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A number of structural alterations have been shown to activate the leukemogenic potential of the ABL oncogene, but there is little understanding of the regulatory mechanisms that are subverted by such changes. We have used directed... more
A number of structural alterations have been shown to activate the leukemogenic potential of the ABL oncogene, but there is little understanding of the regulatory mechanisms that are subverted by such changes. We have used directed mutagenesis to examine a potential regulatory motif in cABL, which could directly influence ABL tyrosine kinase activity. A tyrosine to phenylalanine substitution within the ATP binding fold of the ABL kinase domain is sufficient to activate cABL enzymatic activity, and the mutant protein will alleviate growth factor dependence when expressed in the BA/F3 cell line. This growth promotion is dependent upon the structure of the amino terminus of the protein, and the ABL mutation will cooperate with certain BCR sequences in BCR/ABL fusion proteins to deregulate ABL kinase activity.
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ABSTRACT We have used a methotrexate (MTX)-resistant mouse 3T6 cell line (M50L3), that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of 300, to study the mechanism for turning off DHFR gene expression following... more
ABSTRACT We have used a methotrexate (MTX)-resistant mouse 3T6 cell line (M50L3), that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of 300, to study the mechanism for turning off DHFR gene expression following withdrawal of serum factors or elevation of the intracellular level of cAMP. When resting (G0) M50L3 cells are serum-stimulated to reenter the cell cycle, the level of DHFR activity begins to increase at about the same time the cells begin synthesizing DNA. The increase in enzyme activity is preceded by increases in the synthesis rate of the enzyme, and the content and production rate of DHFR mRNA. These increases, as well as entry into S phase, are blocked when the cells are serum-stimulated in the presence of dibutyryl cyclic AMP (db-cAMP) and theophylline. In this study, we found that when these drugs were added, or the serum stimulus was withdrawn during S phase (20 h following stimulation), the subsequent increase in DHFR level was blocked. Immunoprecipitation of DHFR from pulse-labelled cells showed that both treatments led to a rapid decrease in synthesis rate of the enzyme. The effect on total protein synthesis was much less than on DHFR synthesis. In DNA-excess filter hybridization experiments, we found that the content of cytoplasmic DHFR mRNA decreased in parallel with the synthesis rate of the enzyme. This was due in part to a decrease in the production rate of DHFR mRNA relative to total mRNA. In addition, drug addition or serum withdrawal led to a significant destabilization of DHFR (as well as total) mRNA. About 85% of poly(A)(+) DHFR mRNA was associated with polysomes in resting, growing, or cAMP-treated cells, suggesting that DHFR gene expression was not controlled at the translational level under these conditions.
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... incidence of the Ph chromosome in paediatric (6%) (Chessells et a/. 1979: Priest et al, 1980; Kibeiro et al, 1987) and adult (25%) (Le Beau & Rowley ... adult common ALL, up to 5 5% of cases may be Ph-positive while only 6% of... more
... incidence of the Ph chromosome in paediatric (6%) (Chessells et a/. 1979: Priest et al, 1980; Kibeiro et al, 1987) and adult (25%) (Le Beau & Rowley ... adult common ALL, up to 5 5% of cases may be Ph-positive while only 6% of childhood ALL cases were Ph-positive (Maurer et a ...
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Tumor-specific alterations in oncogenes are thought to play a central role in the development of cancer. An example is the consistent fusion of the bcr gene to the c-abl oncogene on the Ph chromosome in CML. The Ph chromosome can also be... more
Tumor-specific alterations in oncogenes are thought to play a central role in the development of cancer. An example is the consistent fusion of the bcr gene to the c-abl oncogene on the Ph chromosome in CML. The Ph chromosome can also be observed in ALL. About 50% of Ph+ ALL cases, in contrast to CML, do not exhibit chromosomal breakpoints in the major cluster region or mcr (Ph+ mcr- ALL). These cases may have a novel bcr-abl fusion gene instead. We tested this hypothesis in eight Ph+ mcr- ALL patients by amplifying the putative hybrid part of the bcr-abl cDNA, using the polymerase chain reaction method. All cases examined showed the same joining of the first exon of the bcr gene to the c-abl oncogene. Thus, the novel bcr-abl fusion in Ph+ mcr- ALL is the result of a molecularly distinct Ph chromosome. This allows the definition of Ph+ leukemias by their respective bcr-abl oncogene activation. Moreover, the cDNA amplification method we use is a clinically useful tool to screen for bcr-abl oncogene activations in leukemia patients.
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Approximately 5% of children and 10-20% of adults with acute lymphoblastic leukaemia (ALL) have a chromosome translocation t(9;22) which at the cytogenetic level appears identical to that in chronic myeloid leukaemia (CML). The t(9;22)... more
Approximately 5% of children and 10-20% of adults with acute lymphoblastic leukaemia (ALL) have a chromosome translocation t(9;22) which at the cytogenetic level appears identical to that in chronic myeloid leukaemia (CML). The t(9;22) translocation was first recognised in CML patients by its 22q- or Philadelphia (Ph) chromosome. While all Ph positive CML patients so far described have a chromosome 22 breakpoint within the breakpoint cluster region (bcr) located in the 3' part of the phl gene, only some Ph positive ALL patients have breakpoints in bcr. We have cloned the breakpoint of the 9q+ chromosome from the DNA of a Ph positive ALL patient in whom there is no breakpoint in the bcr. The non-chromosome 9 sequences of the breakpoint region are shown to be derived from chromosome 22. The breakpoint in chromosome 22 is shown to be the first intron of the phl gene about 66kb upstream of the bcr. Using probes from this intron, rearrangements were detected in the DNA of two out of ...
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Several groups have recently described methods for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements in B-cell malignancies by polymerase chain reaction (PCR) gene amplification using variable region-(VH) and... more
Several groups have recently described methods for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements in B-cell malignancies by polymerase chain reaction (PCR) gene amplification using variable region-(VH) and joining (JH) region-specific primers. The simplest methods utilize a single VH primer specific for sequences present in most VH regions corresponding to the third framework region (FR3). An alternative approach is to use a panel of VH family-specific primers specific for the first framework regions (FR1). In the course of nucleotide sequence analysis of IgH gene rearrangements amplified using a VH FR1 primer panel, these authors previously observed 3' VH region deletion and/or base mis-matches sufficient to prevent efficient priming from the VH FR3 primer target sequence in a significant minority of cases of B-lineage malignancy. An improved PCR method has therefore been developed by using a panel of seven VH FR1 family-specific primers incorporat...
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Activating ras mutations are frequent (25-60%) in chronic myelomonocytic leukemia (CMML) and in acute myeloid leukemia (AML) (30%), in contrast to chronic myeloid leukemia (CML) in which the incidence is very low (0-3%). This might... more
Activating ras mutations are frequent (25-60%) in chronic myelomonocytic leukemia (CMML) and in acute myeloid leukemia (AML) (30%), in contrast to chronic myeloid leukemia (CML) in which the incidence is very low (0-3%). This might reflect that the leukemic cell in CML is at a level of differentiation in which ras gene activation is not involved or, alternatively, might be due to the presence in CML of the bcrlabl fused gene. We have analyzed the presence of point mutations in codons 12, 13, 59, 61 and 63 of N-, K-, and H-ras genes, in 26 cases of Philadelphia-chromosome-positive, bcrlabl-positive acute leukemia (Ph+ AL), and in eight CMML cases by using the polymerase chain reaction. Aberrant ras genes were detected in a single Ph+ AL case, and in four out of eight CMML patients. The Ph+ AL showing altered ras allele had an unusual point mutation in H-ras gene, substituting leucine for glutamine. This mutation has not been previously found in any hematological disease. Our findings...
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The oviposition behaviour of Gryon gallardoi (Hymenoptera; Scelionidae) on Spartocera dentiventris (Hemiptera; Coreidae) host eggs was investigated in the laboratory. Masses of 12 non-parasitized freshly laid (less than 24 h old) eggs... more
The oviposition behaviour of Gryon gallardoi (Hymenoptera; Scelionidae) on Spartocera dentiventris (Hemiptera; Coreidae) host eggs was investigated in the laboratory. Masses of 12 non-parasitized freshly laid (less than 24 h old) eggs were exposed to 2-5 days old mated females with previous oviposition experience (n = 10). Behaviour was observed for 2 h under the stereomicroscope. The eggs were Then kept individually at 25 degrees +/- 1 degree C/12 h photophase till hatching. The mean number of parasitized eggs was 7.8 +/- 0.81 (mean +/- SE). Five distinct kinds of behaviour were observed: drumming with antennae on the eggs, ovipositor insertion, egg marking, walking and resting. On average, ovipositor insertion was not followed by marking 4.3 +/- 0.76 times per female. In nearly all of these events, parasitism was unsuccessful. Walking and resting were observed less frequently than the other behaviours (1.6 +/- 0.56 and 2.1 +/- 0.48 times/female, respectively). Superparasitism occu...
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Fifty-six patients with de novo acute myeloid leukemia M4/M5 subtypes were studied for rearrangements of the mixed lineage leukemia gene, MLL (also called HRX, Htrx-1, or ALL-1). Ten patients (18%) showed rearrangements of the MLL gene, 9... more
Fifty-six patients with de novo acute myeloid leukemia M4/M5 subtypes were studied for rearrangements of the mixed lineage leukemia gene, MLL (also called HRX, Htrx-1, or ALL-1). Ten patients (18%) showed rearrangements of the MLL gene, 9 in a major breakpoint cluster region within a centromeric 8.3-kb BamHI fragment, whereas rearrangement in one patient was the result of a direct tandem duplication of exons 2-6 of MLL. Analysis of sequences at the duplication junction revealed that the points of MLL fusion within introns 6 and 1 both lie within Alu elements. This suggests the involvement of Alu repeat mediated homologous recombination in MLL self fusion. For the 10 rearranged samples, cytogenetics analysis revealed a normal karyotype in 3, and 3 had abnormalities other than 11q23. Survival analysis of patients revealed no difference between those with rearrangement of MLL and those showing the germ-line configuration.
Research Interests: Cancer, Humans, Child, Female, Male, and 7 moreInfant, Aged, Middle Aged, Adult, Prognosis, Base Sequence, and Molecular Sequence Data
Using a temperature-sensitive mutant of the p210 BCR-ABL gene, transfected into a growth factor-dependent cell line (BaF3), we show that transient BCR-ABL kinase expression increases single cell and clonogenic resistance to apoptosis... more
Using a temperature-sensitive mutant of the p210 BCR-ABL gene, transfected into a growth factor-dependent cell line (BaF3), we show that transient BCR-ABL kinase expression increases single cell and clonogenic resistance to apoptosis arising from genotoxic damage induced by ionizing radiation and VP-16/etoposide. This effect is achieved in the absence of any detectable changes in the levels of BCL-2, BAX or BCL-x proteins and is independent of proliferative, MAP kinase-dependent effects of BCR-ABL kinase. In contrast to parental cells that transiently arrest in G2 and then apoptose, p210 BaF3 cells show a pronounced and sustained G2 arrest following radiation coupled with enhanced phosphorylation of cdc2. A cell cycle block in early M phase induced by the mitotic spindle poison, nocodazole, does not provide protection from apoptosis. Reversal of G2 arrest by caffeine abolishes the protective effect of BCR-ABL kinase. These data provide further insight into the transforming propertie...
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Breakpoints on chromosome 11 at band q23 have been observed in patients with primary or secondary leukaemia. Recent data have shown that these breakpoints are clustered in a approximately 15kb region of a gene named HRX. This gene product... more
Breakpoints on chromosome 11 at band q23 have been observed in patients with primary or secondary leukaemia. Recent data have shown that these breakpoints are clustered in a approximately 15kb region of a gene named HRX. This gene product has homology to the Drosophila trithorax gene product, which suggests it may play a role in regulating transcription control. Disruption of HRX as a result of chromosomal translocation is thought to contribute to the leukaemogenic process; this may occur in utero giving rise to infant acute leukaemia or may be induced by epipodophyllotoxic drugs resulting in secondary leukaemia. Translocations of 11q23 can involve a number of different partner chromosomes. The reciprocal genes on chromosomes 4q21, 9p22 and 19p13 have been recently cloned and are predicted to encode proline and serine rich proteins. Of particular interest is the high degree of homology observed between the genes on 9p22 and 19p13, which suggests that they too may have an important r...
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The Philadelphia translocation commonly observed in chronic myeloid leukaemia (CML) and a proportion of cases of acute leukaemia results in the creation of a chimeric fusion protein, BCR-ABL. The fusion protein exhibits an elevated... more
The Philadelphia translocation commonly observed in chronic myeloid leukaemia (CML) and a proportion of cases of acute leukaemia results in the creation of a chimeric fusion protein, BCR-ABL. The fusion protein exhibits an elevated tyrosine kinase activity as compared to normal ABL. Using a temperature sensitive mutant of p210 BCR-ABL (ts-p210) we find that the primary effect of BCR-ABL expression in an IL-3 dependent cell line is to prolong survival following growth factor withdrawal; only a small proportion of cells remain viable and rapidly evolve to complete growth factor independence. During passage in the presence of IL-3 at the temperature permissive for kinase activity, ts-p210 expressing cultures become dominated by completely growth factor independent cells within 10-30 days. There is also a significant difference between BCR-ABL and IL-3 mediated signalling with respect to the MAP kinase pathway; in contrast to IL-3 stimulation or v-ABL expression, BCR-ABL does not signal...
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Five cloned genes encoding the mouse ribosomal protein L30 were isolated from a recombinant DNA library and characterized by restriction mapping and nucleotide sequence analysis. Only one of these genes has introns and is expressed; the... more
Five cloned genes encoding the mouse ribosomal protein L30 were isolated from a recombinant DNA library and characterized by restriction mapping and nucleotide sequence analysis. Only one of these genes has introns and is expressed; the others are inactive processed pseudogenes. The expressed gene consists of five exons and four introns spanning 2,723 nucleotides. Transcripts of this gene are processed into the mature L30 mRNA by pathways that exhibit both constraints and flexibility with regard to the order of intron excision. The L30 mRNA which is 457 to 468 nucleotides in length excluding the polyadenylic acid tail, exhibits some microheterogeneity at its 3' end and encodes a basic protein of 115 amino acids. The 5' portion of the rpL30 gene has some novel features which are remarkably similar to the previously characterized mouse rpL32 gene. These include homologous sequences in the -60 to -340 region, the absence of a good TATA consensus sequence, and the presence of a ...
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The chromosome 22 derivative, the Philadelphia (Ph) chromosome, results from a reciprocal translocation t(9;22) (q34;q11) and is associated with chronic myeloid leukemia (CML). The translocation can be identified at the DNA level in... more
The chromosome 22 derivative, the Philadelphia (Ph) chromosome, results from a reciprocal translocation t(9;22) (q34;q11) and is associated with chronic myeloid leukemia (CML). The translocation can be identified at the DNA level in Ph-positive CML by using a probe to the breakpoint cluster region (bcr). In addition, as a result of this translocation an abl-related 210-kd protein with protein tyrosine kinase (PTK) activity is produced. We analyzed 28 cases of Ph-negative CML for rearrangement of the chromosome 22 sequences and found that eight of the 28 show rearrangement of the bcr. When 12 of the Ph-negative cases were independently reviewed, five were indistinguishable from Ph-positive CML on the basis of morphology, peripheral blood film and clinical details. These five also showed bcr rearrangement. The other seven were reclassified as six atypical CML (aCML) and one chronic myelomonocytic leukemia (CMML). None of these seven showed bcr rearrangement. In addition 11 cases of bc...
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Differential screening of a recombinant cDNA library using cDNAs transcribed from poly(A)+ RNA of normal or leukemic leukocytes revealed a number of recombinants homologous to mRNAs characteristic of particular leukemias. The occurrence... more
Differential screening of a recombinant cDNA library using cDNAs transcribed from poly(A)+ RNA of normal or leukemic leukocytes revealed a number of recombinants homologous to mRNAs characteristic of particular leukemias. The occurrence of one of these (pCG14) in high abundance was shown to be sufficiently characteristic of the circulating leukocyte population of chronic granulocytic leukemia (CGL) patients to distinguish them from all other populations of leukocytes. We have now characterized the gene encoding this mRNA and shown that its expression is specific to the granulocyte lineage in hemopoietic cells and is, moreover, limited to a narrow stage of differentiation during granulopoiesis. Our results explain why high levels of pCG14 RNA are characteristic of chronic granulocytic leukemia peripheral blood leukocytes.
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Six nonproductive kappa immunoglobulin genes (kappa- alleles) were cloned and sequenced. The structural abnormalities discerned from sequence analysis were correlated with functional lesions at the level of transcription, RNA processing,... more
Six nonproductive kappa immunoglobulin genes (kappa- alleles) were cloned and sequenced. The structural abnormalities discerned from sequence analysis were correlated with functional lesions at the level of transcription, RNA processing, turnover, and translation. Four kappa- alleles, three containing V kappa genes and one not, are transcribed at normal or even greater than normal rates, the defects in these genes being expressed at various posttranscriptional levels. The other two kappa- alleles, both of which lacked V genes, exhibited greatly depressed yet clearly detectable transcriptional activity. These results are consistent with a hierarchical relationship between enhancer and promoter elements in which the enhancer establishes transcriptional competence at the kappa locus and the promoter (or pseudopromoter) determines the relative level of transcriptional activity. One of the structural abnormalities discovered in this study, a large deletion which removes the entire J kapp...
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The MLL gene, the closest human homologue to the Drosophila trithorax gene, undergoes chromosomal translocation with a large number of different partner genes in both acute lymphoid and acute myeloid leukemias. We have identified a new... more
The MLL gene, the closest human homologue to the Drosophila trithorax gene, undergoes chromosomal translocation with a large number of different partner genes in both acute lymphoid and acute myeloid leukemias. We have identified a new partner gene, EEN, fused to MLL in a case of acute myeloid leukemia. The gene is located on chromosome 19p13, where two other MLL partner genes, ENL and ELL/MEN have also been identified. The deduced protein of 368 aa contains a central alpha-helical region and a C-terminal Src homology 3 (SH3) domain most similar to the C-terminal SH3 domain found in the Grb2/Sem-5/Drk family of genes. Sequence analysis of the fusion MLL/EEN transcript in our patient reveals that exon 6 of MLL is fused to the N-terminal end of EEN, a fusion that would create a chimeric protein that includes the major functional domain of EEN. EEN is expressed in a variety of tissue types and encodes a protein of approximately 46 kDa. The EEN protein is the human homologue of a member of a recently described murine SH3 domain-containing protein family. It is also highly related to a putative gene identified in Caenorhabditis elegans, and a number of similar sequences are present in the EST databases of several species.
Research Interests: Caenorhabditis elegans, Transcription Factors, Multidisciplinary, Sequence Analysis, Drosophila, and 16 moreAcute Myeloid Leukemia, Humans, Female, Animals, C2H2 zinc fingers, Infant, Karyotyping, Proteins, Molecular cloning, Protein Secondary Structure Prediction, S100 protein family, Amino Acid Sequence, Base Sequence, DNA binding proteins, Protein Biosynthesis, and Molecular Sequence Data
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Research Interests: Molecular Evolution, Transcription Factors, DNA, Protein Structure and Function, Drosophila, and 14 moreSequence alignment, Animals, Oncogene, Comparative Analysis, Genome, Genes, Clinical Sciences, Evolutionary Constraint, Genome sequence, Amino Acid Sequence, Fusion Protein, DNA binding proteins, Domain Structure, and Molecular Sequence Data
Inappropriate activation of Abl family kinases plays a crucial role in different human leukaemias. In addition to the well known oncoproteins p190Bcr-Abl and p210Bcr-Abl, Tel-Abl, a novel fusion protein resulting from a different... more
Inappropriate activation of Abl family kinases plays a crucial role in different human leukaemias. In addition to the well known oncoproteins p190Bcr-Abl and p210Bcr-Abl, Tel-Abl, a novel fusion protein resulting from a different chromosomal translocation, has recently been described. In this study, the kinase specificities of the Bcr-Abl and Tel-Abl proteins were compared to the physiological Abl family kinases c-Abl and Arg (abl related gene). Using short peptides which correspond to the target epitopes in known substrate proteins of Abl family kinases, we found a higher catalytic promiscuity of Bcr-Abl and Tel-Abl. Similar to Bcr-Abl, Tel-Abl was found in complexes with the adapter protein CRKL. In addition, c-Crk II and CRKL are tyrosine phosphorylated and complexed with numerous other tyrosine phosphorylated proteins in Tel-Abl expressing Ba/F3 cells. GTPase analysis with a Ras-GTP-specific precipitation assay showed constitutive elevation of GTP-loaded Ras in cells expressing the leukaemic Abl proteins. The mitogenic MAPK/Erk kinases as well as Akt/PKB, a kinase implicated to negatively regulate apoptosis, were also constitutively activated by both Bcr-Abl and Tel-Abl. The results indicate that the leukaemic Abl-fusion proteins have catalytic specificities different from the normal kinases c-Abl and Arg and that Tel-Abl is capable to activate at least some pathways which are also upregulated by Bcr-Abl.
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The Philadelphia (Ph) chromosome breakpoints in chronic myelocytic leukaemia are clustered on chromosome 22 band q11 in a 5.8-kilobase (kb) region designated bcr. The c-abl protooncogene is translocated from chromosome 9 band q34 into bcr... more
The Philadelphia (Ph) chromosome breakpoints in chronic myelocytic leukaemia are clustered on chromosome 22 band q11 in a 5.8-kilobase (kb) region designated bcr. The c-abl protooncogene is translocated from chromosome 9 band q34 into bcr and the biochemical consequence of this molecular rearrangement is the production of an abnormal fusion protein bcr-abl p210 with enhanced protein-tyrosine kinase activity compared to the normal p145 c-abl protein. The Ph chromosome translocation is also seen in some acute lymphoblastic leukaemias with B-cell precursor phenotypes some of which have bcr rearrangement (bcr+) and some do not (bcr-). We present evidence that the Ph+, bcr- leukaemias are associated with a novel p190 abl kinase. We propose that acute lymphoblastic leukaemias that are bcr+, p210+ are probably lymphoid blast crises following a clinically silent chronic phase of chronic myelocytic leukaemia arising in multipotential stem cells whereas bcr-, p190+ cases are de novo acute lymphoblastic leukaemias arising in more restricted precursors.
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A recombinant plasmid library representing polyadenylated RNAs in the leucocytes of a Ph1-positive chronic granulocytic leukaemia (CGL) has been constructed. One recombinant (designated pCG14) isolated from this library contains a DNA... more
A recombinant plasmid library representing polyadenylated RNAs in the leucocytes of a Ph1-positive chronic granulocytic leukaemia (CGL) has been constructed. One recombinant (designated pCG14) isolated from this library contains a DNA sequence complementary to a small polyadenylated RNA that is abundant in RNA from CGL leucocytes. The relative concentrations of pCG14 RNA in the RNAs from a variety of normal and leukaemic leucocytes and human haemopoietic cell lines have been measured with a molecular hybridisation assay. This has shown that pCG14 RNA is 10 to 50 times more abundant in RNA from CGL leucocytes than in the RNAs from these other cells. The data indicate that the occurrence of pCG14 RNA in high abundance is sufficiently characteristic of a CGL leucocyte population to distinguish it from other populations of leucocytes. They suggest that the measurement of the concentrations of specific RNA species in leucocyte RNA by means of molecular hybridisation with cloned complementary DNAs may provide additional markers for the objective classification of human leukaemias which could be particularly useful since the method exploits a criterion different from any currently in use.
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Hantaviruses belong to the family Bunyaviridae characterized by tri-segmented RNA genomes. Depending on the hantavirus species, infection can lead to hantavirus cardiopulmonary or haemorrhagic fever with renal syndrome. In vitro studies... more
Hantaviruses belong to the family Bunyaviridae characterized by tri-segmented RNA genomes. Depending on the hantavirus species, infection can lead to hantavirus cardiopulmonary or haemorrhagic fever with renal syndrome. In vitro studies suggest that pathogenic hantaviruses evade induction of innate antiviral responses, and this ability might determine the virulence in humans. Since reverse genetic systems are not available, in vitro reassortment is currently the only way to culture defined hantavirus variants. Here, we demonstrate for the first time the generation of a reassortant between a pathogenic Old World and a non-pathogenic New World hantavirus in vitro. The reassortant contained the glycoprotein coding M-segment derived from the pathogenic Puumala virus (PUUV) and the other genomic segments coding for the nucleocapsid protein and RNA-dependent RNA-polymerase from Prospect Hill virus (PHV), which is taken as non-pathogenic in humans. Exchange of the M-segment was confirmed by sequencing and virus neutralization test with PUUV-specific sera. Functional analysis of the reassortant and parental viruses revealed characteristic growth kinetics and innate immune responses as determined by expression analyses for lambda interferon and MxA, and by interferon-stimulated response element reporter gene studies. Consistent with previous studies with other pathogenic hantaviruses, PUUV elicited reduced innate responses if compared with PHV. In all these functional assays the reassortant revealed PHV-like phenotypes. Thus, neither the PUUV M-segment nor entry via specific M-segment directed pathways modulated the virus type-specific innate responses. Moreover, the data imply that this approach might be an option for production of attenuated viruses that could be used as vaccines against pathogenic hantaviruses.
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The polymerase chain reaction (PCR) was used to detect clonal rearrangements of the immunological heavy chain gene in frozen samples of human lymphoid tissue. DNA sequences in rearranged genes were amplified using oligomeric primers... more
The polymerase chain reaction (PCR) was used to detect clonal rearrangements of the immunological heavy chain gene in frozen samples of human lymphoid tissue. DNA sequences in rearranged genes were amplified using oligomeric primers predicted from conserved sequences in the variable (VH) and joining (JH) regions. On polyacrylamide gel electrophoresis, polyclonal B cell proliferations showed a "smear", probably due to the variable lengths of the diversity (DH) region genes and the N regions separating the VH and DH and JH regions. In contrast, DNA from B cell lymphomas showed a clear single band in eight out of 10 cases. PCR undertaken on germ line DNA from non-lymphoid tumours showed no detectable bands or smears. The method can be completed within one day of biopsy, compared with several days in the case of conventional DNA blot analysis. Furthermore, it is cheaper, simpler, avoids the need for radioactive materials and requires very small amounts of DNA (about 1 micrograms).
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Page 1. Regulation of Di hydrofolate Reductase Gene Expression in Mouse Fibroblasts during the Transition from the Resting to Growing State LEE F. JOHNSON,' CAROLYN L. FUHRMAN AND LEANNE M. WIEDEMANN Department ...
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The ETV6 (TEL) locus at chromosome band 12p 13 is a major site of translocations in acute leukemia, particularly in childhood acute lymphoblastic leukemia (ALL). In cases with translocations involving ETV6, the normal ETV6 allele is often... more
The ETV6 (TEL) locus at chromosome band 12p 13 is a major site of translocations in acute leukemia, particularly in childhood acute lymphoblastic leukemia (ALL). In cases with translocations involving ETV6, the normal ETV6 allele is often deleted. In addition, loss of heterozygosity of ETV6 is frequently observed in childhood'ALL. Thus, it has been suggested that ETV6 may have an anti-oncogenic role to play, in addition to its oncogenic role. We have described an unusual case of ALL in which ETV6 is found fused to the ABL gene; ABL is normally activated by fusion to the BCR gene in the 9:22 translocation. We expanded the primary cells from this ETV6/ABL rearranged case of ALL in SCID animals and analyzed them for expression of both ETV6/ABL and the normal ETV6 mRNA. We found that both the rearranged and normal ETV6 mRNAs are expressed in the expanded cell population. Furthermore, sequence analysis of the ETV6 PCR product revealed no point mutations which would influence the amino acid sequence. Thus, deletion of the second ETV6 allele is not necessary for the transformation to leukemia by ETV6/ABL.
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Following our recent reports of detecting clonal immunoglobulin and T-cell receptor gene rearrangements by the polymerase chain reaction, we have improved and simplified the technique for use in diagnostic histopathology laboratories and... more
Following our recent reports of detecting clonal immunoglobulin and T-cell receptor gene rearrangements by the polymerase chain reaction, we have improved and simplified the technique for use in diagnostic histopathology laboratories and determined, on coded samples, the sensitivity, specificity, and reproducibility of the modified methodology in distinguishing malignant lymphoma from reactive lymphoid hyperplasia and nonlymphoid tumors. Using only three primer pairs for the immunoglobulin heavy chain and T-cell receptor beta and gamma chain genes on well-characterized lesions of widely varying morphology and immunophenotype, clonal rearrangements were detected in 65% of B-cell lymphomas, and 77-82% of T-cell tumors. Specificity and observer consistency ranged from 93-97%. The method requires very careful control, particularly to avoid misinterpretation of results because of contamination and nonspecific amplification, but in its present form is relatively simple and inexpensive, and gives results on single paraffin-embedded sections within 24 h.
Research Interests: Cryopreservation, T cell receptor, DNA, Humans, Polymerase Chain Reaction, and 14 moreHyperplasia, Clinical Sciences, Molecular cloning, Observer Variation, Cost Benefit Analysis, Diffuse Large B-Cell Lymphoma, Reproducibility of Results, Immunophenotyping, Base Sequence, Formaldehyde, Sensitivity and Specificity, Lymphoid Tissue, Immunoglobulin, and Molecular Sequence Data
A series of T-cell proliferations in peripheral blood, bone marrow, or tissue samples, together with seven T-cell lines, were analysed for clonality. The technique used employs the polymerase chain reaction (PCR) to amplify rearranged... more
A series of T-cell proliferations in peripheral blood, bone marrow, or tissue samples, together with seven T-cell lines, were analysed for clonality. The technique used employs the polymerase chain reaction (PCR) to amplify rearranged T-cell receptor gamma genes, using primers recognising conserved sequences in the variable and joining gene segments. Of the 20 cases of T-cell leukaemia or lymphoma analysed, a clone was detected in 14 (70%): Of seven T-cell lines, a clone was detected in 6 (84%). No positive results were recorded in eight non-T-cell disorders (including nonlymphoid malignancies and reactive disorders). When the results of this technique were combined with the results of our previously published method for the detection of clonally rearranged T-cell receptor-beta (TCR-beta) genes using PCR, 9 of 10 (90%) T-cell tumours were detected. This method uses only four primer combinations in two tubes, and is therefore simple and rapid: it requires no radiolabelling, uses only a small amount of tissue, and can be performed on formalin-fixed, paraffin-embedded tissue.
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In classical t(9;22) translocation, as observed in chronic granulocytic leukemia (CGL), a hybrid DNA unit is produced, including a rearranged PHL gene, previously known as bcr (breakpoint cluster region) plus the translocated c-abl gene... more
In classical t(9;22) translocation, as observed in chronic granulocytic leukemia (CGL), a hybrid DNA unit is produced, including a rearranged PHL gene, previously known as bcr (breakpoint cluster region) plus the translocated c-abl gene from chromosome 9: a hybrid bcr-abl protein, p210 is formed, with increased tyrosine kinase activity. Such DNA rearrangement, with a p210 protein synthesis, is also found in cases of Philadelphia-positive acute lymphoblastic leukemia (ALL), but in apparently similar cases the bcr gene is not rearranged, and a novel p190 abl-related protein can be found; c-abl rearrangement has also been observed.It is thus established that correlations between cytogenetic and molecular events can be found in CGL and ALL, as in other haemopoietic malignancies: translocation and possible rearrangement of the c-abl oncogene seem of particular importance in this case.
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Three major types of mRNA can be expressed as a result of the Philadelphia translocation, dependent on the position of the break within the BCR gene on chromosome 22. In addition, alternative splicing of the mRNA transcribed from the... more
Three major types of mRNA can be expressed as a result of the Philadelphia translocation, dependent on the position of the break within the BCR gene on chromosome 22. In addition, alternative splicing of the mRNA transcribed from the BCR/ABL fusion gene has been reported and it has been suggested that this may play a role in the generation of the acute phase of Philadelphia positive chronic myeloid leukaemia (CML). We have examined the fusion RNA present in 24 cases of chronic phase CML and 21 cases of patients with CML in blast crisis using the polymerase chain reaction. In no case was it possible to detect the presence of the e1a2 junction which encodes the p190 hybrid protein product. We conclude that the acquisition of the p190 does not play a significant role in the generation of the blast crisis of CML. Neither could we detect a significant difference in the number of cases which simultaneously express both b2a2 and b3a2 junction products in samples isolated from chronic phase and blast crisis. In the series analysed by ethidium bromide stained gels, there was, however, an increase in the percentage of cases expressing the b3a2 junction in the mononuclear cells of blast crisis patients as compared to the white blood cells of patients in chronic phase.
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While deletion or mutation of the p53 gene is one of the most common molecular alterations detected in a wide variety of tumours, it has been shown to occur in only a relatively small percentage of the leukaemia cases examined. However,... more
While deletion or mutation of the p53 gene is one of the most common molecular alterations detected in a wide variety of tumours, it has been shown to occur in only a relatively small percentage of the leukaemia cases examined. However, it may be that other components of the p53 pathway are involved. Amplification of the MDM2 gene has recently been demonstrated in human sarcomas resulting in an increase in MDM2 protein levels. This protein can bind to p53 preventing the transactivation of p53 responsive genes, thus mimicking mutation or deletion of p53. We have investigated the prevalence of MDM2 amplification in human leukaemias. 101 leukaemia or lymphoma samples and nine cell lines were studied using Southern blotting. In no case was MDM2 amplification present. We conclude that MDM2 amplification is not a common event in human leukaemias.
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EEN, identified initially as a fusion partner to the mixed-lineage leukaemia gene in human leukaemia, and its related members, EEN-B1 and EEN-B2, have recently been shown to interact with two endocytic molecules, dynamin and synaptojanin,... more
EEN, identified initially as a fusion partner to the mixed-lineage leukaemia gene in human leukaemia, and its related members, EEN-B1 and EEN-B2, have recently been shown to interact with two endocytic molecules, dynamin and synaptojanin, as well as with the huntingtin protein. In the present study, we show that the expression of the EEN gene-family members is differentially regulated. Multiple-spliced variants were identified for EEN-B2. In the brain, EEN-B1 and EEN-B2 mRNA are preferentially expressed in the cerebellar Purkinje and granule cells, dentate gyrus cells, hippocampal pyramidal neurons and cerebral granule cells. The expression patterns of EEN-B1 and EEN-B2 mRNA in the brain overlap with those of dynamin-I/III, synaptojanin-I and huntingtin, whereas the ubiquitous expression of EEN is consistent with that of dynamin-II. In testes, members of the EEN family are co-expressed with testis-type dynamin and huntingtin in Sertoli cells and germ cells respectively. Our results on the overlapping expression patterns are consistent with the proposed interaction of EEN family members with dynamin, synaptojanin and huntingtin protein in vivo. Although all three EEN family members bind to dynamin and synaptojanin, EEN-B1 has the highest affinity for binding, followed by EEN and EEN-B2. We also demonstrate that amphiphysin, a major synaptojanin-binding protein in brain, can compete with the EEN family for binding to synaptojanin and dynamin. We propose that recruitment of the EEN family by dynamin/synaptojanin to clathrin-coated pits can be regulated by amphiphysin.