressed (data not shown). These data suggest that p19ARF and/or p15INK4b and not p16INK4a could be the functional target(s) of the rearrangements in these cases (Gordie et al, submitted for publication).
Jean-Michel Cayuela et al. Blood 1997;90:
©1997 by American Society of Hematology

3. Characterization of the MTS1 bcrα breakpoint cluster.
Characterization of the MTS1 bcrα breakpoint cluster. (A) Restriction map of the MTS1 bcrα region. Only relevant restriction sites are shown. Xb, Xba I; Xh, XhoII; M, Mbo I; E, EcoRI; OL, oligonucleotide. (B) Southern blot analysis of T39p, T117, and T127 rearrangements using the 5′ exon1α probe and Mbo I, XhoII, and Xba I digests. The T39p sample contains approximately 15% nontumoral cells. (C) Reverse view of ethidium bromide–stained gel showing PCR experiments using primers OL17 v OL14 or OL15. Dilutions of DNA from peripheral blood lymphocytes (PBL) from a healthy donor in DNA from the pre-B cell line REH, in which both MTS1 and MTS2 genes are deleted, were included. M, molecular weight marker V (Boehringer Mannheim, Meylan, France); C-, negative control (no DNA). (D) Nucleotide sequence of the MTS1 bcrα breakpoint cluster. Mbo I, Xba I, and EcoRI restriction sites are underlined, and the candidate heptamer targeted by the recombinase activity is boxed. Positions of oligonucleotides used to localize the breakpoints are shown.
Jean-Michel Cayuela et al. Blood 1997;90:
©1997 by American Society of Hematology

4. Cloning and sequencing of 3 breakpoints occurring in MTS1 bcrα.
Cloning and sequencing of 3 breakpoints occurring in MTS1 bcrα. Top: Schematic representation of the 36-kb deletion that brings sequences 5′ to MTS2 exon 1 (MTS2 bcr1 ) upstream to MTS1 exon 1α. (▪) MTS1 exons; (□) MTS2 exons. Vertical arrows represent breakpoints. Insert: Hybridization with junction-specific oligonucleotides after direct PCR amplification of T39p, T117, and T127 breakpoints with OL16 and OL1. PCR products were analyzed in a 2% agarose, 2% NuSieve gel, transferred on a hybond N+ membrane, and successively hybridized with 32P-radiolabeled oligonucleotide specific for junctional sequences of T39p (AJ T39p), T117 (AJ T117), and T127 (AJ T127) and with a common internal oligoprobe, OL15. C-, negative control (no DNA). Bottom: Nucleotide sequence alignment of T39p, T117, and T127 rearrangements with germline chromosome 9 sequences. Canonic heptamers are boxed, and putative N regions are underlined. No consensus nonamer was found. However, highly degenerated nonamers may be present at 23 bp from the MTS2 bcr1 heptamer and at 12 bp from the MTS1 bcrα heptamer. Localization of breakpoints on germline sequences is indicated by arrows.
Jean-Michel Cayuela et al. Blood 1997;90:
©1997 by American Society of Hematology

5. Characterization of the MTS1 bcrβ cluster and cloning of 3 representative rearrangements.
Characterization of the MTS1 bcrβ cluster and cloning of 3 representative rearrangements. (A) Partial map of the DNA region flanked by STS2.3 and STS2.7. Localization of the 2 breakpoints occurring 5′ to MTS1 exon 1β (T73 and T79) and of the 14 breakpoints occurring 3′ to exon 1β is shown. Breakpoints were localized by Southern blot experiments using the MTS1 exon 1β probe and BglII digests, and in certain cases, Sal I, BamHI, EcoRI, and HindIII digests. Bracketed cases show rearranged fragments of identical length in various digests. (B) Top: Schematic representation of MOLT4, T42, and T123 rearrangements. Vertical arrows represent breakpoints. Insert: Hybridization with junction-specific oligonucleotides after direct PCR amplification of the MOLT4 breakpoint using primers OL5 and OL22 and the T42 and T123 breakpoints using primers OL5 and OL20. PCR products were successively hybridized with 32P-radiolabeled oligonucleotides specific for junctional sequences of MOLT4 (AJ MOLT4), T42 (AJ T42), and T123 (AJ T123) and with a common internal oligoprobe, OL6. Bottom: Nucleotide sequence alignment of MOLT4, T42, and T123 rearrangements with chromosome 9 germline sequences. Heptamers are boxed, and putative N regions are underlined. No definite nonamer was found.
Jean-Michel Cayuela et al. Blood 1997;90:
©1997 by American Society of Hematology

6. Localization of the breakpoints occurring 3′ to MTS1 exon 1β by PCR
Localization of the breakpoints occurring 3′ to MTS1 exon 1β by PCR. (A) Partial map of the region located 3′ to MTS1 exon 1β.
Localization of the breakpoints occurring 3′ to MTS1 exon 1β by PCR. (A) Partial map of the region located 3′ to MTS1 exon 1β. The polymorphic dinucleotide CA repeat and the primers OL5 to OL12 used to localize the breakpoints are indicated. Breakpoint-containing regions determined by PCR are represented above by solid bars. (B) Reverse view of an ethidium bromide–stained gel showing representative PCR experiments using primers OL5 v OL7 or OL6. Dilutions of DNA from PBL from a healthy donor in DNA from pre-B cell line REH were included in the experiment. M, molecular weight marker V. One undeleted allele is present in case T84. T128 and T39R could not be studied by PCR, because of the presence of nontumoral cells in the T128 sample and the absence of available material for T39R. (C) Nucleotide sequence of the region located immediately 3′ to MTS1 exon 1β. The position of the relevant oligonucleotides is shown, and candidate heptamers are boxed.
Jean-Michel Cayuela et al. Blood 1997;90:
©1997 by American Society of Hematology