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D Morello

Publications and source records attributed to D Morello.

At least 37 records · Page 2Linked to original sources
PubMed1994

The cis-acting elements known to regulate c-myc expression ex vivo are not sufficient for correct transcription in vivo.

Much of our knowledge about the regulation of the c-myc proto-oncogene expression has come from studies of c-myc gene expression in several well defined ex vivo systems, including differentiation systems and tumor cells. However, very few investigations have been performed to determine the factors and cis-acting sequences that regulate c-myc expression in vivo. In order to obtain information on the sequences required to regulate c-myc gene transcription from the two major P1 and P2 initiation sites in the mouse, we have generated several constructs containing human or murine c-myc genomic sequences with various 5' flanking sequences and derived corresponding transgenic mice. A sensitive S1 nuclease protection assay was performed to analyse and to compare transgene expression with that of the endogenous c-myc mRNA, either in adult organs, or during development. None of the transgenic mice expressed the construct appropriately, although several strains exhibited unexpected expression most probably due to position effects. Our results indicate that the cis-acting elements described to regulate c-myc expression ex vivo are not sufficient to drive the correct expression of c-myc gene in vivo and strongly suggest that additional regulatory elements located upstream from -3500 (with respect to mouse P1 promoter) and downstream 1500 bp from polyadenylation sites are required.

Animals↗
PubMed1993

Transcriptional control of MHC class I and beta 2-microglobulin genes in vivo.

The expression of class I and beta 2-microglobulin (beta 2-m) genes, which encode the H and L chains of the H-2 histocompatibility Ag, respectively, is complex both in the adult mouse and during development. Although they are ubiquitously expressed in the adult, the mRNA levels of these genes are variable from one organ to another, being high in liver, lung, and lymphoid organs and low in brain and testis. During development, both class I and beta 2-m mRNA are poorly expressed. To determine the molecular mechanism, either transcriptional or post-transcriptional, controlling class I and beta 2-m mRNA levels, we have compared their transcriptional activities by performing run-on experiments with nuclei extracted from several embryonic and adult organs. We show that most of the differences observed in H-2 and beta 2-m mRNA steady state levels are the reflection of their different transcriptional activities. These results demonstrate that MHC class I and beta 2-m gene expression in adult organs, as well as during development, is mainly controlled at the transcriptional level.

Animals↗
PubMed1993

H-2/myc, E mu/myc, and c-myc transgenic mice: potent sources of early hematopoietic cell lines.

Activation of the cellular c-myc oncogene appears to be linked to the development of malignancies in a variety of tissues, in particular, hematopoietic tumors. In an attempt to obtain cell lines corresponding to early stages of lymphoid differentiation, we have derived several strains of transgenic mice in which human c-myc genomic sequences were coupled to various regulatory sequences, H-2, E mu, and c-myc. The class I H-2Kb 5' region is active in most adult tissues; the activity of the enhancer E mu of the heavy chain immunoglobulin is restricted to lymphocytes; and the 2-kilobase-long c-myc 5' region has been shown by in vitro experiments to contain most of the sequences required for c-myc regulated expression. Regardless of the construct, H-2/myc, E mu/myc, or c-myc, the transgenes were mainly expressed in lymphoid tissues. However, the quantity of transgene mRNA differed markedly among the transgenic strains, thus providing a situation in which the level of c-myc expression could be correlated with tumor development. Several cell lines were derived and maintained by in vivo transplantation and/or in vitro cultures. All except two of the lines correspond to immature T- or B-lymphoblasts, as revealed both by fluorescence-activated cell sorting and Southern blot analysis; one cell line corresponds to a gamma delta T-cell population, and the other to a bipotential mixed lymphoid and myeloid tumor.

Animals↗
PubMed1993

The 5' and 3' non-coding sequences of the c-myc gene, required in vitro for its post-transcriptional regulation, are dispensable in vivo.

We have previously shown that in vivo the steady-state level of c-myc mRNA in different quiescent organs and its induction in the early stages of hepatic regeneration and after inhibition of protein synthesis are mainly controlled by post-transcriptional mechanisms. In order to localize the target sequences for these mechanisms, transgenic lines expressing various versions of the human c-myc proto-oncogene have been constructed. To avoid all possible transcriptional controls due to the c-myc 5' regulatory region, the c-myc genomic sequences were fused to MHC H-2Kb class I regulatory sequences, which have previously been shown to be able to drive reporter gene expression in most adult tissues. The transgenes contained either all human c-myc genomic sequences or were deleted for one of the sequences which have been shown in in vitro experiments to play a role in c-myc mRNA stabilization, in particular exon 1, intron 1 and the 3' non-coding region. Several independent transgenic lines were derived for each construct. Using S1 nuclease protection analysis, we have monitored H-2K, mouse c-myc and transgene mRNA expression in several quiescent adult organs, at the start of liver regeneration and after inhibition of protein synthesis in each transgenic line. Our results indicate that the 5' non-coding sequences, including exon 1 and intron 1, and the 3' untranslated region are all dispensable in the different aspects of c-myc post-transcriptional regulation.

Animals↗
PubMed1992

Different regulation of class I gene expression in the adult mouse and during development.

The expression of the class I genes encoding for histocompatibility Ag is complex both in adult and during development. Although ubiquitously expressed in the adult, the mRNA level of class I genes is variable from one organ to another. During development, H-2K mRNA expression has two phases: the first from blastocyst to day 11, where H-2K mRNA level is extremely low, and the second, beginning after day 11, when H-2K mRNA expression increases first dramatically (10x) and then progressively to birth. To localize the sequences responsible for the regulation of H-2K gene expression in the adult and during development, we have constructed a series of transgenic strains carrying 1) a 9-kb native H-2K gene, H-2K LF, corresponding to the entire H-2Kb gene with 2 kb of upstream sequences and 3 kb of downstream sequences, and 2) two hybrid constructs linking the same 5'-flanking region of H-2Kb gene to two reporter genes, the human growth hormone and the human c-myc proto-oncogene. Expression of the transgenes was compared with that of the endogeneous H-2K gene in adult organs and during development of the different transgenic strains. In the adult, the three constructs behave almost like the endogeneous H-2K gene, but the H-2K LF construct is the only one whose expression is independent of the integration site and related to the copy number. During development, both fusion genes are barely expressed in the embryo as well as in the extra-embryonic tissues, whereas the H-2K LF transgene expression parallels that of the endogeneous class I gene. Therefore, our results show that H-2K developmental regulatory sequences are not included in the region that controls H-2K mRNA expression in the adult, indicating that H-2K class I gene expression in adult organs and in development is regulated by different mechanisms.

Animals↗
PubMed1992

Unexpected position-dependent expression of H-2 and beta 2-microglobulin/lacZ transgenes.

In order to study sequences involved in the developmentally regulated and tissue-specific expression of the class I Major Histocompatibility Complex (MHC) genes, we have constructed several H-2/lacZ transgenic lines in which the 5' regulatory sequences of the H-2Kb gene are linked to the Escherichia coli beta-galactosidase (lacZ) gene. In five H-2/lacZ lines, the pattern of lacZ expression, detected histochemically varied greatly from line to line. None of the H-2/lacZ transgenes were transcribed in cells normally expressing a high level of endogenous H-2 molecules, although these H-2 regulatory sequences have been shown to be sufficient to drive tissue-specific expression of other reporter genes. Interestingly, when constructs containing 5' beta 2-microglobulin (beta 2m) regulatory sequences linked to lacZ were used to derive transgenic lines, similar results were obtained. A survey of lacZ labeling in H-2/lacZ and beta 2m/lacZ transgenic mice strongly suggests that these transgenes are very sensitive to position effect, lacZ expression being controlled by endogenous chromosomal regulatory elements specific for each insertion site. Here we describe the complex pattern of lacZ expression in the different transgenic lines during development; we discuss the unusual properties of these transgenes and underline their potential use for developmental studies and characterization of genomic sequences involved in spatiotemporal gene expression.

Animals↗
PubMed1990

c-myc, c-fos, and c-jun regulation in the regenerating livers of normal and H-2K/c-myc transgenic mice.

We investigated the mechanisms of regulation of c-myc, c-fos, and c-jun at the early stages of liver regeneration in mice. We show that the transient increase in steady-state levels of c-myc mRNA at the start of liver regeneration is most probably regulated by posttranscriptional mechanisms. Although there was a marked increase in c-myc transcriptional initiation shortly after partial hepatectomy, a block in elongation prevented the completion of most transcripts. To gain further information on the mechanism of regulation of c-myc expression during liver regeneration, we used transgenic mice harboring the human c-myc gene driven by the H-2K promoter. In these animals, the murine c-myc responded to the growth stimulus generated by partial hepatectomy, whereas the expression of the transgene was constitutive and did not change in the regenerating liver. However, the mRNA from both genes increased markedly after cycloheximide injection, suggesting that the regulation of c-myc mRNA abundance in the regenerating liver differs from that occurring after protein synthesis inhibition. Furthermore, we show that in normal mice c-fos and c-jun mRNA levels and transcriptional rates increase within 30 min after partial hepatectomy. c-fos transcriptional elongation was restricted in nongrowing liver, but the block was partially relieved in the regenerating liver. Nevertheless, for both c-fos and c-jun, changes in steady-state mRNA detected after partial hepatectomy were much greater than the transcriptional increase. In the regenerating liver of H-2K/c-myc mice, c-fos and c-jun expression was diminished, whereas mouse c-myc expression was enhanced in comparison with that in nontransgenic animals.

Animals↗
PubMed1990

Differential regulation and expression of jun, c-fos and c-myc proto-oncogenes during mouse liver regeneration and after inhibition of protein synthesis.

In order to obtain information in vivo about the possible relationships between early response gene products, we have analysed the expression of c-myc, c-fos and jun proto-oncogenes in regenerating mouse liver. We show that c-myc, c-fos, jun B, c-jun and jun D mRNA expression is transiently increased soon after partial hepatectomy, jun and fos expression being induced earlier (30 min) than that of c-myc (1-2 h). C-fos, jun B and c-jun mRNA expression is dramatically enhanced (50 fold) while that of jun D and c-myc is weaker (less than 10 fold), but lasts longer. Moreover, the relative contributions of transcriptional and post-transcriptional regulations are unique for each proto-oncogene analysed. These results suggest that following the growth signal delivered by partial hepatectomy, the five proto-oncogenes analysed are all involved in the progression of hepatocytes through G1; however, due to their differential regulation and kinetics, they might play different roles in the changes in gene expression that occur during the transition from quiescence to proliferation. When protein synthesis is inhibited by injection of cycloheximide, the expression of c-myc, c-fos, jun B, c-jun and jun D mRNA is also transiently increased. Although this increase is mainly due to post-transcriptional mechanisms, c-myc, c-jun, jun D and, to a lesser extent, jun B transcription is enhanced, suggesting that labile repressor-like molecules may inhibit transcription of these genes in the quiescent liver. Moreover, the kinetics of c-myc, c-fos and jun mRNA induction are not identical, showing that different components are involved in their turnover or stability.

Animals↗
PubMed1989

Adenovirus type 12 E1A down regulates expression of a transgene under control of a major histocompatibility complex class I promoter: evidence for transcriptional control.

The E1 region of human adenovirus type 12 (Ad12 E1) represses the expression of major histocompatibility complex (MHC) class I genes in transformed primary rodent cells. Conflicting results have been reported as to whether this E1A-mediated repression occurs at a transcriptional or a posttranscriptional level. In the present study, we show that in Ad12 E1-transformed primary baby mouse kidney cells from transgenic mice harboring the human growth hormone gene under control of an H-2K promoter both the expression of the endogenous MHC class I genes and the expression of the transgene are repressed. This experiment, as well as nuclear run-on analyses performed with single-stranded probes, revealed that Ad12 E1A inhibits MHC class I gene expression by repressing its promoter.

Adenovirus Early Proteins↗
PubMed1989

Transgenic mice.

Stable integration into the mouse genome of exogenous genetic information has become, over the past few years, a very potent approach for different aspects of biology. It is a common feature that the integrated exogenous gene (the transgene) is expressed properly both spatially and temporally. Constructing different lines of transgenic mice carrying various versions of a gene, therefore, permits cis acting DNA sequences involved in the specificity of expression to be defined, in the context of the developing animal. This in turn opens the way to a variety of experiments in which a given gene product is targeted to one or another cell type, thus offering some insight into the physiological role of this product. Such a strategy has been used, for example, to address the questions of the role of oncogenes in malignant transformation. The insertion of foreign DNA per se may disrupt the function of endogenous genes, thus creating an insertional mutation. The corresponding affected genes may subsequently be cloned, using the transgene as a tag. Finally, the ability to perform homologous recombination, recently demonstrated with embryonic stem cells that can colonize the germ line of a foreign embryo, should constitute in the near future a unique way to analyse in detail the functioning of the mammalian genome.

Animals↗
PubMed1989

Lymphoproliferative syndrome associated with c-myc expression driven by a class I gene promoter in transgenic mice.

We have produced transgenic mice carrying an H-2K/human c-myc fusion gene. In this construct, the human c-myc proto-oncogene expression is driven by the 5' flanking sequences (including promoter) of the class I H-2Kb gene, which have previously been shown to direct the expression of a marker gene, the human growth hormone (hGH), in most tissues of H-2K/hGH transgenic mice. Comparative analysis, by S1 nuclease mapping, of the H-2K and human c-myc gene expression in different organs of the H-2K/myc mice shows that exogenous c-myc and endogenous H-2K expression is found in most organs examined. However, the liver is a notable exception, for here c-myc expression is very weak. The exogenous c-myc expression is maximal in lymphoid organs of all H-2K/myc transgenic strains. One strain, H-2K/myc 27, hereditarily develops a lymphoproliferative syndrome which eventually leads to death. The H-2K/myc 27 lymphoid tissues are profoundly abnormal: pre-B cells as well as mature B cells are underrepresented in the bone marrow but the thymus as well as lymph nodes are largely infiltrated by B cells. Moreover, in the thymus, the proportions of the different thymic cell populations are altered. However, in the other H-2K/myc transgenic strains, even in those expressing a comparable or even higher level of myc, no pathology has been observed over a period of 20 months. Our results, therefore, demonstrate that constitutive enforced c-myc expression might disturb lymphocyte development, but does not directly lead to malignancy.

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

[(Proto)-oncogenes, cellular growth and development].

In the past few years, one realizes that number of genes whose products are involved in regulating normal cell growth and development are also capable of inducing malignancy. These genes are called oncogenes and include 1 degree the cellular oncogenes (c-onc) or proto-oncogenes and 2 degrees the viral oncogenes (v-onc) which are most likely derived from c-onc and which are the transforming genes of several strains of animal retrovirus. The c-onc are present in the genome of all vertebrate cells and show a remarkable degree of evolutionary conservation, suggesting that they serve essential cellular functions. The analysis of biological properties of both types of oncogenes is of importance to understand the role of proto-oncogenes in normal cell proliferation and/or differentiation and development and to determine how alterations in their structures play a role in malignant transformation. In order to give an overview of the diversity of oncogene activities, several oncogene products involved in growth control, signal transduction, gene expression and development will be described.

Animals↗
PubMed1986

Myelin proteolipid protein (PLP and DM-20) transcripts are deleted in jimpy mutant mice.

The myelin-associated proteolipid protein, PLP, is one of the two major components of the central nervous system (CNS) myelin. We analyze, by using a rat PLP cDNA and S1 nuclease protection experiments, the PLP transcripts in the mouse brain and show that the PLP gene encodes two different but related mRNA transcripts, the PLP and the DM-20 transcripts. On the other hand, we demonstrate that in the jimpy mutant, which is characterized by an abnormal CNS myelination, both these transcripts are partially deleted in the 3' end of their coding region. The deletion is 70 (+/- 5) nucleotides long. Implications of this finding for the synthesis of PLP and DM-20 proteins in the mutant are discussed.

Animals↗
PubMed1986

Transhybridomas from fusion gene transgenic lymphocytes can be used to produce foreign protein of biological interest.

A method is described for achieving the efficient production of proteins of biological interest by the establishment of hybridomas from lymphocytes of transgenic animals carrying a fusion gene having promoter-regulatory sequences functional in lymphocytes fused to the coding sequences of the protein to be produced. While possible improvements in the method are discussed, it is anticipated that the method will ultimately be applicable to the production of any protein of interest.

Animals↗
PubMed1986

Studies on the expression of an H-2K/human growth hormone fusion gene in giant transgenic mice.

Transgenic mice carrying the H-2K/human growth hormone (hGH) fusion gene were produced by microinjecting into the pronucleus of fertilized eggs DNA molecules containing 2 kb of the 5' flanking sequences (including promoter) of the class I H-2Kb gene joined to the coding sequences of the hGH gene. Thirteen transgenic mice were obtained which all contained detectable levels of hGH hormone in their blood. Nine grew larger than their control litter-mates. Endogenous H-2Kb and exogenous hGH mRNA levels were analysed by S1 nuclease digestion experiments. hGH transcripts were found in all the tissues examined and the pattern of expression paralleled that of endogenous H-2K gene expression, being high in liver and lymphoid organs and low in muscle and brain. Thus 2 kb of the 5' promoter/regulatory region of the H-2K gene are sufficient to ensure regulated expression of hGH in transgenic mice. This promoter may therefore be of use to target the expression of different exogenous genes in most tissues of transgenic mice and to study the biological role of the corresponding proteins in different cellular environments.

Animals↗
PubMed1985

Specific expression of hepatitis B surface antigen (HBsAg) in transgenic mice.

Two transgenic mice were obtained that contain in their chromosomes the complete hepatitis B virus (HBV) genome except for the core gene. These mice secrete particles of HBV surface antigen (HBsAg) in the serum. In one mouse, HBV DNA sequences that had integrated at two different sites were shown to segregate independently in the first filial generation (F1) and only one of the sequences allowed expression of the surface antigen. Among these animals the males produced five to ten times more HBsAg than the females. A 2.1-kilobase messenger RNA species comigrating with the major surface gene messenger RNA is expressed specifically in the liver in the two original mice. The results suggest that the HBV sequences introduced into the mice are able to confer a tissue-specific expression to the S gene. In addition, the HBV transgenic mice represent a new model for the chronic carrier state of hepatitis B virus infection.

Animals↗