Fusion mRNA Research Articles

Detection of BCR-ABL transcripts in acute lymphoblastic leukemia in Indian patients.

Thirty-three patients with acute lymphoblastic leukemia (ALL) from India were studied for the presence of BCR-ABL chimeric transcripts, by a seminested cDNA-PCR. We report the presence of BCR-ABL chimeric transcripts in 4/17 (24%) children (under 15 years) and 3/16 (19%) adults (15-50 years). This is in sharp contrast to the published literature from the West where the presence of BCR-ABL has been reported in only 2-5% children and 35% adults. Whether the presence of BCR-ABL fusion mRNA, which is generally an attribute of ALL in adults and of poorer prognosis, may contribute to chemo-incurability in young Indian patients, remains to be seen, as a larger number of patients are studied for treatment outcome and survival on uniform therapy protocols.

Microtubule-associated protein 1 light chain 3 is a fibronectin mRNA-binding protein linked to mRNA translation in lamb vascular smooth muscle cells.

Intimal cushions form in the fetal ductus arteriosus by fibronectin-dependent smooth muscle cell migration which is associated with greater efficiency of fibronectin mRNA translation. We investigated whether the AU-rich element (ARE), UUAUUUAU, in the 3'-untranslated region (3'UTR) of fibronectin mRNA is involved in this mechanism by transfecting smooth muscle cells with plasmids containing the chloramphenicol acetyltransferase coding region with its 3'UTR replaced by fibronectin 3'UTR bearing intact or mutated ARE. More efficient translation of fusion mRNA with intact versus mutated ARE was observed. This effect was amplified in ductus (10.9-fold) compared with nonmigratory, lower fibronectin-producing aorta cells (6.5-fold). Ductus cells transfected with wild-type but not ARE-mutated plasmid reverted to the stellate phenotype of aorta cells associated with reduced fibronectin production. This suggested that plasmid ARE sequesters RNA-binding factors, thereby reducing endogenous fibronectin mRNA translation. We next purified a 15-kD fibronectin ARE-dependent RNA-binding protein and identified it as microtubule-associated protein 1 light chain 3 (LC3). LC3 is present in greater amounts in ductus compared with aorta cells, and overexpression of LC3 in aortic cells by transfection enhances fibronectin mRNA translation to levels observed in ductus cells.

Hyperdiploidy andE2A-PBX1 fusion in an adult with t(1;19)+ acute lymphoblastic leukemia: Case report and review of the literature

The t(1;19)(q23;p13), detected cytogenetically in 5-6% of cases, is one of the most common translocations in childhood acute lymphoblastic leukemia (ALL). Most t(1;19)+ ALLs are pseudodiploid or contain fewer than 50 chromosomes, are classified as pre-B based on expression of cytoplasmic, but not surface, immunoglobulin (clg+/slg-), express a characteristic pattern of cell surface antigens, and contain E2A-PBX1 fusion mRNAs. A minority of cases are early pre-B (clg-/slg-), do not express the characteristic pattern of cell surface antigens, and lack E2A-PBX1 fusion mRNAs. These latter cases are frequently hyperdiploid, with a modal chromosome number of 55-57. The incidence of the t(1;19) in adults with ALL (approximately 3%) appears to be similar to that observed in children, but the genetic and immunophenotypic features of adult t(1;19)+ ALL have not been described extensively. We report a case of t(1;19)+ ALL occurring in a 38-year-old man in the setting of hyperdiploidy > 50. Despite this feature, this case was pre-B, conformed to the classic t(1;19) immunophenotype, and expressed E2A-PBX1 fusion mRNAs. This prompted us to review the published literature on ploidy and genetic features of t(1;19)+ ALLs. Overall, E2A-PBX1 fusion occurred in 95% (102/107) of t(1;19)+ B-lineage ALLs with 50 or fewer chromosomes, 80% of which were pseudodiploid, vs. only 25% (2/8) of t(1;19)+ ALLs with more than 50 chromosomes.

The dominant developmental mutants of tomato, Mouse-ear and Curl, are associated with distinct modes of abnormal transcriptional regulation of a Knotted gene.

The Curl (Cu) and Mouse-ear (Me) mutations of tomato cause two seemingly unrelated developmental syndromes with a wide range of pleiotropic phenotypes. Yet, the distinct morphogenic alterations in shoots, leaves, and inflorescences conferred by the two mutations appear to be caused by unchecked meristematic activity that characterizes dominant mutations in Knotted1 (Kn1)-like genes of monocot plants. We have been unable to separate the two closely linked Cu and Me mutations, and they may lie in the same gene. A homeobox-containing class I Kn1-like gene, TKn2, also maps to the same location. Significantly, the dominant mutations are associated with two aberrant modes of TKn2 transcription. Overexpression of the two in-frame wild-type transcripts of TKn2 is associated with the Cu mutation, whereas misexpression of an abundant and oversized fusion mRNA is associated with the Me mutation. Available molecular evidence strongly suggests that the defective Me-TKn2 transcript is generated via a novel splicing event that merges transcripts of two closely linked genes. The translated fusion product is comprised of most of the 5' end of the adjacent PPi-dependent fructose 6-phosphate phosphotransferase (PFP) transcript spliced in-frame to coding position 64 of the TKn2 transcript, leaving the TKn2 homeobox intact. We suggest that class I Kn1-like genes were selected early during evolution to regulate basic programs of aerial meristems and that subtle alterations in their function may be the basis for the wide diversity in growth parameters of shoot systems, leaves, and inflorescences among plant species.

Fusion of TEL, the ETS-Variant Gene 6 (ETV6), to the Receptor-Associated Kinase JAK2 as a Result of t(9; 12) in a Lymphoid and t(9; 15; 12) in a Myeloid Leukemia

AbstractTranslocations in hematologic disease of myeloid or lymphoid origin with breakpoints at chromosome band 12p13 frequently result in rearrangements of the Ets variant gene 6 (ETV6). As a consequence either the ETS DNA-binding domain or the Helix-Loop-Helix (HLH) oligomerization domain of ETV6 is fused to different partner genes. We show here that a t(9; 12)(p24; p13) in a case of early pre-B acute lymphoid leukemia and a t(9; 15; 12)(p24; q15; p13) in atypical chronic myelogenous leukemia in transformation involve the ETV6 gene at 12p13 and the JAK2 gene at 9p24. In each case different fusion mRNAs were found, with only one resulting in an open reading frame for a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the protein tyrosine kinase (PTK) domain of JAK2. The cloning of the complete human JAK2 coding and genomic sequences and of the genomic junction fragments of the translocations allowed a characterization of the different splice events leading to the various mRNAs. JAK2 plays a central role in non–protein tyrosine kinase receptor signaling pathways, which could explain its involvement in malignancies of different hematologic lineages. Besides hop in Drosophila no member of the JAK family has yet been implicated in tumorigenesis.

Fusion of TEL, the ETS-Variant Gene 6 (ETV6), to the Receptor-Associated Kinase JAK2 as a Result of t(9; 12) in a Lymphoid and t(9; 15; 12) in a Myeloid Leukemia

Translocations in hematologic disease of myeloid or lymphoid origin with breakpoints at chromosome band 12p13 frequently result in rearrangements of the Ets variant gene 6 (ETV6). As a consequence either the ETS DNA-binding domain or the Helix-Loop-Helix (HLH) oligomerization domain of ETV6 is fused to different partner genes. We show here that a t(9; 12)(p24; p13) in a case of early pre-B acute lymphoid leukemia and a t(9; 15; 12)(p24; q15; p13) in atypical chronic myelogenous leukemia in transformation involve the ETV6 gene at 12p13 and the JAK2 gene at 9p24. In each case different fusion mRNAs were found, with only one resulting in an open reading frame for a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the protein tyrosine kinase (PTK) domain of JAK2. The cloning of the complete human JAK2 coding and genomic sequences and of the genomic junction fragments of the translocations allowed a characterization of the different splice events leading to the various mRNAs. JAK2 plays a central role in non–protein tyrosine kinase receptor signaling pathways, which could explain its involvement in malignancies of different hematologic lineages. Besides hop in Drosophila no member of the JAK family has yet been implicated in tumorigenesis.

Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein–Barr nuclear antigen-1 protein to neomycin resistance

Use of EBV-based vector systems has been limited by the requirement to generate EBNA+ cells which are 'permissive' for replication of an oriP-vector. In current constructs, selectable marker and EBNA-1 are not always co-expressed. This is a significant problem since the EBNA-1 gene product can be toxic in some cell types and may be selected against. In this paper, we describe a gene construct that overcomes this limitation. We have exploited the piconaviral internal ribosome entry site to allow the genes for Epstein–Barr nuclear antigen-1 and G-418 resistance to be transcribed as a dicistronic fusion mRNA under the control of the phosphoglucokinase promoter. This construct can be routinely integrated into human cell lines. The presence of EBNA-1 protein was reflected by a large increase in transfection frequencies (1000-fold) using an oriP-based vector which was shown to replicate stably in these cells with no apparent gross rearrangements detected after 8 weeks in culture. Using this system, G-418 resistance should directly reflect integration, as well as expression of the EBNA-1 gene, which, in turn, increases transfection frequencies and stability of EBV-based vector systems and should result in its increased use.

Incidence and Clinical Relevance of TEL/AML1 Fusion Genes in Children With Acute Lymphoblastic Leukemia Enrolled in the German and Italian Multicenter Therapy Trials

The molecular approach for the analysis of leukemia associated chromosomal translocations has led to the identification of prognostic relevant subgroups. In pediatric acute lymphoblastic leukemia (ALL), the most common translocations, t(9; 22) and t(4; 11), have been associated with a poorer clinical outcome. Recently the TEL gene at chromosome 12p13 and the AML1 gene at chromosome 21q22 were found to be involved in the translocation t(12; 21)(p13; q22). By conventional cytogenetics, however, this chromosomal abnormality is barely detectable and occurs in less than 0.05% of childhood ALL. To investigate the frequency of the molecular equivalent of the t(12; 21), the TEL/AML1 gene fusion, we have undertaken a prospective screening in the running German Berlin-Frankfurt-Münster (BFM) and Italian Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) multicenter ALL therapy trials. We have analyzed 334 unselected cases of pediatric ALL patients consecutively referred over a period of 5 and 9 months, respectively. The overall incidence of the t(12; 21) in pediatric ALL is 18.9%. The 63 cases positive for the TEL/AML1 chimeric products ranged in age between 1 and 12 years, and all but one showed CD10 and pre-B immunophenotype. Interestingly, one case displayed a pre-pre–B immunophenotype. Among the B-lineage subgroup, the t(12; 21) occurs in 22.0% of the cases. Fifteen of 61 (24.6%) cases coexpressed at least two myeloid antigens (CD13, CD33, or CDw65) in more than 20% of the gated blast cells. DNA index was available for 59 of the 63 TEL/AML1 positive cases; a hyperdiploid DNA content (≥1.16) was detected in only four patients, being nonhyperdiploid in the remaining 55. Based on this prospective analysis, we retrospectively evaluated the impact of TEL/AML1 in prognosis by identifying the subset of B-lineage ALL children enrolled in the closed German ALL-BFM-90 and Italian ALL-AIEOP-91 protocols who had sufficient material for analysis. A total of 342 children were investigated for the presence of TEL/AML1 fusion gene and 99 cases (28.9%) were positive. The patients expressing the TEL/AML1 fusion mRNA appeared to have a better event-free survival (EFS) than the patients who lacked this chimeric product. Whereas three of the TEL/AML1 positive cases (3.0%) have relapsed to date, 27 patients without TEL/AML1 rearrangement (11.1%) suffered from relapse. To date, the only subset of B-lineage ALL with a favorable prognosis has been the hyperdiploid group (DNA index ≥1.16 <1.6). Our findings reinforce the need to include the molecular screening of the t(12; 21) translocation within ongoing prospective ALL trials to prove definitively its prognostic impact.

Incidence and Clinical Relevance of TEL/AML1 Fusion Genes in Children With Acute Lymphoblastic Leukemia Enrolled in the German and Italian Multicenter Therapy Trials

AbstractThe molecular approach for the analysis of leukemia associated chromosomal translocations has led to the identification of prognostic relevant subgroups. In pediatric acute lymphoblastic leukemia (ALL), the most common translocations, t(9; 22) and t(4; 11), have been associated with a poorer clinical outcome. Recently the TEL gene at chromosome 12p13 and the AML1 gene at chromosome 21q22 were found to be involved in the translocation t(12; 21)(p13; q22). By conventional cytogenetics, however, this chromosomal abnormality is barely detectable and occurs in less than 0.05% of childhood ALL. To investigate the frequency of the molecular equivalent of the t(12; 21), the TEL/AML1 gene fusion, we have undertaken a prospective screening in the running German Berlin-Frankfurt-Münster (BFM) and Italian Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) multicenter ALL therapy trials. We have analyzed 334 unselected cases of pediatric ALL patients consecutively referred over a period of 5 and 9 months, respectively. The overall incidence of the t(12; 21) in pediatric ALL is 18.9%. The 63 cases positive for the TEL/AML1 chimeric products ranged in age between 1 and 12 years, and all but one showed CD10 and pre-B immunophenotype. Interestingly, one case displayed a pre-pre–B immunophenotype. Among the B-lineage subgroup, the t(12; 21) occurs in 22.0% of the cases. Fifteen of 61 (24.6%) cases coexpressed at least two myeloid antigens (CD13, CD33, or CDw65) in more than 20% of the gated blast cells. DNA index was available for 59 of the 63 TEL/AML1 positive cases; a hyperdiploid DNA content (≥1.16) was detected in only four patients, being nonhyperdiploid in the remaining 55. Based on this prospective analysis, we retrospectively evaluated the impact of TEL/AML1 in prognosis by identifying the subset of B-lineage ALL children enrolled in the closed German ALL-BFM-90 and Italian ALL-AIEOP-91 protocols who had sufficient material for analysis. A total of 342 children were investigated for the presence of TEL/AML1 fusion gene and 99 cases (28.9%) were positive. The patients expressing the TEL/AML1 fusion mRNA appeared to have a better event-free survival (EFS) than the patients who lacked this chimeric product. Whereas three of the TEL/AML1 positive cases (3.0%) have relapsed to date, 27 patients without TEL/AML1 rearrangement (11.1%) suffered from relapse. To date, the only subset of B-lineage ALL with a favorable prognosis has been the hyperdiploid group (DNA index ≥1.16 <1.6). Our findings reinforce the need to include the molecular screening of the t(12; 21) translocation within ongoing prospective ALL trials to prove definitively its prognostic impact.

The HRX Proto-oncogene Product Is Widely Expressed in Human Tissues and Localizes to Nuclear Structures

AbstractChromosomal rearrangement of the HRX (MLL, ALL-1, Htrx) gene situated at chromosome band 11q23 is one of the most frequent genetic changes in infant leukemias of myeloid and lymphoid lineage and in treatment-induced secondary leukemias. The HRX gene codes for a predicted 431-kD protein that shows significant homology to the Drosophila trithorax protein, an Hox epigenetic regulator. Typically, the region encoding the HRX gene is rearranged, mostly in reciprocal translocations with a number of partners, resulting in a range of fusion genes. However, this is not the only abnormality affecting HRX because partial duplication of the gene, as well as interstitial deletions, can occur. Despite extensive studies of HRX at the genetic level, the protein products of the HRX gene and their patterns of expression in normal and leukemic cells remain uncharacterized. In this study we analyzed the distribution and localization of HRX proteins in cell lines and human tissues, using both polyclonal and monoclonal antibodies. The specificity of these reagents was confirmed using cells transfected with the HRX-ENL fusion gene. Western blot analyses of protein extracts from cells carrying the t(11; 19) and t(4; 11) translocations showed HRX chimeric proteins whose migrations corresponded to the sizes predicted from analyses of translocation-induced fusion mRNAs expressed by the derivative 11 chromosomes. Immunocytochemical analysis showed a punctate distribution of wild-type and chimeric HRX proteins within cell nuclei, suggesting that HRX localizes to nuclear structures in cells with and without 11q23 translocations. Nuclear staining was found in the majority of tissues studied with the strongest reactivity in cerebral cortex, kidney, thyroid, and lymphoid tissues. Thus, HRX is widely expressed in most cell types including hematopoietic cells, a finding that precludes an immunocytochemical approach for diagnosis of leukemias bearing 11q23 structural abnormalities.

Monoclonal Antibodies Specific to the Acute Lymphoblastic Leukemia t(1; 19)-Associated E2A/pbx1 Chimeric Protein: Characterization and Diagnostic Utility

AbstractNonrandom chromosomal abnormalities are found in most human malignancies, particularly leukemias and lymphomas. A characteristic t(1; 19) (q23; p13.3) chromosomal translocation is detected in 5% of childhood acute lymphoblastic leukemia (ALL) cases. This translocation results in the formation of a fusion gene, which leads to the expression of an oncogenic E2A/pbx1 protein. Breakpoints in the E2A gene almost invariably occur within a single intron, and the identical portion of PBX1 is joined consistently to exon 13 of E2A in fusion mRNA. In this article, we report the development of monoclonal antibodies against E2A/pbx1 fusion protein using a specific peptide that corresponds to the junction region of the protein. The obtained antibodies recognize specifically the chimeric E2A/pbx1 fusion protein and lack cross-reactivities with E2A and pbx1. Immunohistochemical staining and flow cytometric studies show that these antibodies can distinguish t(1; 19)-positive from t(1; 19)-negative leukemic cells. These results indicate that the obtained E2A/pbx1-specific monoclonal antibodies might prove to be valuable diagnostic reagents and important tools for elucidating the mechanisms involved in oncogenesis and progression of t(1; 19)-positive childhood ALL.

A t(6;12)(q23;p13) results in the fusion of ETV6 to a novel gene, STL, in a B-cell ALL cell line.

ETV6 (TEL) is rearranged in various types of hematologic malignancies. The B-cell precursor acute lymphoblastic leukemia (ALL) cell line SUP-B2 has a t(6;12)(q23;p13) involving ETV6 at 12p13 and a submicroscopic deletion of the other ETV6 allele. The reciprocal translocation results in the fusion of ETV6 to a previously unknown gene at 6q23, which we named STL (six-twelve leukemia gene). Both reciprocal fusion transcripts can be detected: On the der(6) chromosome, the ETV6/STL mRNA shows an apparently out of frame fusion of ETV6 at nucleotide 187 to STL, which would result in the addition of 14 amino acids to the first 54 amino acids of ETV6. On the der(12) chromosome three different variants of the STL/ETV6 fusion mRNA could be detected; variable size segments were inserted at the breakpoint between STL and ETV6 exon 3. One of these variants could give rise to a protein in which the first 54 amino acids of ETV6 are replaced by 12 amino acids from one of the STL short open reading frames. Sequence analysis of a 1.4 kb STL cDNA clone from a skeletal muscle library revealed no long open reading frames. This cell line will be very useful in studying the different mechanisms by which alterations of ETV6 contribute to leukemogenesis and in testing the hypothesis that ETV6 might act as a tumor suppressor gene.

A t(6;12)(q23;p13) results in the fusion ofETV6 to a novel gene,STL, in a B-cell ALL cell line

ETV6 (TEL) is rearranged in various types of hematologic malignancies. The B-cell precursor acute lymphoblastic leukemia (ALL) cell line SUP-B2 has a t(6;12)(q23;p13) involving ETV6 at 12p13 and a submicroscopic deletion of the other ETV6 allele. The reciprocal translocation results in the fusion of ETV6 to a previously unknown gene at 6q23, which we named STL (six-twelve leukemia gene). Both reciprocal fusion transcripts can be detected: On the der(6) chromosome, the ETV6/STL mRNA shows an apparently out of frame fusion of ETV6 at nucleotide 187 to STL, which would result in the addition of 14 amino acids to the first 54 amino acids of ETV6. On the der(12) chromosome three different variants of the STL/ETV6 fusion mRNA could be detected; variable size segments were inserted at the breakpoint between STL and ETV6 exon 3. One of these variants could give rise to a protein in which the first 54 amino acids of ETV6 are replaced by 12 amino acids from one of the STL short open reading frames. Sequence analysis of a 1.4 kb STL cDNA clone from a skeletal muscle library revealed no long open reading frames. This cell line will be very useful in studying the different mechanisms by which alterations of ETV6 contribute to leukemogenesis and in testing the hypothesis that ETV6 might act as a tumor suppressor gene.

Detection of bcr-abl Transcripts in Philadelphia Chromosome–Positive Acute Lymphoblastic Leukemia After Marrow Transplantation

AbstractThirty-six patients with Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL) were studied for the presence of the bcr-abl fusion mRNA transcript after an allogeneic matched related (N = 12), partially matched related (N = 4), matched unrelated (N = 14), autologous (N = 5), or syngeneic (N = 1) bone marrow transplant (BMT). Seventeen were transplanted in relapse, and 19 were transplanted in remission. Twenty-three patients had at least one positive bcr-abl polymerase chain reaction (PCR) assay after BMT either before a relapse or without subsequent relapse. Ten of these 23 relapsed after a positive assay at a median time from first positive PCR assay of 94 days (range, 28 to 416 days). By comparison, only 2 relapses occurred in the 13 patients with no prior positive PCR assays; both patients had missed at least one scheduled follow-up assay and were not tested 2 months and 26 months before their relapse. The unadjusted relative risk (RR) of relapse associated with a positive PCR assay compared with a negative assay was 5.7 (95% confidence interval 1.2 to 26.0, P = .025). In addition, the data suggest that the type of bcr-abl chimeric mRNA detected posttransplant was associated with the risk of relapse: 7 of 10 patients expressing the p190 bcr-abl relapsed, compared with 1 of 8 who expressed only the p210 bcr-abl mRNA (P = .02, log-rank test). The RR of p190 bcr-abl positivity compared to PCR-negative patients was 11.2 (confidence interval 2.3-54.8, P = 0.003), whereas a positive test for p210 bcr-abl was apparently not associated with an increased relative risk. In separate multivariable models, PCR positivity remained a statistically significant risk factor for relapse after separately adjusting for donor (unrelated and partially matched v matched, autologous, and syngeneic), remission status at the time of transplant, the presence of acute graft-versus-host disease (GVHD), and type of conditioning regimen (total body irradiation dose of ≤1,200 cGy v <1,200 cGy). The PCR assay appears to be a useful test for predicting patients at high risk of relapse after BMT and may identify patients who might benefit from therapeutic interventions. The finding that the expression of p190 bcr-abl may portend an especially high risk of relapse suggests a different clinical and biologic behavior between p190 and p210 bcr-abl.

Introns act post-transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1.

The expression of the Arabidopsis thaliana PAT1 gene, which encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase, was investigated using translational fusions of the PAT1 promoter to the GUS reporter gene. Independent stably transformed A. thaliana lines containing a single copy of a fusion that includes the entire plastid transit peptide and the first two introns of PAT1 had on average 30 times more GUS enzyme activity than plants transformed with a construct in which GUS was fused a short distance downstream of the PAT1 start codon. Plants containing the construct without introns or leader peptide accumulated undetectable amounts of PAT1-GUS fusion protein and mRNA, even though the transcriptional rate of both fusion constructs was comparable. Fusions containing the entire transit peptide and either of the first two introns yield as much GUS activity as constructs containing both introns, but constructs containing the transit peptide but no introns give rise to much lower levels. Therefore, introns greatly enhance the expression of PAT1-GUS fusions, and they act post-transcriptionally to increase the steady-state level of mRNA.

Rapid sequence analysis of gene trap integrations to generate a resource of insertional mutations in mice.

Gene trapping in murine embryonic stem cells is a proven method for the simultaneous identification and mutation of genes in the mouse. Gene trap vectors are designed to detect insertions within genes through the production of a fusion mRNA transcript, making the identification of the endogenous gene possible by 5' rapid amplification of cDNA ends (RACE). Although the amplification of specific cDNAs can be achieved rapidly, cloning and screening of informative-sized cDNAs has proven to be time consuming. To eliminate the need for cloning, we have developed a method for solid-phase sequencing of 5' RACE products. More than 150 independent gene trap cell lines were analyzed, and sequence information was obtained for every line successfully amplified by RACE. With the vector used in this study, 40% of the cell lines were found to contain properly spliced gene trap events. The remaining lines were either spliced inefficiently or contained deletions of the vector. These results highlight the advantage of sequencing gene trap integrations before further characterization. This work now paves the way for large-scale gene trap screens in mice and should greatly accelerate the functional analysis of the mammalian genome.

Detection of circulating tumor cells in patients with Ewing's sarcoma and peripheral primitive neuroectodermal tumor.

To determine the feasibility of detecting Ewing's sarcoma (ES) or peripheral primitive neuroectodermal tumor (PNET) through a reverse-transcriptase polymerase chain reaction (RT-PCR) of the t(11;22)(q24;q12) fusion transcript in blood and bone marrow samples from patients with these neoplasms. Peripheral-blood (PB) and/or bone marrow aspirate (BM) samples were obtained from 28 patients with ES or PNET at initial presentation or at relapse. Patients were divided into two groups: newly diagnosed patients with nonmetastatic disease and those with metastatic/relapsed disease. RNA was extracted from fractionated BM and PB samples, and RT-PCR was performed for the EWS/HumFLI1 fusion mRNA was transcribed across the t(11;22) breakpoint. Among the 16 patients with nonmetastatic disease, three of 16 were RT-PCR positive for EWS/HumFLI1 RNA in BM and three of 10 were positive in PB. The total number of nonmetastatic patients who were positive in either PB or BM was four of 16 (25%). Among patients with metastatic/relapsed disease, two of six were positive in BM and five of 10 were positive in PB. The total fraction of patients with metastatic/relapsed disease that was positive in either BM or PB was six of 12 (50%). In this study, we show that it is possible to amplify the EWS/HumFLI1 RNA by RT-PCR from the BM and PB of a subset of patients with both nonmetastatic and metastatic ES or PNET, which implies that occult tumor cells are present at these sites. The true biologic and clinical meaning of this information is unknown. However, it does suggest a possible application of RT-PCR for the monitoring of residual disease in patients who are undergoing therapy for ES or PNET. This approach may permit early identification of patients who may benefit from alternative therapy or who may be spared possible overtreatment.

Molecular Mechanism of Polyamine Stimulation of the Synthesis of Oligopeptide-binding Protein

Polyamine stimulation of the synthesis of oligopeptide-binding protein (OppA) was shown to occur mainly at the level of translation by measuring OppA synthesis and its mRNA level. Several artificial oppA genes were constructed by site-directed mutagenesis. These synthesize different kinds of OppA mRNAs: mRNAs differing in the size of 5'-untranslated region; mRNAs having the Shine-Dalgarno (SD) sequence in a different position; mRNAs having different secondary structure in the region of the SD sequence; and fusion mRNAs consisting of the 5'-untranslated region of OppA mRNA and the open reading frame of beta-galactosidase. By measuring the synthesis of OppA or beta-galactosidase from these mRNAs, we found that the 171-nucleotide 5'-untranslated region and 145 nucleotides of the ORF of OppA mRNA are involved in the polyamine stimulation of OppA synthesis. When the secondary structure of the above region of OppA mRNA was analyzed by optimal computer folding, it was shown that the degree of polyamine stimulation of OppA protein synthesis was dependent on the structure of the SD sequence in addition to its position. Loose base pairing of the SD sequence with other regions of the mRNA caused strong polyamine stimulation, while intense base pairing of the SD sequence with other regions of the mRNA resulted in insignificant or weak polyamine stimulation.

NPM/ALK fusion mRNA expression in Hodgkin and Reed-Sternberg cells is rare but does occur: Results from single-cell cDNA analysis

The translocation t(2;5)(p23;q35) leads to the fusion of the nucleophosmin gene (NPM) on chromosome 5q35 to the recently described receptor kinase ALK on 2p23. It is characteristic of a subgroup of CD30+ large-cell anaplastic non-Hodgkin's lymphoma (ALCL). Since some cases of Hodgkin's disease (HD) and ALCL share common features, a common pathogenesis has been proposed in a report of the expression of NPM/ALK fusion mRNA in 11/13 Hodgkin's lymphomas. We approached this question by micro-manipulatory isolation of single Hodgkin and Reed-Sternberg (H-RS) cells and subsequent RT-PCR amplification of NPM/ALK fusion cDNA from these single cells. Specificity of cell selection was shown by the HD-specific pattern of EBV-gene expression in single H-RS cells. In 4 out of 7 cases, NPM/ALK fusion cDNA was detected in the RNA from whole lymph node tissue. In 2 out of 9 cases, NPM/ALK fusion sequences were amplified from single H-RS cells, albeit in a very low frequency (< 5%). These data indicate that NPM/ALK fusion transcripts do not play an early role in the pathogenesis of HD. Whether the rare expression of NPM/ALK is the result of clonal heterogeneity or an indication for clonal evolution and progression toward ALCL can only be answered by the repeated analysis of indicator cases during the course of the disease.

Reversal of the relative expression of cardiac and skeletal alpha1 subunit isoforms of L-type calcium channel during in vitro myogenesis.

Cardiac and skeletal type of excitation-contraction coupling (ECC) are quite different. Those differences could be explained by structural ones in the molecular entities involved in ECC, ie dihydropyridines (DHP) receptors (alpha1 subunit of L-type calcium channels) of the sarcolemma or ryanodine receptors of the sarcoplasmic reticulum membrane. As previously demonstrated by means of electrophysiology, the two types of ECC coexist during the first stages of in vitro development of skeletal muscle, whereas the skeletal type predominates at the later ones. In order to see whether evolution of ECC could be correlated with the one of alpha1 subunit expression, we determined by Northern Blotting which isoforms of alpha1 subunit are expressed during the in vitro myogenesis. mRNA corresponding to the cardiac isoform are present in myoblasts (before fusion), but patch-clamp experiments showed that they are not functional. After fusion, skeletal and cardiac mRNA are coexpressed in myotubes, with different intensities: whereas expression of skeletal mRNA (which are the more intensive) stabilized at the later stages tested, cardiac mRNA decreased. We conclude that evolution in mRNA alpha1 subunit isoforms expression could partly explained evolution of ECC features during in vitro myogenesis.