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Biomedical subjects

J Groffen

Publications and source records attributed to J Groffen.

At least 127 records · Page 7Linked to original sources
PubMed1987

c-K-ras mutations in human carcinomas occur preferentially in codon 12.

A study was carried out to determine the frequency and distribution of mutations in the c-K-ras gene in human carcinoma tissue. The study was done on a total of 51 lung, colon and breast carcinoma tumors using a panel of oligonucleotides coding for the wild type and all possible mutations in codons 12 and 61 of c-K-ras gene. Four of 16 colon carcinomas, two of 27 lung carcinomas and one of eight breast carcinomas were found to contain mutations in codon 12. No mutations were found at position 61. Of the six possible amino acid replacements in codon 12, all but one was represented in the seven mutations identified.

Breast Neoplasms↗
PubMed1986

Gamma delta beta-thalassaemias 1 and 2 are the result of a 100 kbp deletion in the human beta-globin cluster.

The DNA spanning two large deletions in the human beta-globin gene cluster (gamma beta-thalassaemia 1 and 2) has been cloned by cosmid cloning and chromosomal walking. The entire region was mapped and analyzed for the presence of repetitive sequences. The results show that the affected loci have lost almost 100 kb of DNA in a deletion event not involving homologous or repetitive sequences.

Chromosome Deletion↗
PubMed1986

Molecular analysis of both translocation products of a Philadelphia-positive CML patient.

The breakpoint regions of both translocation products of the (9;22) Philadelphia translocation of CML patient 83-H84 and their normal chromosome 9 and 22 counterparts have been cloned and analysed. Southern blotting with bcr probes and DNA sequencing revealed that the breaks on chromosome 22 occurred 3' of bcr exon b3 and that the 88 nucleotides between the breakpoints in the chromosome 22 bcr region were deleted. Besides this small deletion of chromosome 22 sequences a large deletion of chromosome 9 sequences (greater than 70 kb) was observed. The chromosome 9 sequences remaining on the 9q+ chromosome (9q+ breakpoint) are located at least 100 kb upstream of the v-abl homologous c-abl exons whereas the translocated chromosome 9 sequences (22q-breakpoint) could be mapped 30 kb upstream of these c-abl sequences. The breakpoints were situated in Alu-repetitive sequences either on chromosome 22 or on chromosome 9, strengthening the hypothesis that Alu-repetitive sequences can be hot spots for recombination.

Adolescent↗
PubMed1986

Four human carcinoma cell lines with novel mutations in position 12 of c-K-ras oncogene.

We have used synthetic oligonucleotides to probe for mutations affecting amino acid 12 of the c-K-ras gene in human cell line DNA. Of seven carcinoma cell lines tested, four were found to contain a mutation at this position. In each the nucleotide G was replaced with an A resulting in a Gly to Asp substitution in three cases (cell lines A427, A1165 and A1663) and Gly to Ser in the fourth (A549). Neither of these substitutions have been previously reported in either human tumor or human tumor-derived cell line DNA's. These results indicate that association between mutations involving position 12 of the human c-K-ras oncogene and carcinomas may be stronger than previously recognized.

Base Sequence↗
PubMed1986

The chronic myelocytic cell line K562 contains a breakpoint in bcr and produces a chimeric bcr/c-abl transcript.

In the DNAs of all Ph1-positive chronic myelocytic leukemia patients studied to date, a breakpoint on chromosome 22 (the Ph1 chromosome) can be demonstrated with a probe from the bcr (breakpoint cluster region). Although the K562 cell line was established from cells of a chronic myelocytic leukemia patient, we have been unable to detect the Ph1 chromosome by cytogenetic means. Employing a probe from the 5' region of bcr, we have cloned an amplified Ph1 breakpoint fragment from K562. This demonstrates that K562 contains multiple remnants of a Ph1 chromosome with a breakpoint within bcr and thus may serve as a model system for the study of Ph1-positive chronic myelocytic leukemia at a molecular level. The isolation of bcr cDNA sequences shows that parts of bcr encode a protein. Employing K562, we demonstrate the presence of an abnormally sized mRNA species hybridizing to c-abl and to a bcr cDNA probe, indicating the possible consequence of the Ph1 translocation on a transcriptional level in chronic myelocytic leukemia. The isolation and sequencing of a cDNA containing the breakpoint area of this mRNA provide further evidence for its chimeric structure. Cloning of large stretches of chromosomal DNA flanking bcr and c-abl sequences in K562 and identification of the exons participating in the formation of the chimeric mRNA shows that a splice of at least 99 kilobases is made to fuse the 3' bcr exon to the 5' c-abl exon. Furthermore two chimeric cDNAs were isolated containing chromosome 9 sequences that map 43.5 kilobases downstream from the K562 breakpoint. These chromosome 9 sequences neither hybridize to the 8.5-kilobase chimeric c-abl mRNA nor to normal c-abl mRNAs in Hela cells and probably represent incorrect splicing products present in the K562 cell line.

Amino Acid Sequence↗
PubMed1985

Evidence of a new chimeric bcr/c-abl mRNA in patients with chronic myelocytic leukemia and the Philadelphia chromosome.

The hallmark of chronic myelocytic leukemia is the presence of the Philadelphia chromosome (Ph1). In recent studies, we obtained data that strongly suggested the involvement of an oncogene, c-abl, in this type of leukemia. This oncogene, normally located on chromosome 9, is translocated to chromosome 22 as a result of the Ph1 translocation. In addition, we identified a region on chromosome 22, the breakpoint cluster region (bcr), which contains the chromosomal breakpoint in all patients with chronic myelocytic leukemia who are positive for Ph1. Recent studies have suggested that the bcr is part of a gene that is truncated as a consequence of the Ph1 translocation. The deleted part of this gene could be replaced by c-abl sequences; to test this hypothesis we analyzed the RNA of five patients with chronic myelocytic leukemia. All five had chimeric bcr/c-abl messenger RNA, suggesting that the deleterious effects of this disease can be associated with an abnormal chimeric protein encoded by the bcr and the c-abl oncogene.

Adult↗
PubMed1985

C-abl and bcr are rearranged in a Ph1-negative CML patient.

Chromosomal analysis of a patient with chronic myelocytic leukemia (CML) revealed a translocation (9;12) (q34;q21) without a detectable Philadelphia chromosome (Ph1). Using molecular approaches we demonstrate (i) a rearrangement within the CML breakpoint cluster region (bcr) on chromosome 22, and (ii) a joint translocation of bcr and c-abl oncogene sequences to the derivative chromosome 12. These observations support the view that sequences residing on both chromosome 9 (c-abl) and 22 (bcr) are involved in the generation of CML and suggest that a subset of Ph1-negative patients may in fact belong to the clinical entity of Ph1-positive CML.

Adult↗
PubMed1985

Chromosomal localization and characterization of c-abl in the t(6;9) of acute nonlymphocytic leukemia.

Acute nonlymphocytic leukemia associated with the chromosomal translocation t(6;9)(p23;q34) is an entity that is frequently associated with basophilia, which it shares with chronic myelogenous leukemia. The breakpoint on chromosome 9, q34, appears to be cytogenetically identical in both malignancies and is the site of the cellular oncogene c-abl. We investigated the role of c-abl in cells from two patients with the t(6;9) using in situ chromosomal hybridization, Southern hybridization, and in vitro phosphorylation. We showed that c-abl is not translocated from chromosome 9, resulting in a breakpoint that is on the 3' side of this gene. The t(6;9) translocation does not appear to result in the production of an aberrantly sized protein product or in the acquisition of in vitro tyrosine kinase activity. This is in direct contrast to the findings in chronic myelogenous leukemia, in which c-abl is translocated, leading to the production of a structurally altered c-abl protein with activated tyrosine kinase. Lastly, we demonstrated that the cells of one patient contain sequences from chromosome 9 inserted at the junction of a reciprocal translocation between chromosomes 4 and 10 on the 4q+ chromosome. This insertion, which is at least 100 kilobase pairs in length, represents a duplication and translocation of the protein coding region of c-abl.

Adult↗
PubMed1984

Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22.

We have identified and molecularly cloned 46 kb of human DNA from chromosome 22 using a probe specific for the Philadelphia (Ph') translocation breakpoint domain of one chronic myelocytic leukemia (CML) patient. The DNAs of 19 CML patients were examined for rearrangements on chromosome 22 with probes isolated from this cloned region. In 17 patients, chromosomal breakpoints were found within a limited region of up to 5.8 kb, for which we propose the term "breakpoint cluster region" (bcr). The two patients having no rearrangements within bcr lacked the Ph' chromosome. The highly specific presence of a chromosomal breakpoint within bcr in Ph'-positive CML patients strongly suggests the involvement of bcr in this type of leukemia.

Bacterial Proteins↗
PubMed1984

Localization of the human c-sis oncogene in Ph1-positive and Ph1-negative chronic myelocytic leukemia by in situ hybridization.

Oncogenes are a group of evolutionary conserved cellular genes (c-onc) homologous to the transforming genes of oncogenic retroviruses (v-onc). Some of them are localized near the breakpoints of specific chromosomal aberrations occurring in various neoplasms, as for example the Philadelphia translocation, t(9;22)(q34;q11), in chronic myelocytic leukemia (CML). Recently, we localized the human c-abl oncogene to chromosome region 9q34 and demonstrated a translocation of this gene to the Philadelphia chromosome (Ph1,22q-) in various forms of Ph1-positive, but not Ph1-negative, chronic myelocytic leukemia (CML). Another human oncogene, c-sis, is located on chromosome 22 and was recently reported to be transferred to chromosome 9q+ in one CML patient. We have now studied 2 CML patients with classic and variant types of Ph1 translocation, one Ph1-negative case, and a healthy control using in situ hybridization of a c-sis probe to metaphase chromosomes. These studies show that c-sis: (1) is localized to region 22q12.3-q13.1, far away from the breakpoint region 22q11 in CML, (2) segregates with the translocated part of chromosome 22 to different chromosomes in Ph1-positive patients, and (3) remains on chromosome 22 in the Ph1-negative case. Therefore, these data give no support for an active role of the c-sis gene in the generation of CML. Thus, if either of these two oncogenes is involved in the development of Ph1-positive CML, c-abl appears to be the more important one.

Chromosomes, Human, 21-22 and Y↗
PubMed1983

Chromosomal localization of the human c-fms oncogene.

A molecular probe was prepared with specificity for the human cellular homologue of transforming sequences represented within the McDonough strain of feline sarcoma virus (v-fms). By analysis of a series of mouse-human somatic cell hybrids containing variable complements of human chromosomes it was possible to assign this human oncogene, designated c-fms, to chromosome 5. Regional localization of c-fms to band q34 on chromosome 5 was accomplished by analysis of Chinese hamster-human cell hybrids containing as their only human components, terminal and interstitial deleted forms of chromosome 5. The localization of c-fms to chromosome 5 (q34) is of interest in view of reports of a specific, apparently interstitial, deletion involving approximately two thirds of the q arm of chromosome 5 in acute myelogenous leukemia cells.

Animals↗
PubMed1983

c-sis is translocated from chromosome 22 to chromosome 9 in chronic myelocytic leukemia.

By analysis of a series of somatic cell hybrids derived by fusion of either mouse or Chinese hamster cells with leukocytes from different chronic myelocytic leukemia (CML) patients or from normal donors, we have localized the human oncogene, c-sis, on the q11 to qter segment of chromosome 22 and demonstrated its translocation from chromosome 22 to chromosome 9 (q34) in CML.

Animals↗
PubMed1983

Regulation of viral and cellular oncogene expression by cytosine methylation.

Mink cells morphologically transformed by either Snyder-Theilen feline sarcoma virus (ST-FeSV) or Abelson murine leukemia virus (Abelson-MuLV) exhibit relatively high rates of reversion to the nontransformed phenotype. The proviral DNAs are conserved within the revertant lines and have not undergone changes in integration sites due to translocations or other genomic rearrangements. In contrast, expression of well-defined viral-encoded transforming proteins is blocked and elevated levels of phosphotyrosine characteristic of the parental transformed cells are reduced to control levels. Loss of the transformed phenotype is associated with increased cytosine methylation of proviral DNA sequences while levels of methylation resume control levels upon spontaneous retransformation of revertant clones. Following molecular cloning, and transfection to Rat-2 cells, ST-FeSV proviral DNAs from revertant and transformed cells induced similar numbers of transformed foci. Cytosine methylation sites involved in regulation of expression of the major ST-FeSV encoded transforming protein have been localized within the proviral DNA itself rather than in adjacent cellular flanking sequences. In contrast to the v-fes proviral DNA, c-fes, the cellular homolog of the ST-FeSV acquired transforming sequences, is highly methylated in cytosine residues in both transformed and revertant clones. These findings demonstrate regulation of viral oncogene-mediated transformation by cytosine methylation and suggest that expression of cellular homologs of viral oncogenes, such as c-fes, are also subject to regulation at this level.

Animals↗
PubMed1983

Isolation of v-fms and its human cellular homolog.

The integrated form of McDonough FeSV proviral DNA, including cellular flanking sequences, was molecularly cloned from nonproductively transformed Fisher rat cells. Acquired cellular-derived (v-fms) sequences within the cloned proviral DNA were mapped from between 2.6 and 5.5 kb from the 5'LTR. Upon transfection, the cloned proviral DNA was biologically active; it caused induction of the transformed phenotype and the resulting transformed cells expressed the major McDonough FeSV translational product, P170gag-fms at high level. Using a series of molecular probes representing subgenomic regions of the viral v-fms gene, a cosmid library of human lung carcinoma DNA was screened for v-fms homologous sequences. Three cosmid clones containing overlapping v-fms homologous cellular DNA inserts, representing a contiguous region of cellular DNA sequence of approximately 64 kb in length, were isolated. Within this region of human genomic DNA, v-fms homologous sequences are dispersed over a total region of around 32 kb. These represent the entire human cellular homolog of v-fms, are colinear with the viral v-fms transforming gene, and contain a minimum of four intervening sequences. At least 12 regions of highly repetitive DNA sequences have been mapped in close proximity to c-fms coding sequences.

Animals↗
PubMed1983

Transforming genes of avian (v-fps) and mammalian (v-fes) retroviruses correspond to a common cellular locus.

The Gardner (GA) and Snyder-Theilen (ST) isolates of feline sarcoma virus (FeSV) represent genetic recombinants between feline leukemia virus (FeLV) and transformation-specific sequences (v-fes gene) of cat cellular origin. A related transforming gene (v-fps), common to the Fujinami, PRC II, and UR 1 strains of avian sarcoma virus has also been described. Translational products of each of these recombinant virus isolates are expressed in the form of polyproteins exhibiting protein kinase activities with specificity for tyrosine residues. In the present study, v-fes and v-fps homologous sequences of GA-FeSV, ST-FeSV, and Fujinami sarcoma virus (FSV) are defined and these independently derived transforming genes are shown to correspond to a common cellular genetic locus which has remained highly conserved throughout vertebrate evolution.

Animals↗