Transposition of c-abl oncogene in a case of masked Ph chromosome duplicated in blastic phase

Summary A female with chronic myelocytic leukemia (CML) in blastic phase (BP) showed a masked Ph chromosome that had originated by a translocation between chromosomes 8 and 22, with no obvious involvement of chromosome 9. A duplication of the masked Ph and trisomy 13 were present as additional anomalies. The karyotype on peripheral blood unstimulated cultures was 48,XX,t(8;22)(p12;q11),+13,+der(22) t(8;22)/47,XX,t(8;22)(p12;q11),+der(22)t(8;22). While the duplication of the Ph is a frequent finding in BP of CML, we did not find any other case in the literature with duplication of a masked Ph. In situ hybridization with c-abl and bcr probes showed that a 3 bcr sequence was translocated to the der(8) chromosome, while the c-abl oncogene was transposed to the masked Ph.     

HLA-DPB1 supertype-associated protection from childhood leukaemia: relationship to leukaemia karyotype and implications for prevention

Most childhood B cell precursor (BCP) acute lymphoblastic leukaemia (ALL) cases carry the reciprocal translocation t(12;21)(p13;q22) (∼25%), or a high hyperdiploid (HeH) karyotype (30%). The t(12;21) translocation leads to the expression of a novel fusion gene, TEL-AML1 (ETV6-RUNX1), and HeH often involves tri- and tetrasomy for chromosome 21. The presence of TEL-AML1+ and HeH cells in utero prior to the development of leukaemia suggests that these lesions play a critical role in ALL initiation. Based on our previous analysis of HLA-DP in childhood ALL, and evidence from in vitro studies that TEL-AML1 can activate HLA-DP-restricted T cell responses, we hypothesised that the development of TEL-AML1+ ALL might be influenced by the child’s DPB1 genotype. To test this, we analysed the frequency of six HLA-DPB1 supertypes in a population-based series of childhood leukaemias (n = 776) classified by their karyotype (TEL-AML1+, HeH and others), in comparison with newborn controls (n = 864). One DPB1 supertype (GKD) conferred significant protection against TEL-AML1+ ALL (odds ratio (OR), 95% confidence interval (95% CI): 0.42, 0.22–0.81; p < 0.005) and HeH ALL (OR; 95% CI: 0.44, 0.30–0.65; p < 0.0001). These negative associations were almost entirely due to a single allele, DPB1*0101. Our results suggest that DPB1*0101 may afford protection from the development of TEL-AML1+ and HeH BCP ALL, possibly as the result of a DP-restricted immune response to BCP ALL-associated antigen(s), the identification of which could have important implications for the design of prophylactic vaccines.     

Rejoining between 9q+ and Philadelphia chromosomes results in normal-looking chromosomes 9 and 22 in Ph1-negative chronic myelocytic leukemia

Summary Rearrangement of the breakpoint cluster region (bcr) and the chromosomal location of c-abl and 3-bcr were studied in two patients with Philadelphia chromosome (Ph¹)-negative chronic myelocytic leukemia (CML). One patient (patient 1) had a normal karyotype and the other (patient 2), 46,XY,inv(3)(q21q26). Both patients showed the bcr rearrangement by Southern blot analysis with a 1.2 kb 3-bcr probe. In situ hybridization studies demonstrated the location of the homologous sequences of bcr on chromosome 22 in patient 1, and on chromosomes 9 and 22 in patient 2. These findings indicate that the morphologically normal-looking chromosomes 9 and 22 in patient 2 are the result of a retranslocation between chromosomes 9q+ and 22q-, abnormalities which were first formed by a standard Ph¹ translocation.     

Influence of BCR/ABL fusion proteins on the course of Ph leukemias

The hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) is the presence of the Philadelphia chromosome as a result of the t(9;22) translocation. This gene rearrangement results in the production of a novel oncoprotein, BCR/ABL, a constitutively active tyrosine kinase. There is compelling evidence that the malignant transformation by BCR/ABL is critically dependent on its Abl tyrosine kinase activity. Also the bcr part of the hybrid gene takes part in realization of the malignant phenotype. We supposed that additional mutations accumulate in this region of the BCR/ABL oncogene during the development of the malignant blast crisis in CML patients. In ALL patients having p210 fusion protein the mutations were supposed to be preexisting. Sequencing of PCR product of the BCR/ABL gene (Dbl, PH region) showed that along with single-nucleotide substitutions other mutations, mostly deletions, had occurred. In an ALL patient a deletion of the 5th exon was detected. The size of the deletions varied from 36 to 220 amino acids. For one case of blast crisis of CML changes in the character of actin organization were observed. Taking into account the functional role of these domains in the cell an etiological role of such mutations on the disease phenotype and leukemia progression is plausible.     

The first BCR gene intron contains breakpoints in Philadelphia chromosome positive leukemia.

The hallmark of chronic myelogenous leukemia (CML) is a translocation between chromosomes 9 and 22 - the Philadelphia (Ph') translocation. The translocation is also found in acute lymphocytic leukemia (ALL) albeit in a lower percentage of patients. The breakpoint on chromosome 22 is located within the BCR gene: in CML, breakpoints are clustered within 5.8 kb of DNA, the major breakpoint cluster region (Mbcr). In ALL, breakpoints have been reported within the Mbcr but also in more 5' regions encompassing the BCR gene. To characterize the latter breakpoints, we have molecularly cloned and mapped the entire gene, which encompasses approximately 130 kb of DNA. Mbcr negative, Ph'-positive ALL breakpoints were not distributed at random within the gene but rather were found exclusively within the 3' half of the first BCR gene intron. In contrast to the Mbcr, which is limited to a region of 5.8 kb, this part of the intron has a size of 35 kb. Translocation breakpoints in this region appear to be specific for ALL, since it was not rearranged in clinically well-defined CML specimens nor in any other tumor DNA samples examined.     

bcr rearrangement and C-abl gene expression in Ph1-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts

bcr gene rearrangement and c-abl gene expression were analyzed in a patient with Philadelphia chromosome (Ph1)-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts. These data were compared with those from a patient with chronic myelogenous leukemia (CML) in mixed crisis. The leukemic cells of both patients showed immuno-phenotypic profiles such as non-T, non-B common ALL with some MPO-positive leukemic cells and rearranged JH genes. On analysis of molecular events associated with the Ph1 chromosome, the leukemic cells of a patient with CML in mixed crisis showed bcr rearrangement and an 8.5-kb bcr-abl chimeric mRNA, but those of a patient with Ph1-positive hybrid acute leukemia showed no 8.5-kb bcr-abl mRNA, as previously reported in a number of Ph1-positive acute lymphoblastic leukemia (ALL) cases. These results revealed that the molecular event found in Ph1-positive ALL is not only restricted to lymphoid lineage but may play an important role in the proliferation of the myeloid lineage.     

Translocation events demonstrated by molecular,in situ hybridization and chromosome pairing analyses in highly asymmetric somatic hybrid plants

Cytological analyses show rearranged chromosomes in some highly asymmetric nuclear hybrids obtained after fusion of mesophyll protoplasts ofNicotiana plumbaginifolia (wild type) with γ-irradiated (100 krad), kanamycin-resistant mesophyll protoplasts ofPetunia hybrida. Molecular, cytogenetic andin situ hybridization analyses performed on the asymmetric somatic hybrid P₁, previously identified as having a clearly metacentric chromosome besides a nearly completeNicotiana chromosome complement, are reported. Meiotic analysis andin situ hybridization experiments using ribosomal DNA as a probe showed that this metacentric chromosome represents a translocation of a chromosome fragment onto chromosome 9 ofN. plumbaginifolia. Southern hybridization with an rDNA probe showed that onlyNicotiana-specific rDNA was present.In situ hybridization experiments, using total genomic DNA ofP. hybrida as a probe, demonstrated that the translocated fragment representedPetunia DNA.     

A novel translocation,t(9;21)(q13;q22) rearranging the RUNX1 gene in acute myelomonocytic leukemia

A novel translocation t(9;21)(q13;q22) associated with trisomy 4 has been detected in a patient with acute myelomonocytic leukemia (AML,M4) in relapse. The chromosomal translocation results in rearrangement of the RUNX1 gene at 21q22. The DNA sequence rearranged on chromosome 9 remains unidentified. The diversity of the partners involved in translocations implicating RUNX1 suggests that the functional consequences of the abnormality are more due to the truncation of RUNX1 than to the identity of its partner in the rearrangement.     

The complex translocation (9;14;14) involving IGH and CEBPE genes suggests a new subgroup in B-lineage acute lymphoblastic leukemia

Many subtypes of acute lymphoblastic leukemia (ALL) are associated with specific chromosomal rearrangements. The complex translocation t(9;14;14), a variant of the translocation (14;14)(q11;q32), is a rare but recurrent chromosomal abnormality involving the immunoglobulin heavy-chain (IGH) and CCAAT enhancer-binding protein (CEBPE) genes in B-lineage ALL (B-ALL) and may represent a new B-ALL subgroup. We report here the case of a 5-year-old girl with B-ALL, positive for CD19, CD38 and HLA-DR. A direct technique and G-banding were used for chromosomal analysis and fluorescentin situ hybridization (FISH) with BAC probes was used to investigate a possible rearrangement of the IGH andCEBPE genes. The karyotype exhibit the chromosomal aberration 46,XX,del(9)(p21),t(14;14)(q11;q32). FISH with dual-color break-apartIGH-specific and CEPBE-specific bacterial artificial chromosome (BAC) probes showed a complex t(9;14;14) associated with a deletion of cyclin-dependent kinase inhibitor 2A (CDKN2A) and paired box gene 5 (PAX5) at 9p21-13 and duplication of the fusion gene IGH-CEBPE.     

Rearrangement,amplification, and assortment of mitochondrial DNA molecules in cultured cells of Brassica campestris

Summary We compared Brassica campestris mitochondrial and chloroplast DNAs from whole plants and from a 2-year-old cell culture. No differences were observed in the chloroplast DNAs (cpDNAs), whereas the culture mitochondrial DNA (mtDNA) was extensively altered. Hybridization analysis revealed that the alterations are due entirely to rearrangement. At least two inversions and one large duplication are found in the culture mtDNA. The duplication element is shown to have the usual properties of a plant mtDNA high frequency recombination repeat. The culture mtDNA exists as a complex heterogeneous population of rearranged and unrearranged molecules. Some of the culture-associated rearranged molecules are present in low levels in native plant tissue and appear to have sorted out and amplified in the culture. Other mtDNA rearrangements may have occurred de novo. In addition to alterations of the main mitochondrial genome, an 11.3 kb linear mtDNA plasmid present in whole plants is absent from the culture. Contrary to findings in cultured cells of other plants, small circular mtDNA molecules were not detected in the B. campestris cell culture.     

The genetic analysis of F1 hybrid larvae between female Trichinella spiralis and male Trichinella britovi

Hybrids between female Trichinella spiralis and male Trichinella britovi were constructed. Then, hybrid genotype was characterized by DNA markers including mitochondrial cytochrome c oxidase subunit I (CO I) gene, the gene encoding the 43-kDa excretory–secretory (ES) protein, and genomic DNA fragments specific for T. spiralis and T. britovi identified from random amplified polymorphism DNA (RAPD). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial CO I gene revealed that all hybrids carried a T. spiralis pattern. The same analysis of the gene encoding the 43-kDa ES protein showed that each hybrid carried both T. spiralis and T. britovi gene type simultaneously. In the analysis of genomic DNA using RAPD-derived PCR primers, some hybrids carried T. spiralis and T. britovi-specific RAPD markers, while others carried the RAPD marker of T. spiralis only.     

Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis

Summary The mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light. We have isolated from a S. pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9 192-containing cell population. Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene. We inactivated the repair function of the cloned fragment by a single insertion of the S. pombe ura4 gene. This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating that the rad9 gene does not encode an essential cellular function. In addition, the rad9-192 mutant population is as radiosensitive as the disruption mutant, indicating that rad9 gene function is severely if not totally inhibited by the molecular defect responsible for the rad9-192 phenotype. DNA sequence analysis of rad9 reveals an open reading frame of 1,278 bp, interrupted by three introns 53 bp, 57 bp, and 56 by long, respectively, and ending in the termination codon TAG. This gene is capable of encoding a protein of 426 amino acids, with a corresponding calculated molecular weight of 47,464 daltons. No significant homology was detected between the rad9 gene or its deduced protein sequence and sequences previously entered into DNA and protein sequence data banks.     

Humanbcr-abl gene has a lethal effect on embryogenesis

The chimaericbcr-abl oncogene is thought to have a crucial role in the development or maintenance of chronic nyelogenous leukaemia. To study this oncogene in a more direct way, thebcr-abl gene encoding the P210 protein under control of thebcr gene promoter was introduced into fertilized one-cell embryos, which were then re-implanted into foster mothers. Our data, obtained after several experiments, demonstrate that no live transgenic progeny could be obtained using thisbcr-abl construct. Thebcr gene is expressed in the course of embryogenesis and thebcr-abl gene product appears to have a pleiotropic lethal effect during this period of development. In concordance, several gross abnormalities were observed while no evidence of neoplastic formation was found. These results suggest that thebcr-abl encoded protein severely affects the process of normal embryogenesis.     

Nucleotide sequence of the rci gene encoding shufflon-specific DNA recombinase in the IncI1 plasmid R64: homology to the site-specific recombinases of integrase family

Summary Shufflon is a novel type of DNA rearrangement in which four DNA segments are flanked by seven 19-bp repeat sequences. The site-specific recombination between any inverted repeats results in an inversion of the DNA segment(s) either independently or in groups. The recombination is mediated by a gene designated rci. We have determined the nucleotide sequence of the rci gene and found that it encodes a basic protein with 384 amino acid residues. The rci gene was fused with lacZ and its gene product was identified by Western blot analysis. The Rci protein shows regional homologies to the site-specific recombinases encoded by the bacteriophage genomes, including those of , 80, P22, P2, 186, P4 and P1.     

Translocation t(1; 14) and rearrangement of the gene for the alpha chain of the T-cell receptor in acute T-lymphoblastic leukemia

A t(1; 14) (p32; q11) translocation has been found by the cytogenetic study of a T cell acute lymphoblastic leukemia patient. Molecular hybridization with a probe (D14S7) which recognizes a fragment of the alpha chain T receptor gene has shown a rearranged EcoRI 11.0 kb band in addition to the germline 3 and 8 kb bands. This translocation infrequently described until now is a new example of an alpha chain gene rearrangement in T cell malignancy. Moreover the breakpoint on chromosome 1 may also involve the newly isolated protooncogene, L-myc, normally localized on the chromosomal band 1 p32.     

A flower-specific cDNA encoding a novel thionin in tobacco

Summary A gene library of Bacillus subtilis chromosomal DNA was screened for genes capable of reverting the growth defects of the Escherichia coli secA51(Ts) mutant at 42° C. A B. subtilis gene, designated csaA, was found to phenotypically suppress not only the growth defects of the E. coli mutant, but also to relieve the detrimental accumulation of precursors of exported proteins. The csaA gene encoded a protein of 15 kDa (137 amino acids) and was likely to be the distalmost member of an operon. No similarity to csaA was found among DNA or protein sequences deposited in databases. In contrast to other homologous or heterologous suppressors of the E. coli secA51(Ts) mutation, the csaA gene did not exert pleiotropic effects on either the E. coli sec Y24(Ts) or lep9(Ts) mutations. However, it restored the ability of a SecB-deficient mutant to grow on complex medium. It is proposed that CsaA serves as a molecular chaperone for exported proteins or alternatively acts by stabilizing the SecA protein.     

O-acetyl sialic acid specific IgM in childhood acute lymphoblastic leukaemia

Initial studies have revealed an enhanced surface expression of O-acetylated sialoglycoconjugates (O-AcSGs) on lymphoblasts concomitant with high titres of IgG in childhood Acute Lymphoblastic Leukaemia (ALL) (Mandal C, Chatterjee M, Sinha D, Br J Haematol 110, 801–12, 2000). In our efforts to identify disease specific markers for ALL, we have affinity-purified IgM directed against O-AcSGs that reacts with three disease specific O-AcSGs present on membrane proteins derived from peripheral blood mononuclear cells (PBMC) of ALL patients. Antibody specificity towards O-AcSGs was confirmed by selective binding to erythrocytes bearing surface O-AcSGs, decreased binding with de-O-acetylated BSM and following pretreatment with O-acetyl esterase. Competitive inhibition ELISA demonstrated a higher avidity of IgM for O-AcSG than IgG. Flow cytometry demonstrated the diagnostic potential of purified O-AcSA IgM as binding was specific with ALL patients and minimal with other haematological disorders and normal individuals. It therefore may be adopted as a non-invasive approach for detection of childhood ALL. Taken together, the data indicates that carbohydrate epitopes having terminal O-AcSA 2 6 GalNAc determinants induce disease specific IgG and IgM, potentially useful molecular markers for childhood ALL.     

Frequent epigenetic inactivation of KIBRA,an upstream member of the Salvador/Warts/Hippo (SWH) tumor suppressor network,is associated with specific genetic event in B-cell acute lymphocytic leukemia

The WW-domain containing protein KIBRA has recently been identified as a new member of the Salvador/Warts/Hippo (SWH) pathway in Drosophila and is shown to act as a tumor suppressor gene in Drosophila. This pathway is conserved in humans and members of the pathway have been shown to act as tumor suppressor genes in mammalian systems. We determined the methylation status of the 5' CpG island associated with the KIBRA gene in human cancers. In a large panel of cancer cell lines representing common epithelial cancers KIBRA was unmethylated. But in pediatric acute lymphocytic leukemia (ALL) cell lines KIBRA showed frequent hypermethylation and silencing of gene expression, which could be reversed by treatment with 5-aza-2'-deoxycytidine. In ALL patient samples KIBRA was methylated in 70% B-ALL but was methylated in &lt; 20% T-ALL leukemia (p = 0.0019). In B-ALL KIBRA methylation was associated with ETV6/RUNX1 {t(12;21) (p13;q22)} chromosomal translocation (p = 0.0082) phenotype, suggesting that KIBRA may play an important role in t(12;21) leukemogenesis. In ALL paired samples at diagnosis and remission KIBRA methylation was seen in diagnostic but not in any of the remission samples accompanied by loss of KIBRA expression in disease state compared to patients in remission. Hence KIBRA methylation occurs frequently in B-cell acute lymphocytic leukemia but not in epithelial cancers and is linked to specific genetic event in B-ALL.