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K B Marcu

Publications and source records attributed to K B Marcu.

At least 55 records · Page 3Linked to original sources
PubMed1989

Mode of c-myc gene regulation in folic acid-induced kidney regeneration.

We have examined the contributions of transcriptional and post-transcriptional control mechanisms for the regulated activation of the c-myc proto-oncogene in regenerating kidney tissue in vivo. The c-myc gene is activated in kidney tubule cells which are induced to proliferate as a consequence of folic acid-induced renal injury in vivo. We show that c-myc transcriptional initiation is induced in a co-ordinate fashion with a strong block to transcriptional elongation within the gene's first exon. Therefore, transcription is not effectively induced throughout the c-myc gene in these regenerating renal cells. In contrast, initiation of c-fos gene transcription was induced while the degree of transcriptional blockage within the gene's first exon remained constant. The steady-state levels of myc transcripts, originating from the gene's P1 and P2 start sites, accumulated to very high levels within 4-6 hr which were maintained for at least 24 hr after folic acid treatment. We conclude that post-transcriptional control mechanisms are largely responsible for the dramatic induction of c-myc mRNAs in regenerating kidney tissue in vivo.

Animals↗
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↗
PubMed1989

Tissue-specific post-transcriptional regulation of c-myc expression in normal and H-2K/human c-myc transgenic mice.

We show that the steady-state levels of c-myc mRNAs vary considerably in different organs of normal adult mice, maximal expression being observed in lymphoid organs and minimal expression in liver and brain. Nuclear run-on analysis of c-myc gene transcription in adult liver and spleen reveals that the difference in c-myc gene expression in these two organs is due to differential post-transcriptional control. Moreover, these nuclear run-on assays indicate that no premature termination of c-myc gene transcription takes place in the nuclei of the three adult tissues analysed. In fetal liver development, we observe a decrease in c-myc mRNA, but this is not due to changes in transcriptional activity implicating post-transcriptional regulatory mechanisms. Our studies of c-myc gene expression in organs of H-2K/myc transgenic mice, harboring an H-2K promoter driven human c-myc gene, confirm that the in vivo c-myc regulation is mainly post-transcriptional and shows that sequences shared by the murine and human c-myc proto-oncogenes are involved in this control.

Animals↗
PubMed1988

The joining of germ-line V alpha to J alpha genes replaces the preexisting V alpha-J alpha complexes in a T cell receptor alpha, beta positive T cell line.

To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCR alpha and beta genes in the progeny of the CD3+, CD4-/CD8- M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing V alpha-J alpha rearrangements by joining an upstream V alpha gene to a downstream J alpha segment. Both the productively and nonproductively rearranged alleles in the M14T line can undergo secondary rearrangements while its TCR beta genes are stable. These secondary recombinations are usually productive, and new forms of TCR alpha polypeptides are expressed in these cells in association with the original C beta chain. Developmental control of this V alpha-J alpha replacement phenomenon could play a pivotal role in the thymic selection of the T cell repertoire.

Alleles↗
PubMed1988

Abnormal recombination of Igh D and J gene segments in transformed pre-B cells of scid mice.

Studies of Ig and TCR genes in transformed lymphocytes of scid mice have revealed aberrant DNA rearrangements. Here we present a more detailed analysis of the Igh gene recombination in nine scid pre-B cell lines transformed by Abelson murine leukemia virus. We found 85% of the rearranged Igh alleles to contain abnormal Dh-Jh deletions of varying size. All of these deletions encompassed Jh elements and extended into the Igh enhancer region, occasionally involving the switch (S) region of the C mu gene. Some of these rearrangements removed most of the Dh elements, but none appeared to extend to the Vh genes. DNA sequence analysis of the two abnormally rearranged Igh alleles in one pre-B cell line showed that no Dh or Jh coding sequences were retained at the recombination sites though heptamer-like (CACTGTG) recognition signal sequences were present in the absence of nonamer (GGTTTTTGT) recognition signal sequences. These results imply that a deregulated recombinase activity may be responsible for the abnormal Dh-Jh deletions and the absence of Vh-Dh joining in established lines of Abelson murine leukemia virus-transformed scid pre-B cells.

Animals↗
PubMed1988

Rearrangement and expression of T cell receptor and immunoglobulin loci in immortalized CD4-CD8- T cell lines.

Injection of newborn mice with mixtures of wild-type moloney murine leukemia (Mo-MuLV) virus and other recombinant retroviruses harboring the myc oncogene alone or in combination with the H-ras oncogene resulted in a 100% incidence of lymphatic leukemias from which permanent cell lines could be established in vitro. These cells are immunoglobulin (Ig)-, Thy-1+BP- and CD8-CD4- indicating that they are early thymocytes. Such transformed pre-T lines lack retroviral myc and ras genes but occasionally possess proviral insertion near to their endogenous myc and pim genes. We show that both Ig heavy chain (Igh) and T cell receptor (TcR) genes are rearranged in most of these lines. In some cases, a primary recombination was followed by a secondary rearrangement at the same locus. We show that VT gamma genes can rearrange outside of their known cluster suggesting that TcR gamma diversification in such pre-T cells may be different to that in more mature T cells. Ig D-JH recombinations may precede TcR gene recombination in these early T cell lines, and some but not all express sterile Cmu transcripts. Some of these lines express surface heterodimers that appear composed of alpha and beta chains that can be immunoprecipitated with a monoclonal anti-T3 antibody but not with the anti-V beta 8 monoclonal antibody F23.1. This established pre-T cell line represents novel biological material for the dissection of T cell development and function analogous to A-MuLV transformed pre-B cells.

Animals↗
PubMed1988

Rapid induction of IgM-secreting murine plasmacytomas by pristane and an immunoglobulin heavy-chain promoter/enhancer-driven c-myc/v-Ha-ras retrovirus.

A retroviral vector, RIM, containing murine c-myc under the control of immunoglobulin heavy-chain gene promoter and enhancer elements and v-Ha-ras driven by a Moloney murine leukemia virus long terminal repeat induced IgM-secreting plasmacytomas in 28% of adult and 83% of 3-week-old pristane-conditioned mice with mean latency periods of 60-70 days. In contrast, the same vector only harboring c-myc or v-Ha-ras was virtually ineffective. RIM-induced plasmacytomas expressed retroviral myc and ras genes while their endogenous c-myc alleles were unrearranged and transcriptionally inactive. These plasmacytomas were clonal as each possessed a unique immunoglobulin heavy-chain joining region rearrangement and a single recombinant provirus. Moloney murine leukemia helper virus did not play an obligatory role in tumorigenesis since insertions of Moloney murine leukemia proviruses were found in only 6 of 24 plasmacytomas induced in adult mice. Taken together, these findings support the view that the v-Ha-ras oncogene can cooperate with an activated myc gene in pristane plasmacytomagenesis.

Animals↗
PubMed1988

Translocated c-myc genes produce chimeric transcripts containing antisense sequences of the immunoglobulin heavy chain locus in mouse plasmacytomas.

Immunoglobulin heavy chain gene antisense transcripts contribute to the expression of translocated c-myc genes in several murine plasma cell tumors. These novel, chimeric transcripts comprise 5-50% of steady-state c-myc mRNA. Two transcripts isolated as cDNA clones use the normal splice donor and acceptor sites within the c-myc first intron. Another cDNA clone has the potential for encoding two types of c-myc proteins. The significance of immunoglobulin heavy chain gene antisense transcripts and transcriptional competence of the immunoglobulin heavy chain locus for c-myc expression is discussed.

Animals↗
PubMed1988

Multiple negative elements upstream of the murine c-myc gene share nuclear factor binding sites with SV40 and polyoma enhancers.

We previously identified a 716 bp DNA segment, 424 to 1140 bp 5' of the murine c-myc gene, which exhibited the properties of a transcriptional 'dehancer' because it negated the effects of the SV40 enhancer (Remmers et al., 1986). Here, we show that this 716 bp 'dehancer region' is composed of multiple negative elements each of which functions at a distance of > 1 Kb to inhibit the SV40 enhancer. One of these negative elements displays lymphoid cell specificity. HeLa cell nuclear factor binding sites are found within these negative effector sequences. One of these binding sites is related to that of the Py EF-C transcription factor which recognizes a short dyad symmetry element within the polyoma enhancer. Interestingly, a dimer of the Py EF-C recognition sequence exhibited a strong 'dehancer effect' in the pSV2CAT expression vector. These negative elements may function by interfering with transcription factors required for SV40 enhancer activity.

Animals↗
PubMed1988

Two transient increases in c-myc gene expression during neuroectodermal differentiation of mouse embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cells of the P19 line can be induced to differentiate into neurons, astrocytes and fibroblasts by exposure to retinoic acid (RA), whereas treatment of the EC cells with dimethyl sulfoxide (DMSO) leads to differentiation into mesodermal tissues, including cardiac and skeletal muscle. In RA- but not DMSO-treated cultures, the level of c-myc mRNA underwent two transient increases. The concentration of c-myc mRNA in RA-treated cultures increased 3-fold after 3 h in the presence of the drug, returned to normal by 9 h, increased again 5-fold from 48 to 96 h, and finally decreased below pre-treatment values by 144 h. Increased levels of c-myc protein were observed at the times of elevated c-myc mRNA. Nuclear run-on assays of the c-myc gene and measurements of c-myc mRNA stability indicated that both transcriptional and post-transcriptional mechanisms contribute to the modulated expression of the c-myc gene. The two transient increases in c-myc expression suggest a role for the c-myc protein in the differentiation of cells along neuroectodermal lineages.

Animals↗
PubMed1987

Absence of somatic mutation in the variable region of MPC 11 variants expressing a different heavy chain isotype.

Somatic mutation of immunoglobulin variable regions contributes to the diversity of the immune response. Some investigators have postulated that somatic mutation is coupled to isotype switching; others have presented evidence that the two processes are not linked. By using in vitro variants of the MPC 11 mouse myeloma cell line that produce heavy chains of different isotypes, we have examined several VH regions at the nucleotide level using a variety of different sequencing procedures. We have found no evidence of somatic mutation and conclude that the processes of somatic mutation and isotype switching may be independent.

Animals↗
PubMed1987

Immunoglobulin heavy chain switch region recombination within a retroviral vector in murine pre-B cells.

We have employed a retroviral vector, ZN(Smu/S gamma 2b)tk1, as a substrate for detecting the presence of immunoglobulin heavy chain constant region (CH) gene switch (S) recombination activity in murine pre-B cells. ZN(Smu/S gamma 2b)tk1 contains a neomycin (neo) resistance gene in addition to the herpes simplex virus thymidine kinase (Htk) gene which is positioned between murine Smu and S gamma 2b sequences. Stable acquisition of the ZN(Smu/S gamma 2b)tk1 vector was selected in G-418 and switch region recombination within these proviruses was selected by resistance to the drug bromodeoxyuridine (BUdR). Fluctuation analyses of ZN(Smu/S gamma 2b)tk1 infected 18-8tk- and 38B9tk- pre-B lines revealed Htk gene inactivations with apparent frequencies of 5 X 10(-5) and 1 X 10(-5) events/cell/generation, respectively, while G-418 resistant Ltk- fibroblasts lost the HTK phenotype at an apparent rate of 4 X 10(-8). Southern blot analysis demonstrated that switch recombination caused the deletion of the Htk gene in all pre-B clones examined while the loss of Htk in Ltk- clones was not mediated by S region recombination. In 21 out of 24 pre-B clones, the recombinations involved the tandemly repetitive portions of the Smu and S gamma 2b sequences. These results demonstrate that the CH gene S region segments inserted into ZN(Smu/S gamma 2b)tk1 are sufficient for B-cell-specific recombination/deletion within the S region tandem repeats.

Animals↗
PubMed1987

Contributions of transcriptional and post-transcriptional mechanisms to the regulation of c-myc expression in mouse erythroleukemia cells.

Chemically induced differentiation of mouse erythroleukemia (MEL) cells leads to complex changes in c-myc mRNA levels. Within 1-2 hr after the addition of the inducer hexamethylene bisacetamide (HMBA), c-myc mRNA levels decrease 10- to 20-fold and remain low until 12-24 hr, at which time the mRNA reaccumulates to its original level. Thereafter as the cells undergo terminal differentiation, c-myc mRNA again declines to a low level. We have investigated the regulation of these changes by measuring c-myc gene transcription and mRNA turnover. We find that the early rapid decline in c-myc mRNA is due to an increase in the block to elongation of transcription within the c-myc first exon. Effective c-myc transcription is then restored after 2 hr of HMBA treatment to the level present in uninduced cells and is maintained throughout the remainder of the differentiation program. These results demonstrate that, except for the rapid decline in c-myc mRNA immediately following inducer treatment, all subsequent regulation of message levels occurs through post-transcriptional mechanisms. Studies of c-myc mRNA turnover suggest that some post-transcriptional regulation is nuclear.

Acetamides↗