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J F Mushinski

Publications and source records attributed to J F Mushinski.

At least 73 records · Page 4Linked to original sources
PubMed1991

Mouse protein kinase C-delta, the major isoform expressed in mouse hemopoietic cells: sequence of the cDNA, expression patterns, and characterization of the protein.

A complementary DNA (cDNA) of 2559 bp which encode all 674 amino acids of mouse protein kinase C-delta (PKC-delta) has been isolated from a cDNA library prepared from ABPL-2, a mouse myeloid tumor. The library was screened with a partial PKC-delta cDNA clone that had been created by polymerase chain reaction (PCR) amplification of ABPL-2 RNA using primers that are conserved among all rat PKC isozymes. This approach proved to be a distinct improvement over screening with synthetic oligonucleotides. Similar sets of cDNAs prepared from other hemopoietic cell lines were screened with this PKC-delta cDNA and with probes for the other PKC isoforms. These experiments revealed that the major isoform of PKC expressed in hemopoietic cells is PKC-delta. PKC-delta protein was purified from ABPL-3, a mouse myeloid tumor which expressed principally the delta isoform of PKC. The protein eluted from a hydroxylapatite column in the same position as PKC-beta and -epsilon would elute, if present. The kinase activity of purified PKC-delta showed strict dependence on the presence of phospholipids, but showed no activation by Ca2+.

Amino Acid Sequence↗
PubMed1991

Nonhomologous recombination at sites within the mouse JH-C delta locus accompanies C mu deletion and switch to immunoglobulin D secretion.

Plasma cells secrete immunoglobulins other than immunoglobulin M (IgM) after a deletion and recombination in which a portion of the immunoglobulin heavy-chain locus (IgH), from the 5'-flanking region of the mu constant-region gene (C mu) to the 5'-flanking region of the secreted heavy-chain constant-region gene (CH), is deleted. The recombination step is believed to be targeted via switch regions, stretches of repetitive DNA which lie in the 5' flank of all CH genes except delta. Although serum levels of IgD are very low, particularly in the mouse, IgD-secreting plasmacytomas of BALB/c and C57BL/6 mice are known. In an earlier study of two BALB/c IgD-secreting hybridomas, we reported that both had deleted the C mu gene, and we concluded that this deletion was common in the normal generation of IgD-secreting cells. To learn how such switch recombinations occur in the absence of a switch region upstream of the C delta 1 exon, we isolated seven more BALB/c and two C57BL/6 IgD-secreting hybridomas. We determined the DNA sequences of the switch recombination junctions in eight of these hybridomas as well as that of the C57BL/6 hybridoma B1-8. delta 1 and of the BALB/c, IgD-secreting plasmacytoma TEPC 1033. All of the lines had deleted the C mu gene, and three had deleted the C delta 1 exon in the switch recombination event. The delta switch recombination junction sequences were similar to those of published productive switch recombinations occurring 5' to other heavy-chain genes, suggesting that nonhomologous, illegitimate recombination is utilized whenever the heavy-chain switch region is involved in recombination.

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PubMed1991

DNA repair in the c-myc proto-oncogene locus: possible involvement in susceptibility or resistance to plasmacytoma induction in BALB/c mice.

This report describes an unexpected difference in the efficiency of removal of UV-induced DNA damage in the c-myc locus in splenic B lymphoblasts from two inbred strains of mice. In cells from plasmacytoma-resistant DBA/2N mice, 35% of UV-induced damage in the regulatory and 5' flank of c-myc is removed by 12 h. However, in cells from plasmacytoma-susceptible BALB/cAn mice, damage is not removed from this region. In the protein-encoding region and 3' flank of c-myc as well as in two dihydrofolate reductase gene fragments, UV damage is repaired with similar efficiency in B lymphoblasts from both strains of mice. Furthermore, in the protein-encoding portion and 3' flank of c-myc, damage is selectively removed from only the transcribed strand. No repair is detected in the nontranscribed strand. In contrast, DNA repair in the 5' flank of c-myc is not strand specific; in DNA from DBA/2N cells, UV damage is rapidly removed from both the transcribed and nontranscribed strands. In BALB/cAn cells no repair was detected in either strand in the 5'flank, consistent with the results with double-stranded, nick-translated probes to this region of c-myc. In addition to the repair studies, we have detected post-UV-damage formation: in most of the genes studied, we find that additional T4 endonuclease-sensitive sites are formed in the DNA 2 h after irradiation. Our findings provide new insights into the details of gene-specific and strand-specific DNA repair and suggest that there may be close links between DNA repair and B-cell neoplastic development.

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PubMed1991

The v-rel and c-rel proteins exist in high molecular weight complexes in avian and murine cells.

We reported previously that the v-rel protein (p59v-rel) exists in a high molecular weight complex with at least four other proteins in the cytoplasm of v-rel-transformed chicken pre-B lymphoid cells (Simek, S. & Rice, N.R., J. Virol., 62, 4730-4736, 1989). One of these proteins is the chicken c-rel protein, but the identities of the others (of about 36 kDa, 115 kDa, and 124 kDa) are unknown. In this report we extend that observation to additional v-rel-transformed cell lines of both pre-B and B cell phenotypes. We also introduced and expressed v-rel in several other avian cell lines (a chicken T cell line, chick embryo fibroblasts, and quail fibroblasts) and found that in these cells p59v-rel was complexed with the same proteins as observed in the v-rel-transformed cells. Thus, the associated proteins are not limited to pre-B cells, but occur and complex with p59v-rel in B cells, T cells, and fibroblasts. We next examined five uninfected avian cells and tissues and found that, with only one exception, p75c-rel was complexed with p36, p115, and p124. Thus, in most cases complex formation is not limited to or dependent on the presence of the transforming v-rel protein, but also occurs with the normal c-rel protein. To determine whether a mammalian c-rel protein is similarly associated with other proteins, we screened murine cell lines for the presence of c-rel mRNA. In agreement with our earlier findings, we found the highest expression in mature B cells, although several pre-B and myeloid cell lines were also strongly positive. Using one of the B cell lines, we detected the murine c-rel protein. We found that, like its avian counterpart, it is a protein of about 75 kDa and is associated with proteins of 36 kDa and 115 kDa. Sephacryl S-400 chromatography revealed that both the avian and murine complexes are of high molecular weight, with an average size of about 400 kDa.

Animals↗
PubMed1991

Mouse bcl-3: cDNA structure, mapping and stage-dependent expression in B lymphocytes.

Human B-cell chronic lymphocytic leukemias (CLLs) are malignancies of mature B lymphocytes. A subset of these tumors is associated with a non-random t(14; 19) translocation (Ueshima et al., 1985). Recently a gene (bcl-3) has been identified in the region adjacent to the chromosome 19 breakpoint in this translocation (McKeithan et al., 1987; Ohno et al., 1990). We now report the isolation of cDNA clones of mouse bcl-3. The mouse bcl-3-coding region is 1746 bp long and exhibits 80% identity with human bcl-3 at both the nucleotide and amino acid level. The bcl-3 locus maps to the proximal end of mouse chromosome 7, which is syntenic to human chromosome 19. The bcl-3 probe readily detects particularly abundant amounts of a 1.8 kb mRNA in mouse tumors consisting of follicular center mature B cells and large pre-B cells, but not in small pre-B cells. The bcl-3 pattern of expression is distinctive in the spectrum of B-cell maturation in that bcl-3 transcripts are particularly abundant in B-cell lines immortalized just prior to Ig switch. The bcl-3 pattern of expression also bears close resemblance to that of bcl-2 (Gurfinkel et al., 1987), which is frequently associated with human B follicular lymphomas [t(14; 18)] and some chronic lymphocytic leukemias (Adachi et al., 1989; 1990; Adachi & Tsujimoto, 1989).

Amino Acid Sequence↗
PubMed1990

The in vivo generation of murine IgD-secreting cells is accompanied by deletion of the C mu gene and occasional deletion of the gene for the C delta 1 domain.

Mature, resting rodent, and primate B lymphocytes express two membrane Ig isotypes, IgM and IgD. Although membrane IgD production by these cells is regulated at a transcriptional level, and does not require deletion of the C mu gene, C mu has been deleted in all of the IgD-secreting tumor cells that have been studied. These IgD-secreting tumors, which include two mineral oil-induced plasmacytomas and three IgD-switch variants of an IgM-secreting hybridoma, might not, however, be representative of the rare IgD-secreting cells generated in response to an immune stimulus. A recent study of mice injected with a goat antibody to mouse IgD has demonstrated the generation of a relatively large secretory IgD response in these animals. We have now produced hybridomas by fusing spleen cells from these mice with a non-Ig-secreting plasmacytoma. Two of these hybridomas, KWD-1 and KWD-2, secrete IgD and express cell membrane IgD. Both of these hybridomas were found to have deleted the C mu gene. KWD-2 produces a delta-chain mRNA and a delta-chain protein similar in size to those previously reported for normal secreted mouse IgD; however, KWD-1 synthesizes a secretory delta-chain mRNA that is approximately 0.25 kb smaller than the KWD-2 secretory delta-chain mRNA and secretes IgD with a delta-chain that is approximately 21 kDa smaller than the secretory delta-chain of KWD-2. ELISA studies with epitope-defined anti-delta mAb indicate that KWD-2 has both delta Fc (C delta 3) [corrected] and delta Fd (C delta 1) [corrected] determinants, whereas KWD-1 has delta Fc but not delta Fd. These studies also demonstrate that the Ag-binding site of KWD-1 is not deleted because KWD-1 specifically binds goat IgG. Northern blot analyses with exon-specific probes indicate that while both KWD-1 and KWD-2 synthesize kappa-chain mRNA and delta-chain mRNA that includes the VH, C delta hinge, and C delta 3 exons, the C delta 1 exon is present only on the KWD-2 delta-chain mRNA. Southern blot analysis confirms that the C delta 1 exon has been deleted in KWD-1, but not KWD-2. We have previously noted that a secretory delta-chain mRNA that is similar in size to that produced by KWD-1 accounts for approximately 25% of the splenic secretory delta-chain mRNA produced by goat anti-mouse IgD antibody-injected mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

DNA repair in the c-myc locus.

We have studied DNA repair after UV damage in the murine c-myc locus. It appears that a region in B cells upstream of the murine c-myc gene is repaired with a different efficiency in plasmacytoma-resistant DBA/2N mice than in plasmacytoma-susceptible BALB/cAn mice. The region just upstream of c-myc is inefficiently repaired in B lymphoblasts derived from BALB/cAn mice. In contrast, this same region of c-myc is efficiently repaired in B lymphoblasts derived from DBA/2N mice. DNA fragments located in the coding region of c-myc and in another gene, dihydrofolate reductase (DHFR), are repaired with equal efficiency in cells from these two strains of mice. It is possible that repair efficiency of the 5' flank of c-myc may be involved in tumor susceptibility of the mouse strain.

Animals↗
PubMed1990

Pvt-1 transcripts are found in normal tissues and are altered by reciprocal(6;15) translocations in mouse plasmacytomas.

The mouse Pvt-1 (for plasmacytoma variant translocation) region maps to a chromosome 15 breakpoint region that is frequently interrupted by "variant" reciprocal chromosome translocations, rcpt(6;15), in plasmacytomas. This region lies several hundred kilobases (kb) 3' of the mouse c-myc gene (Myc) which is deregulated in both rcpt(6;15) and rcpt(12;15) plasmacytomas. rcpt(12;15) translocations apparently activate c-myc directly by interrupting the gene itself, but the mechanism causing c-myc deregulation in tumors bearing rcpt(6;15) translocations remains unknown. The indirect activation of c-myc by Pvt-1 interruption has remained an appealing possibility, but heretofore it has not been possible to establish such a connection. Furthermore, no genes from the Pvt-1 locus have been shown to be transcribed in normal tissues or in tumors with rcpt(6;15) translocations. We report the isolation of a cDNA clone, Pvt-1-1, from mouse spleen mRNA that is specific to the Pvt-1 region. This cDNA probe detects low levels of large (ca. 14 kb) RNA transcripts in normal mouse tissues. In plasmacytomas with rcpt(6;15) translocations, the Pvt-1 transcripts are elevated in abundance and truncated in size. Both changes are apparently induced by the chromosomal translocation. Expression of 14-kb Pvt-1 RNA is elevated in B-cell tumor lines that express immunoglobulin light chain genes; thus, we postulate that these translocations are facilitated by the increased DNA accessibility of immunoglobulin kappa light chain and Pvt-1 genes when they are simultaneously expressed at certain times during B-cell ontogeny.

Amino Acid Sequence↗
PubMed1990

Alternative splicing of RNAs transcribed from the human c-myb gene.

An alternative splicing event in which a portion of the intron bounded by the vE6 and vE7 exons with v-myb homology is included as an additional 363-nucleotide coding exon (termed E6A or coding exon 9A) has been described for normal and tumor murine cells that express myb. We show here that this alternative splicing event is conserved in human c-myb transcripts. In addition, another novel exon (termed E7A or coding exon 10A) is identified in human c-myb mRNAs expressed in normal and tumor cells. Although the myb protein isoform encoded by murine E6A-containing mRNA is larger than the major c-myb protein, the predicted products of both forms of human alternatively spliced myb transcripts are 3'-truncated myb proteins that terminate in the alternative exons. These proteins are predicted to lack the same carboxy-terminal domains as the viral myb proteins encoded by avian myeloblastosis virus and E26 virus. The junction sequences that flank these exons closely resemble the consensus splice donor and splice acceptor sequences, yet the alternative transcripts are less abundant than is the major form of c-myb transcripts. The contribution that alternative splicing events in c-myb expression may make on c-myb function remains to be elucidated.

Amino Acid Sequence↗
PubMed1990

v-myc and v-raf act synergistically to induce B-cell tumors in pristane-primed adult BALBC mice.

In a variety of systems, evidence is accumulating which suggests that neoplastic transformation requires the action of two or more genes such as mutated or over-expressed proto-oncogenes. To determine whether the cytoplasmic serine/threonine kinase oncogene raf could complement a deregulated myc gene and induce tumors in adult mice, BALBC mice were primed with an intraperitoneal (ip.) injection of mineral oil (pristane) and then given an ip. injection of a retroviral construct, J1, J2 or J5, which expresses either v-raf (J1), v-myc (J5) or both (J2). The J1 virus induced no tumors in 150 days in 38 mice, except for 5 helper virus-associated T-cell lymphomas. Under identical conditions the J5 virus, which expresses only v-myc, induced exclusively monocytic neoplasms in 93% of 15 mice. The J2 virus expresses both v-myc and v-raf and caused equal numbers of monocytic and B cell tumors in 66% of 30 mice. Under these conditions, it appears that v-raf expression acts synergistically with v-myc to induce the transformation of B cells, which neither oncogene could do alone. The J3 virus, which originally contained a complete v-myc and an inactivated v-raf, can induce tumors of later stage B cells (plasmacytomas, Potter et al., 1987). Recent studies of virus recovered from these plasmacytomas (called the J3V1 virus, Troppmair et al., 1989) show that the J3 virus has undergone deletions which have reactivated v-raf in a mutated form. Only J3V1, not J3, induced tumors in vivo. Our data presented here corroborate Troppmair et al. and extend Potter et al. (1987) which reported that J3 (presumably J3V1) induced 10% myeloid tumors and 90% plasmacytomas. In light of the discovery, our J2 and J3 data indicate that in combination with the same form of v-myc, different forms of v-raf induce different spectra of tumors.

Animals↗
PubMed1989

The human immunoglobulin C mu-C delta locus: regulation of mu and delta RNA expression during B cell development.

Whether the immunoglobulin (Ig) heavy chain genes C mu and C delta are expressed singly or in combination, their transcripts undergo differentiation-specific alterations in membrane (M) versus secreted (S) forms as well as in abundance. To better understand this regulation, we have cloned cDNAs for human delta m and delta s to establish the 3' end of the C mu-C delta transcription unit. Steady state mRNA levels and transcription rates were then analyzed in normal and transformed human B cells representing different maturation and activation states. The ratio of micron/microsecond RNA and of delta m/delta s RNA correlated with developmental stage, with a higher ratio at earlier stages. Steady state ratios of total mu/delta RNA paralleled ratios of C mu/C delta nascent transcription, suggesting no major posttranscriptional control for differential expression. However, at all developmental stages, transcription termination occurred downstream of the micron exons, suggesting a strong posttranscriptional regulatory component for production of secreted versus membrane forms of mu RNA. The relative abundance of mature delta S RNA was considerably higher in the human than in the mouse, correlating with the increased levels of circulating IgD in the former species. Stimulation of human splenocytes with mitogens did not increase delta RNA; in fact, splenocytes activated with pokeweed mitogen were nearly devoid of delta RNA, and Staphylococcus aureus Cowan I caused only a minor change.

Amino Acid Sequence↗
PubMed1989

Altered myc gene transcription and intron-induced stabilization of myc RNAs in two mouse plasmacytomas.

Accumulation of unusually high amounts of larger-than-normal c-myc mRNAs occurs in two mouse plasmacytomas, TEPC 1165 and TEPC 2027. Southern blot and DNA sequence analyses showed that both tumors have undergone translocations of immunoglobulin heavy chain loci to positions 5' of the c-myc gene promotors resulting in removal of DNA sequences encoding a negative transcriptional regulatory element. In contrast to other mouse plasmacytomas, TEPC 1165 and TEPC 2027 rearranged myc genes show increased transcription, partially explaining their abundance of myc RNA. Similar to other mouse plasmacytomas, the abundance of myc RNA in TEPC 1165 and TEPC 2027 is also influenced by increased stability of structurally atypical myc RNAs. Two myc mRNAs are found in TEPC 2027, a 2.4 kb species including all 3 myc exons and a 4.0 kb species with the 3 exons plus the first intron. The two major myc mRNAs in TEPC 1165, 3.0 and 3.9 kb species, also include all three myc exons plus portions of the first intron. S1 nuclease protection analyses show that the 5' initiation and 3' untranslated (UT) regions of the unusual TEPC 1165 RNAs are normal showing that the size differences arise solely from inclusion of first intron sequences in the large myc RNAs. DNA sequence analysis showed that the presence of first intron sequences in the large myc RNAs is due to mutations affecting the splice donor region at the 3' end of exon 1 in both tumors. SDS-PAGE analysis of immunoprecipitated TEPC 1165 and TEPC 2027 myc proteins showed them to be of normal electrophoretic mobility but no more abundant than in a pre-B cell line 18-81 that contains at least 10 fold less myc RNA. The 4.0 kb myc mRNA of TEPC 2027 is atypically stable while the 2.4 kb myc mRNA undergoes normal rapid turnover within the same cell, demonstrating that the presence of first intron sequences in the large myc RNA stabilizes it despite the presence of 3' UT and putative exon 1 destabilizing sequences. These results show that myc intron 1 sequences can counteract the effect of 3' UT region destabilizing sequences in myc RNA and suggest that the increased myc RNA stability noted in TEPC 1165 and TEPC 2027 is largely due to the presence of the intron 1 sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

A chronic inflammatory response. Its role in supporting the development of c-myb and c-myc related promonocytic and monocytic tumors in BALB/c mice.

This study demonstrates that an inflammatory response caused by the injection of pristane into the peritoneal cavity of mice provides a useful system for rapid induction of myeloid tumors by retroviruses. Two such tumors, which developed in the peritoneal cavity with average latencies of 68 to 71 d and incidences of greater than 50%, are 1) the McML, mature monocyte-macrophage tumors induced by retroviral constructs containing exons 2 and 3 of c-myc cDNA, and 2) the MML, promonocytic tumors induced by Moloney murine leukemia virus infection and its integration into the c-myb locus. Development of both neoplasms is clearly dependent on the intense i.p. inflammatory response, inasmuch as mice given the viruses and not pristane fail to develop these tumors. Although both types of tumors appear in the peritoneal cavity, the MML tumors that arise by i.v. injection of Moloney murine leukemia virus may actually originate via infection, and perhaps transformation, of precursor hemopoietic cells outside the peritoneal cavity, followed by migration of the cells to the peritoneal cavity. This is suggested by the fact that i.v.v but not i.p. injection of virus is an efficient method of producing these particular myeloid tumors. Although both McML and MML tumors require the inflammatory environment for their development, treatment of mice with a nonsteroid anti-inflammatory drug, indomethacin, has no effect on McML monocyte/macrophage tumors but completely prevents the development of the MML promonocyte tumors.

Animals↗