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Biomedical subjects

J Groffen

Publications and source records attributed to J Groffen.

At least 91 records · Page 5Linked to original sources
PubMed1991

Identification of a human gamma-glutamyl cleaving enzyme related to, but distinct from, gamma-glutamyl transpeptidase.

We have cloned a 2.4-kilobase cDNA from a human placental cDNA library by using a gamma-glutamyl transpeptidase [GGT; gamma-glutamyltransferase, (5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] probe. The deduced amino acid sequence of this cDNA, GGT-rel, exhibited an overall similarity of 39.5% with human GGT. Sequences that could represent a heavy and a light chain, analogous to GGT, as well as a putative transmembrane region were identified in GGT-rel. Transfectants overexpressing GGT-rel were tested for their ability to catalyze cleavage of the gamma-glutamyl moiety from natural and synthetic substrates for GGT. Experiments with glutathione added to the medium suggested that GGT-rel could hydrolyze the gamma-glutamyl moiety. More definitive evidence was obtained in experiments in which this protein converted leukotriene C4 to leukotriene D4. However, GGT-rel did not convert synthetic substrates that are commonly used to assay GGT. Our results indicate that GGT can no longer be considered the only enzyme capable of cleaving the gamma-glutamyl linkage of leukotriene C4 and, most likely, of other natural compounds.

Amino Acid Sequence↗
PubMed1991

Aspartylglycosaminuria in the Finnish population: identification of two point mutations in the heavy chain of glycoasparaginase.

Aspartylglycosaminuria is an inherited lysosomal storage disease caused by deficiency of glycoasparaginase (EC 3.5.1.26) and occurs with higher frequency among Finns than other populations. We have purified human glycoasparaginase and determined about 90% of the amino acid sequence of its light subunit and greater than 70% of that of its heavy subunit by Edman degradation and mass spectrometry. Additional sequence data were obtained from the cloning and subsequent nucleotide analysis of a cDNA corresponding to the normal human glycoasparaginase gene. The enzyme is encoded by a single mRNA as a single polypeptide that is posttranslationally processed to generate the subunits and is glycosylated. After preparing first-strand cDNA from leukocyte and fibroblast total RNA, we used the polymerase chain reaction to amplify the glycoasparaginase cDNA of eight Finnish aspartylglycosaminuria patients. We demonstrate that the Finnish patients' mRNA sequence differed from the normal sequence by two single-base changes six nucleotides apart from one another in the heavy chain of glycoasparaginase. The first change resulted in the replacement of arginine by glutamine (R161Q), whereas the second change resulted in a cysteine to serine substitution (C163S). Both mutations resulted in novel restriction endonuclease sites and were present in all eight Finnish aspartylglycosaminuria patients originating from different pedigrees, but they were absent from Finnish and non-Finnish controls and a non-Finnish case of aspartylglycosaminuria. These results indicate molecular homogeneity in aspartylglycosaminuria alleles in the Finnish population.

Amino Acid Sequence↗
PubMed1991

A complex chromosome rearrangement forms the BCR-ABL fusion gene in leukemic cells with a normal karyotype.

Chromosome in situ hybridization studies showed that the normal karyotype of leukemic cells from a patient with Ph1-negative, BCR-positive chronic myeloid leukemia (CML) concealed a complex t(9;22;20)(q34;q11;p13). The close association of 5'-BCR and 3'-ABL was demonstrated by field inversion gel electrophoresis, and in situ hybridization showed that BCR-ABL was located on the short arm of chromosome 20. Our findings further indicate that chromosome rearrangement is the cause of BCR-ABL gene fusion in leukemic cells that show a normal karyotype. Results from in situ hybridization studies were consistent with formation of the t(9;22;20) by a two step chromosomal rearrangement, but field inversion gel electrophoresis results indicated a more complex rearrangement.

Aged↗
PubMed1991

Human bcr-abl gene has a lethal effect on embryogenesis.

The chimaeric bcr-abl oncogene is thought to have a crucial role in the development or maintenance of chronic myelogenous leukaemia. To study this oncogene in a more direct way, the bcr-abl gene encoding the P210 protein under control of the bcr 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 this bcr-abl construct. The bcr gene is expressed in the course of embryogenesis and the bcr-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 the bcr-abl encoded protein severely affects the process of normal embryogenesis.

Animals↗
PubMed1991

Nuclear and cytoplasmic location of the FER tyrosine kinase.

The location of the FER protein within the cell was investigated by using subcellular fractionation and immunofluorescence. FER was found in the cytoplasm and in the nucleus, where it was associated with the chromatin fraction. Its ubiquitous expression and its subcellular location indicate that it may be involved in key regulatory processes.

Amino Acid Sequence↗
PubMed1991

Expression of the chronic myelogenous leukemia-associated p210bcr/abl oncoprotein in a murine IL-3 dependent myeloid cell line.

We have studied the effect of a replication-defective murine retroviral vector expressing the chronic myelogenous leukemia associated oncoprotein p210bcr/abl in murine IL-3 dependent myeloid 32D C13(G) cells. This cell line can be induced to differentiate along either the granulocytic or monocytic lineages thus permitting an independent assessment of the effect of p210bcr/abl on growth and differentiation. Cells expressing p210bcr/abl displayed a complete non-autocrine abrogation of IL-3 dependence and an enhanced response to an activity in FBS which is not IGF-I or IGF-II. During the first few generations following infection with the bcr/abl vector, cells became larger with an increased fraction of cells in G2/M and monocyte/macrophage markers were expressed. Four cytoplasmic proteins phosphorylated in response to IL-3 in the parental cell line with apparent molecular weights of 98, 70, 62, and 52 Kd were amongst those constitutively phosphorylated in p210bcr/abl expressing cells. These results suggest that the functional substitution of IL-3 by p210bcr/abl is due to constitutive activation of proteins involved in IL-3 signal transduction. Alterations of cell differentiation, cell cycle and growth which cannot be attributed to IL-3 like effects indicate that p210bcr/abl has pleiotropic effects involving several other pathways of cellular regulation.

Cell Cycle↗
PubMed1991

Localization of the TRK proto-oncogene to human chromosome bands 1q23-1q24.

A FER-related sequence isolated from a human genomic library was found to be homologous to TRK. In situ hybridization of a 0.92 kb probe, isolated from this sequence, localized the TRK gene to the long arm of chromosome 1 within bands 1q23-1q24. This is a significantly more proximal location of TRK than the 1q32-1q41 site published recently (Miozzo et al., 1990).

Amino Acid Sequence↗
PubMed1991

Molecular insights into the Philadelphia translocation.

The Ph chromosome was the first specific karyotype abnormality associated with a particular neoplastic disease in humans. For many years it was suspected that chromosome abnormalities might cause cancer by alteration of specific genes or their expression. Significant recent developments in our understanding of the molecular consequences of the Ph translocation strengthen that assumption. The Ph translocation generates a hybrid gene consisting of 5' regulatory, promotor, and exon sequences of the bcr gene on chromosome 22 fused to 3' exons and polyadenylation/termination sequences of the ABL proto-oncogene from chromosome 9. It is well established that fusion of bcr and abl genes plays a crucial role in the pathogenesis of CML and ALL. Molecular methods can therefore be used as diagnostic tools to detect the Ph chromosome. Presently, the model of oncogenesis provided by our knowledge of how the abl proto-oncogene becomes activated as a result of the Ph translocation is one of the clearest models of oncogene activation. Despite the progress made, many areas remain to be explored. One important question is, how the hybrid protein is involved in leukemia. Research aimed at investigating the normal function of abl and bcr may be important in efforts to understand their abnormal functioning in leukemia and to increase our understanding of the disease.

Animals↗
PubMed1990

Unique organization of the human BCR gene promoter.

The promoter of the human BCR gene, regulating the transcription of the chimeric BCR/ABL mRNA in leukemia, has been isolated and characterized. A region of 1.1 kb immediately 5' to the transcription start site was analyzed in detail by sequencing, DNase 1 footprinting, gel retardation and functional studies. These experiments localized a minimal promoter to a 650 bp sequence, composed of 270 bp of 5' flanking sequences and 380 bp of exon 1 transcribed sequences. The promoter region includes a TTTAA box, one Sp1 site and a novel protein-binding sequence absolutely necessary for efficient transcription in vivo. Six additional protein-binding regions were identified more to the 5'. Of these, one is found in an inverted repeat in the 3' coding and splice donor region of BCR exon 1.

Animals↗
PubMed1990

Ph-negative chronic myeloid leukemia: molecular analysis of ABL insertion into M-BCR on chromosome 22.

Leukemic cells from a patient with Ph-negative chronic myeloid leukemia (CML) had a normal karyotype. M-BCR was rearranged and chromosome in situ hybridization showed an ABL insertion between 5' and 3' M-BCR on an apparently normal chromosome 22. The association of 5' BCR and 3' ABL at the 5' junction of the chromosome 9 insert was typical of that found for the BCR-ABL fusion gene in other patients with the standard t(9;22) and CML. With an M-bcr-3' probe, we cloned and characterized a 3' junction fragment. Field inversion gel electrophoresis and chromosome in situ hybridization studies using a probe isolated from genomic DNA 5' of the junction showed that 3' M-BCR was joined to a region of chromosome 9q34 rich in repetitive sequences and lying some distance 3' of ABL. The chromosome 9 insert was at least 329 kilobases long and included 3' ABL and a larger portion of chromosome 9q34. Our results allowed us to exclude transposon- or retroviral-mediated insertion of ABL into chromosome 22. Instead, we favored a two-translocation model in which a second translocation reconstituted a standard t(9;22)(q34;q11) but left the chromosome 9 insert, including 3' ABL, in chromosome 22.

Chimera↗
PubMed1990

Rearrangement of the human ABL oncogene in a glioblastoma.

A number of protooncogenes have been implicated in human tumorigenesis. The ABL oncogene is consistently rearranged and activated as a consequence of the translocation t(9;22) that gives rise to the Philadelphia chromosome in chronic myeloid leukemia and in some cases of acute lymphoblastic leukemia. Here we describe rearrangement of ABL in a different type of malignancy. The glioblastoma cell line A172 lacks germline alleles of ABL. A recombination event, presumably followed by a duplication, has created two ABL alleles in which exon 11 is joined to chromosome 16 sequences. Although the main body of ABL exons was still present, two considerably shortened ABL mRNAs of 3.8 and 2.8 kilobases were detected; the 3.8-kilobase mRNA hybridized exclusively to an exon IB probe. Neither mRNA hybridized to an ABL probe encompassing part of the tyrosine kinase domain. Thus, the cell line A172 is able to survive in the absence of a functional ABL gene product, indicating that the role of ABL is unlikely to be "housekeeping."

Base Sequence↗
PubMed1990

Acute leukaemia in bcr/abl transgenic mice.

The Philadelphia chromosome, widely implicated in human leukaemia, is the result of a reciprocal translocation t(9;22) (q34;q11) in which the abl oncogene located at 9q34 is translocated to chromosome 22q11, where it is fused head-to-tail with 5' exons of the bcr gene. In acute lymphoblastic leukaemia, some patients have a breakpoint within the major breakpoint cluster region of the bcr gene, whereas others have the break within its first intron. This second type of translocation results in the transcription of a 7.0-kilobase chimaeric bcr/abl messenger RNA translated into a bcr/abl fusion protein, p190, which has an abnormal tyrosine kinase activity and is strongly autophosphorylated in vitro. We have generated mice transgenic for a bcr/abl p190 DNA construct and find that progeny are either moribund with, or die of acute leukaemia (myeloid or lymphoid) 10-58 days after birth. This finding is evidence for a causal relationship between the Philadelphia chromosome and human leukaemia.

Animals↗
PubMed1990

A genetic linkage map of mouse chromosome 10: localization of eighteen molecular markers using a single interspecific backcross.

Interspecific mouse backcross analysis was used to generate a molecular genetic linkage map of mouse chromosome 10. The map locations of the Act-2, Ahi-1, Bcr, Braf, Cdc-2a, Col6a-1, Col6a-2, Cos-1, Esr, Fyn, Gli, Ifg, Igf-1, Myb, Pah, pgcha, Ros-1 and S100b loci were determined. These loci extend over 80% of the genetic length of the chromosome, providing molecular access to many regions of chromosome 10 for the first time. The locations of the genes mapped in this study extend the known regions of synteny between mouse chromosome 10 and human chromosomes 6, 10, 12 and 21, and reveal a novel homology segment between mouse chromosome 10 and human chromosome 22. Several loci may lie close to, or correspond to, known mutations. Preferential transmission of Mus spretus-derived alleles was observed for loci mapping to the central region of mouse chromosome 10.

Animals↗
PubMed1990

The human tyrosine kinase gene (FER) maps to chromosome 5 and is deleted in myeloid leukemias with a del(5q).

A novel member of the SRC tyrosine kinase gene family was recently isolated and characterized (Hao et al., 1989). This FES/FPS-related gene, named FER, lacks the transmembrane and extracellular domains which characterize tyrosine kinases with receptor function. Expression of FER in a wide range of cell types indicates a general role in intracellular signalling or differentiation processes. We have now mapped FER to chromosome 5q14----q23 using in situ hybridization techniques and suggest a more precise location within bands 5q21----q22. This region lies adjacent to a complex domain of growth factors and receptors, many involved in regulation of haematopoiesis. FER maps within a critical segment frequently deleted from chromosome 5 in patients with acute myeloid leukemia or myelodysplastic syndromes and was shown to be deleted in two such patients. It also maps close to the familial polyposis coli locus at 5q22.

Chromosome Deletion↗
PubMed1990

Characterization of the human ABL promoter regions.

Gel retardation and DNAase 1 footprinting experiments have been performed to characterize the promoter sequences of exon 1a and 1b of the human ABL gene. Several Sp1 motifs and CCAAT boxes are found to be protected by nuclear proteins in the 1b promoter but none of the 7 reported Sp1 sites in 1a were found to bind protein. Multiple sets of initiation sites seem to exist in the 1b promoter region which may represent individual initiation sites, distributed over a DNA region of up to 700 bp. Starting with the most distal initiation site, 1a and 1b ABL promoter sequences show a high degree of homology, suggesting that one is derived from the other. However, multiple evolutionary changes in the 1a promoter sequence indicate that type 1a ABL expression may be differently regulated than 1b.

Abelson murine leukemia virus↗