Chimeric Plasmid Research Articles

Interactions of Thyrotropin-Releasing Hormone, Phorbol Ester, and Forskolin-Sensitive Regions of the Rat Thyrotropin-β Gene

Our previous studies demonstrated TRH stimulation of TSH beta gene transcription in rat pituitary cell cultures and in transient expression assays, with the TRH-sensitive region located between -1.3 kilobases and -204 basepairs (bp) relative to the major transcriptional start site. Using nuclear runoff and transient expression assays, we have analyzed the interactions among TRH, the phorbol ester 12-myristate 13-acetate (PMA), and the adenylate cyclase activator forskolin on TSH beta gene transcription. In cultured pituitary cells, TSH beta gene transcription was stimulated by 2 h of 10(-9) M TRH (2- to 4-fold), 100 nM PMA (2- to 6-fold), or 2 microM forskolin (1.5- to 2.5-fold) treatment, with additive interactions among all three effectors. Chimeric plasmids containing various 5'-flanking portions of the TSH beta gene and both transcriptional start sites, fused to the chloramphenicol acetyltransferase (CAT) gene, were transfected into the clonal pituitary GH3 cell line to delineate DNA sequences conferring this regulation. Transfected TSH beta CAT constructs containing TSH beta gene sequences from -2100/+27I150, -1295/+27I150, and -520/+27I150 expressed CAT enzyme activity which was stimulated by 24 h of TRH (2- to 3-fold), PMA (3- to 6-fold), or forskolin (1.5- to 3-fold) treatment, similar to observations in normal pituitary cells. In addition, a CAT expression vector construct containing only upstream TSH beta gene sequences from -703 to -85 bp, fused to the heterologous thymidine kinase promoter (tkCAT), exhibited similarly stimulated transcription in a transfection assay in response to TRH, PMA, and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid-inducible expression of a glutamine synthetase-CAT-encoding fusion plasmid after transfection of intact chicken retinal explant cultures

In this communication we demonstrate that gene transfer methodology can be applied to study gene expression in intact retinal explant cultures. The appropriate enzyme activity is observed in extracts obtained after electroporation of embryonic day-10 chicken retina with plasmids containing the chloramphenicol acetyltransferase-encoding or γ-galactosidase-encoding reporter genes under transcriptional control by the Rous sarcoma virus long terminal repeat. Similar results are obtained using Ca · phosphate-mediated gene transfer. Moreover, it has been previously established that glucocorticoid hormones stimulate transcription of glutamine synthetase (Glns) mRNA in embryonic retina. We report here that, based on the results of gene transfer experiments with chimeric plasmids containing 5′ - flanking DNA derived from the cloned chicken Glns- encoding gene ( Glns), essential glucocorticoid response elements residebetween approx. 1.3 kb and 2.5 kb upstream from the Glns transcription start point. These data show that transfection of explant cultures can provide a useful approach to the study of gene expression in complex systems.

Identification ofCis-Acting Promoter Elements Important for Expression of the Mouse Glycoprotein Hormone α-Subunit Gene in Thyrotropes

The glycoprotein hormone alpha-subunit gene is expressed in a cell-specific manner in the anterior pituitary and placenta. Previous studies have shown that the region between -178 to -111 is indispensable for placental-specific expression of the human alpha-subunit gene. Using gene transfer techniques with chimeric luciferase plasmids, this report identifies regions of the mouse alpha-subunit promoter that are important for transcriptional activation in primary thyrotropic cells. Transient expression of a series of 5' flanking DNA deletions resulted in stepwise reductions of basal promoter activity between -480 to -417 (4-fold), -254 to -177 (5-fold), and -177 to -120 (3.5-fold). DNase-I protection analysis with nuclear extracts from thyrotropic tumor cells revealed specific protein-DNA interactions within each of these functionally defined regions. These were mapped to positions -474 to -452, -447 to -419, -213 to -170, and -158 to -101 within the 5' flanking region. In contrast, in mouse fibroblast L-cells no significant difference in alpha-subunit promoter activity was found by deleting the region from -480 to -177. However, a 3-fold decrease, similar to that found in primary thyrotropes, was found by deleting the region from -177 to -120. Further, a smaller region between -138 and -122 was the only area detected by the DNase-I protection assay using L-cell nuclear extracts. Thus, several cis-acting promoter elements located up-stream of position -177 are important for expression in thyrotropes. These elements also bind nuclear factors present in thyrotropes but not in nonpituitary fibroblasts and, therefore, differ from those mediating expression of the human alpha-subunit gene in the placenta.

Conjugative transfer of a shuttle plasmid forEscherichia colitoMycobacterium smegmatis

The chimeric plasmid pMY10 containing the origin of replication of pAL5000, the origin of replication of pBR322, the origin of transfer of pRK2 and a kanamycin resistance gene was constructed and succesfully transferred by conjugation from Escherichia coli harbouring the helper plasmid pRK4.24 into Mycobacterium smegmatis. This is the first report of conjugative transfer of plasmid between E. coli and an acid fast organism.

Use of the tyrosinase gene from Streptomyces to probe promoter sequences for Escherichia coli

We have constructed a promoter-probe vector utilizing the expression of a promoter-less tyrosinase derived from Streptomyces plasmid pIJ702. The vector, pMX100, has single sites for EcoRI, KpnI, BamHI, XbaI, SalI, and SphI for cloning promoter sequences. When the tac promoter was inserted into pMX100, E. coli harboring the chimeric plasmid produced the melanin pigment.

Conjugative transfer of a shuttle plasmid for Escherichia coli to Mycobacterium smegmatis

The chimeric plasmid pMY10 containing the origin of replication of pAL5000, the origin of replication of pBR322, the origin of transfer of pRK2 and a kanamycin resistance gene was constructed and succesfully transferred by conjugation from Escherichia coli harbouring the helper plasmid pRK4.24 into Mycobacterium smegmatis. This is the first report of conjugative transfer of plasmid between E. coli and an acid fast organism.

The human oxytocin gene promoter is regulated by estrogens.

Gonadal steroids affect brain function primarily by altering the expression of specific genes, yet the specific mechanisms by which neuronal target genes undergo such regulation are unknown. Recent evidence suggests that the expression of the neuropeptide gene for oxytocin (OT) is modulated by estrogens. We therefore examined the possibility that this regulation occurred via a direct interaction of the estrogen-receptor complex with cis-acting elements flanking the OT gene. DNA-mediated gene transfer experiments were performed using Neuro-2a neuroblastoma cells and chimeric plasmids containing portions of the human OT gene 5'-glanking region linked to the chloramphenicol acetyltransferase gene. We identified a 19-base pair region located at -164 to -146 upstream of the transcription start site which is capable of conferring estrogen responsiveness to the homologous as well as to a heterologous promoter. The hormonal response is strictly dependent on the presence of intracellular estrogen receptors, since estrogen induced stimulation occurred only in Neuro-2a cells co-transfected with an expression vector for the human estrogen receptor. The identified region contains a novel imperfect palindrome (GGTGACCTTGACC) with sequence similarity to other estrogen response elements (EREs). To define cis-acting elements that function in synergism with the ERE, sequences 3' to the ERE were deleted, including the CCAAT box, two additional motifs corresponding to the right half of the ERE palindrome (TGACC), as well as a CTGCTAA heptamer similar to the "elegans box" found in Caenorhabditis elegans. Interestingly, optimal function of the identified ERE was fully independent of these elements and only required a short promoter region (-49 to +36). Our studies define a molecular mechanism by which estrogens can directly modulate OT gene expression. However, only a subset of OT neurons are capable of binding estrogens, therefore, direct action of estrogens on the OT gene may be restricted to a subpopulation of OT neurons.

The human cytomegalovirus immediate early enhancer-promoter is responsive to activation by the adenovirus-5 13S E1A gene

The stimulatory effects of the 13S adenovirus E1A gene product on the human cytomegalovirus (HCMV) major immediate early (IE) enhancer were examined. Chimeric plasmids containing cloned portions of the HCMV major IE enhancer-promoter positioned upstream of the chloramphenicol acetyltransferase gene (cat) were cotransfected into HeLa cells with the plasmid p13S-wt which contained a cDNA encoding the adenovirus 13S E1A gene product. CAT expression from chimeric plasmids containing at least one copy of the HCMV 19 base pair (bp) repetitive motif was stimulated 10-fold in the presence of p13S-wt. The 19-bp motif contains a potential binding site for the cellular transcription factor ATF/CREB. Deletion analysis indicated that the ATF/CREB site was crucial for E1A-mediated stimulation. Insertion of a synthetic oligonucleotide homologous to a 19-bp motif and containing an ATF/CREB binding site into an HCMV chimera lacking ATF/CREB motifs conferred E1A responsivity on HCMV promoter-mediated CAT expression whereas insertion of a similar oligonucleotide containing a change of two bases in the sequence of the ATF/CREB site did not. Measurement of CAT-specific RNA verified the results of the CAT enzyme experiments. The ATF/CREB motif may be a target for stimulation of HCMV gene expression through either viral or cellular transcription factors.

Characterization of rabbit cardiac sarco(endo)plasmic reticulum Ca2(+)-ATPase gene.

We have isolated three overlapping genomic clones extending over 39 kilobases (kb), which encodes the rabbit cardiac sarco(endo)plasmic reticulum Ca2(+)-ATPase gene (SERCA2). S1 nuclease mapping and primer extension analysis of the 5' end of the cardiac/slow-twitch (SERCA2a) and smooth/non-muscle (SERCA2b) mRNAs showed that both transcripts are initiated from the same transcription initiation site, located 528 base pairs (bp) upstream of the translation initiation codon AUG. The putative promoter revealed a "TATA box" like element at -24 bp and a "CAAT box" at -78 bp relative to the cap site. A number of DNA sequence elements that could bind trans-acting factors were also found within the 1.8 kb of DNA sequence upstream from the transcription initiation site. To determine the DNA sequences governing transcriptional regulation, we have stably transfected the myogenic cell line C2C12 with a plasmid containing the putative promoter and 946 bp upstream sequence of the SERCA2 gene, coupled to the chloramphenicol acetyltransferase gene. Our results show that this chimeric plasmid construct exhibits appropriate activation and coordinate expression with the endogenous SERCA2 gene during the terminal differentiation of myoblasts into myotubes, suggesting that it contains the promoter and upstream sequence elements required for the regulated expression of the SERCA2 gene.

Specific Region in Hormone Binding Domain Is Essential for Hormone Binding and Trans-Activation by Human Androgen Receptor

Complementary DNA (cDNA) clones encoding human-androgen receptors (haR) were isolated using synthetic oligonucleotides homologous to the human glucocorticoid, estradiol, progesterone, and aldosterone receptors as probes to screen a human testis lambda gt11 cDNA library. One of the receptor proteins (hARa) produced in vitro bound the [3H]dehydrotestosterone ([3H]DHT) with high affinity and selectivity similar to the human androgen receptor present in target tissues and cells. A second cDNA clone (hARb) encoding an identical amino terminal and DNA binding domains, but differing by four amino acids at the hormone binding domain, did not bind [3H]DHT with high affinity when incubated with protein expressed by in vitro transcription-translation. Cotransfection of hARa in an expression vector with mouse mammary tumor virus (MMTV)-bacterial chloramphenicol acetyltransferase chimeric plasmids, followed a hormone-dependent trans-activation, defining the binding affinity of hARa between 5 x 10(-10) and 1 x 10(-9) M for [3H]DHT. A similar cotransfection experiment with hARb indicated a KD of hARb for [3H]DHT to be above approximately 10(-8) M. The deduced primary structures of hARa and hARb contain the viral erbA homologous region found in other steroid, thyroid, and vitamin receptors and is identical to the hAR sequences reported by others. The amino acid sequence differs at the Gly stretch (16 Gly instead of 27, 24 or 23) of the N-terminal domain and in hARb, the sequence reads I.F.F.F.F.L.L (816-822) instead of K.F.F.D.E-L (816-821) in the hARa and other reported hAR sequences. The difference of four amino acids in the steroid binding domain of hARb is associated with altered DHT binding and thus a lack of trans-activation by way of AR responsive elements in MMTV-long terminal repeat. The interaction of hARa and hARb with synthetic responsive elements by gel-retardation assay and their responsiveness in trans-activation by calcium phosphate coprecipitation demonstrates that hARb can inhibit trans-activation by hARa in this system.

Regulation of Elastin Gene Expression: Evidence for Functional Promoter Activity in the 5′-Flanking Region of the Human Gene

Analysis of nucleotide sequences in the 5'-flanking region of the human elastin gene has revealed several unusual features, suggesting that regulation of elastin gene expression is complex. To identify any cis-acting regulatory promoter elements, a 35-kb fragment of DNA (CosE) was isolated from a human genomic cosmid library by hybridizations with a human elastin cDNA. Southern blots of EcoRI digests of CosE DNA, utilizing a 5'-end labeled 21-mer oligonucleotide corresponding to the signal sequence of elastin, revealed the presence of a single 7.8-kb genomic fragment. Partial dideoxynucleotide sequencing of this EcoRI genomic subclone revealed that it extended approximately 2.5 kb 3' of the translation initiation site (ATG), encompassing exon 1 and a portion of the first intron, while the remaining DNA encompassed the 5'-flanking region. Exonuclease III digestion (3'----5') was performed to remove sequences of the first intron and first exon, including the ATG site. One clone, approximately 5 kb in size, had the 3' end located 14 bp upstream of the ATG site. A 462-bp 3' portion of this 5-kb fragment was subcloned into a Bluescript/CAT chimeric plasmid (pBS0CAT) to generate an elastin gene promoter/CAT reporter gene construct (pEP6CAT). Transient transfection experiments with pEP6CAT using human skin fibroblasts, human HT-1080, mouse NIH-3T3, or freshly isolated neonatal rat aortic smooth muscle cells revealed significant CAT activity in each cell line. These results suggest that the 5'-flanking region of the elastin gene contains the cis-acting regulatory elements necessary for transcription. The chimeric plasmid pEP6CAT provides a means to study the transcriptional control of elastin gene expression by exogenous affector molecules, as well as in human dermatologic diseases.

Metallothioniens with interdomain hinges expanded by insertion mutagenesis

Specific peptides of varying lengths were inserted between the two metal cluster domains of metallothionein (MT), which normally are spanned by only three amino acids, Lys-Lys-Ser. These interdomain expansions were made to test if such structural alterations would affect MT function. These constructs were engineered by inserting defined oligonucleotides of up to four tandem repeats of dodecanucleotides and hexanucleotides into an Alu-1 endonuclease cleavage site, which separates the two exonic regions of an MT-coding sequence from Chinese hamster ovary cells, MT-2. The native and altered sequences were cloned into a high expression Escherichia coli-yeast shuttle vector and used to transform yeast cells whose endogenous MT genes had been previously deleted. Using metal resistance as a biological marker, all constructs were shown to be functional in rendering the host cells resistant to either copper or cadmium. As the inserts, by nature of their amino acid sequence, could add flexibility to the otherwise compact molecule, the two domains apparently are active independently. The level of activity, however, diminished with the length of the insert. Determinations for copy number of the chimeric plasmids and MT mRNAs in the transformed cells showed that the replicational and transcriptional capacity of the long and short constructs were equivalent.

Cytomegalovirus infection stimulates expression of monocyte-associated mediator genes.

Monocytes and tissue macrophages play important roles in host defense against virus infections and, in the case of human cytomegalovirus (HCMV) and HIV, may also be the reservoir for latent disease. Because these cells can also rapidly respond to most infections by secretion of inflammatory mediators, we were interested in determining if HCMV infection could have a direct activating effect on macrophage cytokine production. To do this, we primarily investigated the influence of HCMV infection on IL-1 beta-mRNA expression in peripheral blood monocytes and the promyelocytic cell line, ML-3 as well as the inflammatory response genes TNF-alpha, MAD-9, MAD-6, and MAD-2 in the promyelocytic ML-3 cell line. Exposure of ML-3 cells to the virus prior to induction of differentiation had little influence on mediator gene expression. However, induction of the macrophage phenotype by pretreatment of ML-3 cells with the phorbol ester, PMA, followed by HCMV challenge, resulted in a greatly extended period of expression of IL-1 beta, TNF-alpha, MAD-9, and CSF-1 but not MAD-6 and MAD-2. Constitutively expressed genes such as lysozyme and actin were not similarly modulated. Both RNA dot-blot and in situ hybridization studies demonstrated that infection of human peripheral blood monocytes with HCMV leads to sustained expression of IL-1 beta mRNA for up to 96 h, which contrasted markedly with mock-infected or LPS-stimulated monocytes. Flow cytometric analysis of the intracellular levels of IL-1 beta protein in ML-3 cells indicated that not only was there more protein produced in infected cells, but that the majority of the cells had responded. Enhanced levels of the intracellular form of IL-1 beta in monocytes was confirmed by Western blot analysis. Cotransfection experiments were performed using IL-1 beta-CAT chimeric plasmids together with plasmids encoding HCMV-immediate-early gene region products. Transactivation of the IL-1 beta gene by region 2 of the immediate-early gene was observed in ML-3 cells that had been induced to differentiate prior to transfection. No stimulation of IL-1 beta promoter activity was observed in ML-3 cells that were undifferentiated prior to transfection. In summary, HCMV infection, although not leading to productive infection, nonetheless may contribute to the pathology of the infection through enhancement of monocyte inflammatory mediator gene expression with subsequent stimulation of protein synthesis.

The segregation of the 2 mu-based yeast plasmid pJDB248 breaks down under conditions of slow, glucose-limited growth.

The stability of the 2 mu-based yeast plasmid pJDB248 in Saccharomyces cerevisiae S150-2B(cir0) was investigated in glucose-limited chemostat culture. Plasmid-free cells were detected by loss of (plasmid-encoded) leucine prototrophy and confirmed by colony hybridization. The plasmid was considerably more stable at a high dilution rate (0.12 h-1) than at a lower dilution rate (0.05 h-1). The average plasmid copy number in the cells retaining the plasmid remained constant at approximately 50 in the high dilution rate culture whereas it rose to almost 600 in the slow dilution rate culture. However, in both cultures the overall plasmid level in the total population remained constant, indicating that plasmid segregation breaks down at the low growth rate. Similar experiments on the native 2 mu plasmid demonstrated high stability and no significant differences between the high and low growth rate cultures. It is postulated that the difference in behaviour between the native and chimeric plasmids is related to an interaction between the growth conditions and the loss of the D gene product.

Polyomavirus genome and polyomavirus enhancer-driven gene expression during myogenesis

The mRNAs for myogenic functions are coordinately transcribed with polyomavirus (Py) early mRNA during in vitro differentiation of mouse C2 myoblast cells. Sequence analysis shows that the A domain of the Py enhancer includes an E1A-like consensus sequence that is also found in the 5' upstream region of two genes expressed during myoblast differentiation: alpha-actin and myosin light chain. Therefore, the coordinate expression of such genes with Py early mRNA may be activated by a common cellular regulatory factor. In the present work, we report that C2 cells surviving Py infection are unable to differentiate and do not express alpha-actin and myosin light-chain mRNAs. Hybrids between such Py-resistant myoblast cells and the parental cells exhibited dominance of the permissibility to Py growth and of the expression of myogenic mRNAs. In C2 cells transiently transfected with a chimeric plasmid (pSVPy12CAT) harboring the bacterial chloramphenicol acetyltransferase (CAT) gene driven by the Py enhancer-promoter region, the CAT gene was expressed irrespective of their stage of differentiation. Moreover, undifferentiated stably transfected cells expressing the CAT gene restricted viral growth. Py-resistant C2 myoblasts transiently transfected with pSVPy12CAT also expressed the CAT gene driven by the Py enhancer. This contradictory finding is similar to results previously obtained by other investigators with cloned genes specific for myogenic functions, and it may be explained by a structural difference between the pSVPy12CAT and the Py genomic organizations in which the viral enhancer operates.

Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum.

We have used a combination of nucleotide and N-terminal-amino-acid-sequence analyses to determine the primary structure of the 37-kilodalton (kDa) endoflagellar outer layer, or sheath, protein. Initially, a lambda gt11 clone (designated lambda A34) expressing a portion of the 37-kDa protein was selected from a Treponema pallidum genomic library with a murine monoclonal antibody (H9-2) directed against an epitope of the 37-kDa protein. The insert from lambda A34 provided a probe with which a chimeric plasmid (pR14) encoding all but the nine N-terminal amino acids of the entire protein was selected from a T. pallidum(pBR322) genomic library. The nine N-terminal amino acids determined by amino acid sequencing were combined with the DNA sequence encoded by pR14 to determine the primary structure of the entire 37-kDa protein; the combined sequence made up a polypeptide with a calculated molecular mass of 36,948 Da. Approximately one-third of the deduced sequence was confirmed by N-terminal amino acid analysis of tryptic peptides from the purified 37-kDa protein. Repeated attempts to clone upstream portions of the gene (flaA) by using a variety of strategies were unsuccessful, suggesting that unregulated expression of the intact sheath protein or of its most amino-terminal portions is toxic in Escherichia coli. These studies should provide the basis for further molecular investigations of the endoflagellar apparatus and of treponemal motility.

RNA polymerase II transcription termination is mediated specifically by protein binding to a CCAAT box sequence.

A region in the adenovirus major late promoter (MLP) containing a CCAAT consensus sequence can direct transcription termination of RNA polymerase II, a mechanism that possibly prevents transcriptional interference from upstream genes. Using a chimeric plasmid template that contains the MLP directing expression of the simian virus 40 early region, we showed that an inserted oligonucleotide containing only 13 base pairs of MLP sequences, including the CCAAT box, is capable of inducing transcription termination in an orientation-dependent, position-independent manner. Point mutations within the CCAAT-specific protein-binding site abolished this effect, while a base substitution outside of this region did not affect termination. These data suggest that termination is mediated by a CCAAT box-binding protein. Several other transcription factor-binding sites do not, however, cause termination, suggesting that this may be a relatively specific property of a CCAAT-binding protein.

RNA polymerase II transcription termination is mediated specifically by protein binding to a CCAAT box sequence.

A region in the adenovirus major late promoter (MLP) containing a CCAAT consensus sequence can direct transcription termination of RNA polymerase II, a mechanism that possibly prevents transcriptional interference from upstream genes. Using a chimeric plasmid template that contains the MLP directing expression of the simian virus 40 early region, we showed that an inserted oligonucleotide containing only 13 base pairs of MLP sequences, including the CCAAT box, is capable of inducing transcription termination in an orientation-dependent, position-independent manner. Point mutations within the CCAAT-specific protein-binding site abolished this effect, while a base substitution outside of this region did not affect termination. These data suggest that termination is mediated by a CCAAT box-binding protein. Several other transcription factor-binding sites do not, however, cause termination, suggesting that this may be a relatively specific property of a CCAAT-binding protein.

A novel strategy for producing chimeric bispecific antibodies by gene transfection

We have used genetic manipulation to produce chimeric bispecific antibodies. Plasmids containing variable regions of immunoglobulin from a murine hybridoma secreting anti-hepatitis B surface antigen were joined to human constant regions. These chimeric plasmids were introduced into transfectomas, secreting chimeric antibodies to iodo-hydroxy-nitrophenyl, by electroporation. Transfectomas secreting bispecific antibodies were identified. This approach has advantages over the fusion of hybridomas or chemical linking of two antibody molecules and will enable the use of bispecific antibodies in vivo.

Transcription and messenger RNA processing upstream of bacteriophage T4 gene 32.

Bacteriophage T4 gene 32 lies at the 3' end of a complex transcription unit which includes genes 33, 59, and several open reading frames. In the course of an infection, four major transcripts are synthesized from this unit: two overlapping polycistronic transcripts about 3800 and 2800 nucleotides in length, and two monocistronic gene 32 transcripts about 1150 and 1100 nucleotides in length. These transcripts are made at different times in infection and the polycistronic transcripts have segmental differences in stability. Messenger RNA processing yields a 1025 nucleotide monocistronic gene 32 transcript, and a 135 nucleotide transcript containing part of the gene 59 coding sequence. Processing depends on Escherichia coli encoded ribonuclease E. This pattern of transcription and processing leads to the synthesis of gene 32 mRNA throughout infection, whereas transcripts encoding the upstream genes are present only early in infection. The 3800 nucleotide polycistronic transcript initiates at a promoter that does not require T4 encoded factors for activity. However, full-length synthesis of this transcript depends on the T4 mot gene product. The region upstream of gene 32 also contains four E. coli-like promoters that are active on chimeric plasmids in uninfected cells, but inactive in bacteriophage T4. The location of these cryptic T4 promoters is intriguing in that they lie near the 5' ends of open reading frame B, gene 59 and gene 32. They could play a role in phage development under particular conditions of growth or in bacterial hosts other than those examined here.