Long Terminal Repeat Promoter Research Articles

Construction and functional characterization of mutants of the bovine leukaemia virus trans-activator protein p34tax.

The p34tax protein [p38tax, p34, p38(XBL), XBL-I] of bovine leukaemia virus (BLV) activates transcription from the BLV long terminal repeat (LTR) promoter. To analyse the functional properties of this protein, inframe insertions and internal deletions were systematically introduced in a plasmid-encoded copy of the p34tax gene. The abilities of wild-type and mutant genes to activate gene expression from the LTR promoter linked to the chloramphenicol acetyltransferase gene and to inhibit trans-activation by the wild-type protein were studied. The trans-activating activity of 14 of the 18 mutants tested was completely abolished, but four mutants each containing a lesion in the internal portion of the polypeptide retained activity. Taken together, these results suggest the presence of an internal region of the polypeptide where structural integrity is less strictly required for the functional activity of this protein. Among the mutants incompetent in the transactivation assay, only two with mutations in the N-terminal region of the polypeptide inhibited transactivation by the wild-type protein in a dose-dependent manner. These results facilitate understanding of the physiological function of the tax protein family.

Rapid activation and subsequent down-regulation of the human immunodeficiency virus type 1 promoter in the presence of Tat: possible mechanisms contributing to latency

The mechanism of induction of gene expression of the human immunodeficiency virus type 1 long terminal repeat (LTR) by the Tat transactivator protein was studied in a cell fusion assay. Tat causes a rapid activation of both transcription from the LTR and accumulation of hybrid LTR-chloramphenicol acetyltransferase mRNAs. Approximately 4 h after induction by Tat, expression from the LTR promoter is down-regulated, resulting in a decrease in the accumulation of LTR mRNA. This down-regulation of expression occurs in the continued presence of Tat. Protein synthesis inhibitors can block this down-regulation; therefore, the postinduction repression of expression is dependent upon de novo protein synthesis. We propose that a labile cellular protein(s) is responsible for the low levels of human immunodeficiency virus type 1 expression, possibly contributing to the establishment of a latent state of viral expression.

Mapping the transcriptional transactivation function of simian virus 40 large T antigen

T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T antigen. Mutants with alterations at various locations within the N-terminal 85 amino acids transactivated the RSV LTR promoter less well than did wild-type T antigen. Most of these were also partially defective in their ability to transactivate the SV40 late promoter. Two mutants with lesions in the DNA-binding domain that were unable to bind to SV40 DNA were completely defective for transactivation of both promoter, while a third mutant with a lesion in the DNA-binding domain which retained origin-binding activity transactivated both promoters as well as did wild-type T antigen. Only a low level of transactivation was seen with mutant T antigens which had lesions in or near the zinc finger region (amino acids 300 to 350). Mutations which caused defects in ATPase activity, host range/helper function, binding to p53, binding to the retinoblastoma susceptibility protein, or nuclear localization had little or no effect on transactivation. These results suggest that N-terminal portion of T antigen possesses an activation activity. The data are consistent with the idea that the overall conformation of T antigen is important for transactivation and that mutations in other regions that reduce or eliminate transactivation do so by altering the conformation or orientation of the N-terminal region so that its ability to interact with various targets is diminished or abolished.

HIV-1 indicator cell lines

A simple quantitative bioassay for infectious HIV-1 has been developed. The assay is based on adherent CD4+ HeLa cell lines stably transfected with episomal vectors carrying the Escherichia coli beta-galactosidase gene under the control of the HIV long terminal repeat (LTR) promoter. HIV infection of these cell lines transactivates the LTR promoter inducing beta-galactosidase production. Infected cells and virus foci can be stained dark blue by the addition of the chromogenic substrate X-Gal. Alternatively, a readily automatable quantitative enzyme assay can be performed on the infected cultures. Because of its simplicity the bioassay may be useful for routine quantification of HIV-infected cultures, plaque purification, virus neutralization studies and for the screening of antiviral agents.

Differences in replication and cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) are not determined by long terminal repeats (LTR)

The growth properties of molecular clones of a highly cytopathic Zairian HIV1-NDK and prototype viruses were compared to correlate genetic variations with biological changes. The cloned HIVi-NDK retained the highly replicating cytopathic phenotype and formed larger syncytia than the prototype. One of the major differences in the alignment of the nucleotide sequence of the HIVi-NDK and HIV1-BRU prototypes was localized in the negative regulatory element (NRE) of the long terminal repeat (LTR). In a chloramphenicol acetyl transferase (CAT) assay, we failed to detect a significant difference between LTR promoter activity of the prototype and HIVl-NDK, suggesting that the LTR of both phenotypes had a similar function. The complete recombinant provinus DNA molecules bearing HIVi LTR derived from one phenotype and the rest of the genomes from the other phenotype were constructed and transfected. The high cytopathogenicity of both the original and the chimeric viruses was correlated with the high speed of virus replication. Cytopathogenicity, morphology of syncytia, and replication kinetics of the recombinant viruses were determined by the functions coded within an internal part of HIVi genome, covering the gag to env region, which were, however, not within LTR.

Platelet-activating factor activates HIV promoter in transfected SH-SY5Y neuroblastoma cells and MOLT-4 T lymphocytes

Transfected gene constructs comprising the long terminal repeat (LTR) sequence of the human immunodeficiency virus (HIV) genome spliced to an assayable reporter gene have made possible the evaluation of a lipid mediator, platelet-activating factor (PAF), as a potential HIV transcriptional regulatory molecule. We assessed the activation of the HIV LTR promoter sequence linked to the chloramphenicol acetyltransferase (CAT) reporter gene (HIV-CAT) by PAF in both a human neural (SH-SY5Y neuroblastoma) and a human leukocytic (MOLT-4 T-lymphocyte) cell line. PAF activated expression of the HIV-CAT construct in both the SH-SY5Y and MOLT-4 T-cell lines. PAF-induced CAT activity was approximately six to seven times higher in the SH-SY5Y cells than in the MOLT-4 cells. Preincubation of cells with the specific PAF antagonist BN 52021 completely inhibited CAT expression in both cell lines. The biologically inactive PAF precursor lyso-PAF did not activate CAT expression. Assays for CAT mRNA demonstrated an increase after PAF treatment, an effect that was completely inhibited by BN 52021, and which was not elicited by lyso-PAF. These results show that PAF represents a potential cellular mediator evoking the expression of the HIV genome.

Exploitation of a Rapid and Sensitive Assay to Analyse Transactivation of the Human Immunodeficiency Virus type 1 (HIV-1) Long Terminal Repeat

Transregulation of the promoter within the 5′ long terminal repeat (LTR) of the human immunodeficiency virus (HIV) provirus determines the level of replication of HIV in latently, persistently or acutely infected cells. To measure rapidly the degree of transactivation of the HIV-1 LTR by various cellular and viral effectors, stably transformed cell lines containing integrated copies of the HIV LTR promoter (−122 to +80, relative to the major mRNA cap site) linked to the Escherichia coli lac Z gene were prepared by co-selection for pSV2 neo-mediated G418 resistance. One cell clone, RS 3/7, containing about 40 integrated copies of the recombinant LTR- lacZ gene was analysed further. RS 3/7 cells expressed high levels of β-galactosidase in response to co-transfection with plasmids expressing the HIV-1 transactivator, tat, infection with low multiplicities of herpes simplex virus type 1 (HSV-1), transfection with a plasmid expressing the HSV-1 immediate-early (IE) protein, ICPO, and by incubation with medium containing sodium butyrate. β-galactosidase activity was also induced by incubation of RS 3/7 cells with medium containing full length tat polypeptide. The cysteine analogue, D-penicillamine, previously reported as a potent inhibitor of tat-mediated transactivation (Chandra et al., 1988), was of limited efficacy in RS 3/7 cells transfected with tat-expressing plasmids. This cell line will be of value in identifying additional transactivators of the HIV-1 LTR, and in the selection of inhibitors of such effectors.

Sequence-specific DNA binding of the proto-oncoprotein ets-1 defines a transcriptional activator sequence within the long terminal repeat of the Moloney murine sarcoma virus.

The ets proto-oncogene family is a group of sequence-related genes whose normal cellular function is unknown. In a study of cellular proteins involved in the transcriptional regulation of murine retroviruses in T lymphocytes, we have discovered that a member of the ets gene family encodes a sequence-specific DNA-binding protein. A mouse ets-1 cDNA clone was obtained by screening a mouse thymus cDNA expression library with a double-stranded oligonucleotide probe representing 20 bp of the Moloney murine sarcoma virus (MSV) long terminal repeat (LTR). The cDNA sequence has an 813-bp open reading frame (ORF) whose predicted amino acid sequence is 97.6% identical to the 272 carboxy-terminal amino acids of the human ets-1 protein. The ORF was expressed in bacteria, and the 30-kD protein product was shown to bind DNA in a sequence-specific manner by mobility-shift assays, Southwestern blot analysis, and methylation interference. A mutant LTR containing four base pair substitutions in the ets-1 binding site was constructed and was shown to have reduced binding in vitro. Transcriptional efficiency of the MSV LTR promoter containing this disrupted ets-1 binding site was compared to the activity of a wild-type promoter in mouse T lymphocytes in culture, and 15- to 20-fold reduction in expression of a reporter gene was observed. We propose that ets-1 functions as a transcriptional activator of mammalian type-C retroviruses and speculate that ets-related genes constitute a new group of eukaryotic DNA-binding proteins.

The long terminal repeat region of the mouse mammary tumour virus contains multiple regulatory elements

Mouse mammary tumour virus (MMTV) is the major aetiologic agent of mouse mammary tumour formation. The expression of this virus is regulated by steroid hormones and cell type specific factors. The nucleotide sequence that controls the steroid hormone response has already been localized between -202 and -59 upstream of the start of transcription in the long terminal repeat (LTR) region of the proviral DNA. Through transfection experiments in three different cultured mouse cell lines (NIH3T3, NMuMG and GR), we have investigated which sequences in the MMTV LTR play a role in the cell type specific expression at the proviral promoter. We have identified two elements on the MMTV LTR from -631 to -560 and from -428 to -364 that have the potential to influence expression at the MMTV LTR promoter. The -631 to -560 element mediated a negative response in all the cell types we studied whereas the -428 to -364 element had negative effects in the mouse fibroblast NIH3T3 and the normal mouse mammary gland cell NMuMG but not in the mouse mammary tumour epithelial GR cells. The -428 to -364 element therefore contributes to the cell type specific expression at MMTV LTR promoter. We have also identified another regulatory element between -1094 and -739 that had a slight positive regulatory effect at the MMTV LTR promoter but greatly enhanced expression at a foreign promoter when present at this promoter in an orientation that is the reverse of its own orientation in the MMTV LTR. This orientation-dependent effect was only observed in the mouse mammary epithelial cells NMuMG and GR but not in mouse fibroblastic cell line NIH3T3. This element may be important in regulating the expression of neighbouring genes in a cell type specific manner. These results show that the MMTV LTR contains multiple regulatory elements necessary for the control of expression at its own promoter and the expression of neighbouring genes.

Isolation and analysis of a rat genomic clone containing a long terminal repeat with high similarity to the oncomodulin mRNA leader sequence

The structure of a novel long terminal repeat (LTR) from an intracisternal A particle (IAP) DNA element in the rat (Sprague-Dawley) genome was determined. This LTR has a total length of 313 base pairs (bp). Several structural features typical for retroviral LTR promoters were identified, including a "CCAAT" box, a "TATA" box, a polyadenylation signal, and a polyadenylation site. The LTR is flanked by 3-bp inverted repeats, and it consists of the three typical LTR regions, U3, R, and U5. U3 contains 213 bp, R 46 bp, and U5 54 bp, which is within the usual size range of IAP LTRs. A sequence of 60 bp in the U3 region reveals considerable similarity to a murine IAP LTR U3 element, which is known to interact with nuclear proteins. A sequence of 69 bp in the U5 and R regions has 83 and 93% similarities to an endogenous retroviral LTR from Syrian hamster and to the cDNA leader sequence of (Buffalo) rat oncomodulin, respectively. Oncomodulin is an "EF-hand" Ca2+-binding protein and appears in many human and rodent tumors and in cells with tumor-like properties but not in normal tissues. We postulate that in the rat the tumor-specific expression of oncomodulin is controlled by a retroviral LTR promoter.

Use of a glucocorticoid-inducible promoter for expression of herpes simplex virus type 1 glycoprotein gC1, a cytotoxic protein in mammalian cells.

Abundant expression of herpes simplex virus type 1 glycoprotein gC (gC1) in transfected mammalian cells has not previously been achieved, possibly because gC1 protein is toxic to cells. To approach this problem, the gC1 coding sequence was placed under the control of the weak but inducible glucocorticoid-responsive promoter from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). As controls to evaluate for gC1 cytotoxicity, the MMTV LTR promoter was used to express glycoprotein gD1, and a strong, constitutive promoter from the Moloney murine sarcoma virus LTR was used to express gC1. L cells were transfected with these constructs, and a clone expressing gC1 from the inducible MMTV LTR promoter was analyzed. In the absence of glucocorticoid (dexamethasone) stimulation, only a low level of gC1 mRNA expression was detected; after overnight stimulation with dexamethasone, transcription increased approximately 200-fold. Abundant gC1 protein that was functionally active in that it bound complement component C3b, was produced. From passages 5 through 26 (70 cell population doublings), the gC1-producing clone became less responsive to overnight dexamethasone stimulation. The block to gC1 expression occurred at the level of transcription and was associated with hypermethylation of the MMTV LTR DNA. Treatment of the clone with 5-aza-2'-deoxycytidine partially reversed the block in gC1 protein production. Late-passage cells assumed a gC1-negative phenotype that appeared to offer a selective growth advantage, which suggested that gC1 was cytotoxic. Several findings support this view: (i) some cells expressing gC1 after overnight stimulation with dexamethasone assumed bizarre, syncytial shapes; (ii) continuous stimulation with dexamethasone for 5 weeks resulted in death of most cells; (iii) cells transfected with gC1 under the control of the strong Moloney murine sarcoma virus promoter assumed bizarre shapes, and stable gC1-expressing clones could not be established; and (iv) cells induced to express gD1 retained a normal appearance after overnight stimulation or 15 weeks of continuous stimulation with dexamethasone. The inducible MMTV LTR promoter is useful for expressing gC1 and may have applications for expressing other cytotoxic proteins.

Use of a Glucocorticoid-Inducible Promoter for Expression of Herpes Simplex Virus Type 1 Glycoprotein gC1, a Cytotoxic Protein in Mammalian Cells

Abundant expression of herpes simplex virus type 1 glycoprotein gC (gC1) in transfected mammalian cells has not previously been achieved, possibly because gC1 protein is toxic to cells. To approach this problem, the gC1 coding sequence was placed under the control of the weak but inducible glucocorticoid-responsive promoter from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). As controls to evaluate for gC1 cytotoxicity, the MMTV LTR promoter was used to express glycoprotein gD1, and a strong, constitutive promoter from the Moloney murine sarcoma virus LTR was used to express gC1. L cells were transfected with these constructs, and a clone expressing gC1 from the inducible MMTV LTR promoter was analyzed. In the absence of glucocorticoid (dexamethasone) stimulation, only a low level of gC1 mRNA expression was detected; after overnight stimulation with dexamethasone, transcription increased approximately 200-fold. Abundant gC1 protein that was functionally active in that it bound complement component C3b, was produced. From passages 5 through 26 (70 cell population doublings), the gC1-producing clone became less responsive to overnight dexamethasone stimulation. The block to gC1 expression occurred at the level of transcription and was associated with hypermethylation of the MMTV LTR DNA. Treatment of the clone with 5-aza-2'-deoxycytidine partially reversed the block in gC1 protein production. Late-passage cells assumed a gC1-negative phenotype that appeared to offer a selective growth advantage, which suggested that gC1 was cytotoxic. Several findings support this view: (i) some cells expressing gC1 after overnight stimulation with dexamethasone assumed bizarre, syncytial shapes; (ii) continuous stimulation with dexamethasone for 5 weeks resulted in death of most cells; (iii) cells transfected with gC1 under the control of the strong Moloney murine sarcoma virus promoter assumed bizarre shapes, and stable gC1-expressing clones could not be established; and (iv) cells induced to express gD1 retained a normal appearance after overnight stimulation or 15 weeks of continuous stimulation with dexamethasone. The inducible MMTV LTR promoter is useful for expressing gC1 and may have applications for expressing other cytotoxic proteins.

Transcriptional activation of homologous viral long terminal repeats by the human immunodeficiency virus type 1 or the human T-cell leukemia virus type I tat proteins occurs in the absence of de novo protein synthesis.

The genomes of human retroviruses [human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus (HTLV-I)] encode positive trans-activator proteins, named tat. In the presence of tat, the transcriptional activity of the homologous HIV-1 or HTLV-I long terminal repeat (LTR) promoter is markedly increased. We have constructed mammalian cell lines that contain stably integrated copies of a HIV-1 or a HTLV-I LTR-chloramphenicol acetyltransferase (CAT) gene. When presynthesized HIV-1 or HTLV-I tat proteins were separately introduced into these cells in the presence of cycloheximide, we found a strong increase in the steady-state expression of the homologous viral LTR. Nuclear "run-on" assays verified that this tat-mediated enhancement, occurring in the absence of de novo cellular protein synthesis, was due to increased transcriptional initiation at the LTR promoter. We conclude that one aspect of transcriptional trans-activation of viral LTR by the HIV-1 and HTLV-I tat proteins does not require the production of new cellular proteins.

DNA sequences outside the receptor-binding sites differently modulate the responsiveness of the mouse mammary tumour virus promoter to various steroid hormones.

Glucocorticoids, progestins and androgens all induce the transcription of the mouse mammary tumour virus (MMTV) DNA upon binding of their respective receptors to the hormone response element (HRE). This element is located between -202 and -59 5' upstream of the start of transcription on the MMTV long terminal repeat (LTR) region. The HRE contains four repeats of the hexanucleotide 5'-TGTTCT-3' to which the steroid hormone receptors are thought to bind. To investigate the contribution of the individual receptor-binding sites and neighbouring sequences to the steroid hormone action at the MMTV LTR promoter, we mutated various regions of the HRE and studied their response in transfection experiments. Each of the four receptor-binding sites was found to contribute substantially to the overall induction of transcription by all the various steroid hormones tested. This indicates that each individual receptor-binding site on the HRE is important for maximum hormone response. Additionally, we identified four separate sequences outside the receptor binding sites that differentially modulated the response of the MMTV LTR promoter to various steroids. One of these sequences binds the cellular factor, NFI. Thus the interaction of trans-acting factors with sequences outside the hormone receptor-binding sites controls the hormone response of the MMTV LTR promoter.

Introduction and expression of the interleukin 2 receptor (Tac) gene in hematopoietic stem cells with retrovirus vectors.

Retrovirus vectors provide an efficient carrier for introducing a gene into hematopoietic stem cells although expression of the inserted gene is not always successful. We constructed and compared three retrovirus vectors which carried cDNA encoding the light chain (Tac) of the interleukin 2 receptor under the control of different promoters; long terminal repeat (LTR) of murine retroviruses, the early promoter of simian virus 40 (SV40) and the promoter of the class I antigen gene of the major histocompatibility complex. We made three constructs containing these promoters. A first construct did not contain any additional promoter but LTR. A second and a third constructs contained the SV40 and the class I antigen gene promoters, respectively, in addition to LTR. The LTR of retrovirus vectors is derived from MoMuLV except that the U3 region of the 3'LTR of the third construct is derived from myeloproliferative sarcoma virus (MPSV). The second and third constructs were used for infection of bone marrow stem cells as the first construct was less efficient in expression of the interleukin 2 receptor in fibroblasts. Hematopoietic stem cells infected with the recombinant viruses were transplanted into lethally irradiated mice, and the expression of the transduced gene in hematopoietic progenitor cells was analyzed. Analysis of RNA isolated from spleen colonies showed that substantial amounts of interleukin 2 receptor mRNA were made by the construct containing the class I gene promoter and MPSV LTR. However, we could not detect any transcripts from the constructs containing MoMuLV LTR and SV40 early region promoter.

Transcription from a spleen necrosis virus 5' long terminal repeat is suppressed in mouse cells

To determine the block(s) to spleen necrosis virus (SNV) replication in mouse cells, we studied the expression of a dominant selectable marker, neo, or a gene whose product is easily assayed, the chloramphenicol acetyltransferase (cat) gene, in SNV-derived and murine leukemia virus-derived vectors. Using transient (CAT) and stable (Neor phenotype) transfection assays, we showed that the SNV promoter was used in mouse cells only when the 3' SNV long terminal repeat (LTR) was absent. Infection of mouse cells with recombinant SNV viruses was 1% as efficient as infection of permissive dog (D17) cells. The SNV proviruses in mouse cells appeared normal by Southern blot analysis, indicating that their integration probably occurred by normal mechanisms. S1 nuclease analyses of Neor mouse cell clones, each harboring a single recombinant SNV provirus, showed that the selected (internal) promoter was active, but that the 5' SNV LTR promoter was not. However, in the rare (less than 10(-6)) Neor colonies in which expression of the 5' LTR was selected, both promoters were active. Thus, the block to SNV infection of mouse cells is at least at two levels; one is a 100-fold-decreased efficiency at some step(s) up to and including integration, and the other is at transcription.

Identification of p40x-responsive regulatory sequences within the human T-cell leukemia virus type I long terminal repeat.

Distinct transcriptional regulatory sequences located within the upstream sequences required for p40x trans-activation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) were chemically synthesized and cloned upstream of the basal HTLV-I LTR promoter. Plasmids containing a single 21-base-pair (bp) repeat were weakly inducible by p40x. The level of trans-activation by p40x was increased when two (30-fold) or three (40-fold) 21-bp repeats were present in the upstream control region. In the mutant containing two 21-bp repeats, the upstream 21-bp repeat could be positioned in either the sense (30-fold) or the antisense (16-fold) orientation. Plasmids containing a 51-bp repeat element, which included a single 21-bp repeat, were induced to levels similar to that obtained with the 21-bp repeat sequence alone. Template DNAs containing a single copy of the HTLV-I sequences between -117 and -160 were stimulated approximately 10-fold by p40x when one copy of the 21-bp element was located downstream.

The hormone response element of the mouse mammary tumour virus DNA mediates the progestin and androgen induction of transcription in the proviral long terminal repeat region.

Mouse mammary tumour virus (MMTV) gene expression has been shown to be regulated by glucocorticoids. A hormone response element (HRE) located between -202 and -59 upstream of the start of transcription in the long terminal repeat (LTR) region of the proviral DNA is required for this induction. We have investigated the role played by the HRE in the induction of MMTV LTR transcription by other classes of steroid hormones. Chimaeric constructs containing the HRE and the authentic LTR promoter linked to an indicator gene or the HRE linked to an otherwise hormone insensitive promoter directing the transcription of an indicator gene, were transfected into the human mammary tumour cell line T47D. Transcription at the MMTV LTR promoter or at the previously hormone-insensitive promoter was induced by progestins and androgens but not by oestradiol in transfected cells that contained functional receptors for these hormones. These results identify the HRE as the cis-acting element that mediates the progestin and androgen induction of MMTV LTR transcription. The HRE is therefore a DNA element that is required not just for glucocorticoid but also for progesterone and androgen induction of MMTV LTR transcription.

Transcriptional interference and termination between duplicated alpha-globin gene constructs suggests a novel mechanism for gene regulation.

The interesting possibility that transcriptional interference can occur between eukaryotic genes was raised by studies on the avian leukosis retrovirus (ALV) which showed that deletion of the promoter in the 5' long terminal repeat (LTR) activates the 3' LTR promoter, linked to a downstream gene. These observations provide a molecular explanation for the fact that insertional oncogenesis by the ALV promoter is invariably associated with either a rearranged or deleted 5' LTR sequence. This letter extends these findings to chromosomal RNA polymerase II genes by studying transcriptional interference between duplicated alpha-globin gene constructions. I demonstrate that transcriptional interference causes substantial inhibition of the downstream alpha gene by transcription of the upstream alpha gene. Furthermore, this inhibition is alleviated by placing transcriptional termination signals between the two alpha genes. Because many eukaryotic genes may be arranged in tandem on a chromosome, these observations suggest that transcriptional termination is an important mechanism for preventing interference between adjacent genes. The selective use of termination signals may provide a novel way of regulating the activity of eukaryotic genes.

The long terminal repeat of the intracisternal A particle as a target for transactivation by oncogene products.

It has been shown recently that the c-mos oncogene becomes activated in myeloma XRPC-24 via insertion of an intracisternal A particle (IAP) long terminal repeat (LTR). The inserted LTR serves as a promoter from which transcription of the 3' rearranged c-mos initiates. The insertion is in a head-to-head orientation such that the transcriptional orientations of the IAP and the 3' rearranged c-mos are opposite. It has already been shown that this IAP LTR has two promoters, one transcribing the IAP genome and the other transcribing the rearranged c-mos. Since the IAP genomes are actively transcribed in mouse myelomas but not in normal cells, it was interesting to test whether transcriptional activation of the IAP occurs in the presence of active oncogene products, especially nuclear ones. The 5' LTR of the IAP inserted in myeloma XRPC-24 was chosen as a convenient model to test the effect of viral and cellular oncogene products. These included simian virus 40 (SV40) large-T antigen, the adenovirus early 1A (E1A) gene product, the myc gene product, and p53. The LTR was coupled to the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in two orientations, and the levels of CAT directed by the LTR promoters were assayed in either the presence or the absence of the oncogene products. The levels of CAT directed by the 5' LTR promoter transcribing the IAP were significantly elevated in the presence of SV40 large-T antigen, the adenovirus E1A and myc gene products, and p53. The promoter transcribing the rearranged c-mos was transactivated by SV40 large-T antigen and the adenovirus E1A gene product. The results indicate that oncogene products may have an important role in turning on promoters of other genes. The IAP LTR may serve as a useful model for studying the effect of various gene products on promoters which are known to be activated in the malignant state.