RESUMO
The MOLT-16 cell line was established from the leukemic cells of a patient with T-cell acute lymphoblastic leukemia and contains a t(8;14)(q24;q11) resulting in juxtaposition of sequences downstream of the MYC gene on chromosome 8 and the J region of the T-cell receptor alpha chain gene (TCRA) on chromosome 14. The reciprocal translocation involved a complex rearrangement with two chromosome breakpoints within the TCRAJ region on chromosome 14, resulting in inversion of a 1.4 kb DNA fragment between the two breakpoints. The 5' border of the inversion joints with another segment of chromosome 14, whereas the 3' border joins with a region of chromosome 8 located at least 257 kb downstream of MYC. Extensive deletions have occurred on both chromosomes 8 and 14 in conjunction with the translocation. To investigate the possible involvement of the V(D)J recombinase in this translocation, we analyzed the nucleotide sequences surrounding the translocation breakpoints. The breakpoint on chromosome 14 occurs between a segment coding for a TCRAJ sequence and its hepatamer-nonamer signal. Heptamer-nonamer consensus sequences are also identified on chromosome 8 adjacent to the breakpoint. Inserted N and P nucleotides are observed at the breakpoint junctions.
Assuntos
Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 8/genética , DNA Nucleotidiltransferases/genética , Leucemia-Linfoma de Células T do Adulto/genética , Translocação Genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Inversão Cromossômica , Mapeamento Cromossômico , Clonagem Molecular , Sondas de DNA , DNA de Neoplasias/análise , Deleção de Genes , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/genética , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Análise de Sequência de DNA , Células Tumorais Cultivadas , VDJ RecombinasesRESUMO
Translocations involving chromosome band 11q23, found in acute lymphoid and myeloid leukemias, disrupt the MLL gene. This gene encodes a putative transcription factor with homology to the zinc fingers and other domains of the Drosophila trithorax gene product and to the "AT-hook" motif of high mobility group proteins. To map potential transcriptional activation or repression domains of the MLL protein, yeast GAL4 DNA-binding domain and MLL hybrid protein-expressing plasmids were cotransfected with chloramphenicol acetyltransferase reporter plasmids in a transient transfection system. We found that MLL contains a strong activation domain and a repression domain. The former, located telomeric (3') to the breakpoint region, activated transcription 18-fold to > 200-fold, depending on the promoter and cell line used for transfection. A repression domain that repressed transcription 4-fold was located centromeric (5') to the breakpoint region of MLL. The MLL AT-hook domain protein was expressed in bacteria and was utilized in a gel mobility shift assay to assess DNA-binding activity. The MLL AT-hook domain could bind cruciform DNA, recognizing structure rather than sequence of the target DNA. In translocations involving MLL, loss of an activation domain with retention of a repression domain and a DNA-binding domain on the der(11) chromosome could alter the expression of downstream target genes, suggesting a potential mechanism of action for MLL in leukemia.
Assuntos
Cromossomos Humanos Par 11 , Proteínas de Ligação a DNA/genética , Leucemia Aguda Bifenotípica/genética , Proto-Oncogenes , Fatores de Transcrição , Translocação Genética , Animais , Sítios de Ligação , Cloranfenicol O-Acetiltransferase/biossíntese , Mapeamento Cromossômico , DNA/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Deleção de Genes , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes de Insetos , Células HeLa , Histona-Lisina N-Metiltransferase , Humanos , Mutagênese , Proteína de Leucina Linfoide-Mieloide , Proteínas Recombinantes de Fusão/biossíntese , Transcrição Gênica , Transfecção , Dedos de ZincoRESUMO
HSB-2 is a cell line derived from a patient who had T-cell acute lymphoblastic leukemia (T-cell ALL) with a t(1;7)(p34;q34). We used a genomic probe from the T-cell receptor beta (TCR beta) locus (7q34) to identify DNA rearrangements in HSB-2. Two rearranged BglII DNA fragments were cloned, and one of these clones was shown to contain the translocation breakpoint on the derivative chromosome I [der(I)]. We used a probe derived from this clone to isolate an unrearranged phage clone encompassing the breakpoint at Ip34. The restriction map of this clone was compared to the published maps of known protooncogenes located at Ip32-34. By restriction mapping, Southern blot analysis, and DNA sequencing we showed that the translocation breakpoint on chromosome I is located within the first intron of the LCK gene. The LCK gene codes for p56lck, a member of the SRC family of cytoplasmic tyrosine protein kinases. There are two classes of LCK transcripts (type I and type II), each expressed from a distinct promoter, and each having a unique 5' untranslated region (UTR); the protein coding regions of the two classes are identical. The breakpoint in the t(1;7) separates the two LCK promoters and juxtaposes the constant region of the TCR beta locus with the proximal promoter and with the protein-coding region of the LCK gene on the der(I) chromosome.
