Transcriptional Enhancer Element Research Articles

TaqMan systems for genotyping of disease-related polymorphisms present in the gene encoding apolipoprotein E.

Polymorphisms of the gene encoding apolipoprotein E have been implicated in the pathogenesis of peripheral and coronary artery disease and neurodegenerative disorders such as sporadic and late-onset familial forms of Alzheimer's disease. We have developed TaqMan assay systems for the single nucleotide polymorphisms -219G/T, located in the promoter of the apolipoprotein E gene, 113G/C, present in the transcriptional enhancer element of intron 1, 334T/C, determining Cys or Arg as amino acid residue 112 of mature apolipoprotein E, and 472C/T, determining Arg or Cys as residue 158. The accuracy of genotype determination with the TaqMan systems was demonstrated by analyses with restriction endonucleases. We determined the genotypes of the apolipoprotein E polymorphisms in 2349 study subjects. The genotypes were distributed as: -219GG = 27.3%, -219GT = 49.1%, and -219TT = 23.6% (p = 0.435); 113GG = 41.3%, 113GC = 45.2%, and 113CC = 13.5% (p = 0.343); 334TT = 73.4%, 334TC = 24.7%, and 334CC = 1.9% (p = 0.539); 472CC = 86.3%, 472CT=12.8%, and 472TT= 0.9% ( p = 0.004) (Hardy-Weinberg equilibrium estimates are given in parentheses). The allele combinations which define the three major isoforms of apolipoprotein E, namely apoE2, apoE3, and apoE4, had the following allele frequencies: 334T/472T (epsilon2; 112Cys/158Cys) = 7.3%, 334T/472C (epsilon3; 112Cys/158Arg) = 78.4%, and 334C/472C (epsilon4; 112Arg/158Arg) = 14.2%, respectively. ApoE genotypes were distributed as: epsilon2epsilon2 = 0.9%, epsilon2epsilon3 = 11.2%, epsilon2epsilon4 = 1.6%, epsilon3epsilon3 = 61.3%, epsilon3epsilon4 = 23.1%, and epsilon4epsilon4 = 1.9% (p = 0.014). The TaqMan assays allow for fast and sensitive genotyping and are especially suitable for studies including large numbers of participants.

Amphotropic murine leukemia virus replication in human mammary epithelial cells and the formation of cytomegalovirus-promoter recombinants.

Amphotropic murine leukemia virus (MLV) can replicate in human cells and is a potential contaminant in vector preparations for human gene transfer studies. We have recently shown that replication of amphotropic MLV in specific human sarcoma and lymphoma lines is possible in the absence of the viral 75-bp transcription enhancer elements. Here, we have tested the replication of an amphotropic MLV, MLV-(MOA), and an enhancer-deficient mutant of this virus in human breast carcinoma-derived cell lines. The proviral expression plasmids use a cytomegalovirus (CMV) promoter for the initial transcription of virus RNA. We found that all cells analyzed are permissive for replication of MLV-(MOA). Enhancer-deficient virus is unable to replicate. However, in two lines the replication defect can be rescued by the spontaneous insertion of a CMV promoter and enhancer into the U3 region. This recombinant virus MLV-(RCMV) replicates with kinetics similar to that of MLV-(MOA) but is restricted to specific cell lines. The potential formation of RCMV recombinants during MLV vector preparation must be considered.

Regulated T-cell development: a victim of multiple conspiracies.

Over about 400 million years there has been a conserved subdivision of T cells into those bearing a γδ T-cell receptor (TCR) and those bearing an αβ TCR. No one is sure why this is, but the two cell types have some interesting distinguishing characteristics, particularly their antigen specificities and their anatomical homing.1 In addition, the development of γδ cells seems always to precede that of αβ T cells. This is a good thing for γδ cells, because the TCRδ locus lies embedded within the TCRα locus, and when TCR Vα and Jα gene segments rearrange, they delete the TCRδ genes. Unchecked, this would substantially reduce the chances of making γδ cells.

Insertion of polydeoxyadenosine-rich sequences into an intergenic region increases transcription in Chlamydomonas reinhardtii chloroplasts.

In this study, chloroplast transformation in Chlamydomonas reinhardtii was used to insert a tract of polydeoxyadenosine, which is known to influence DNA structure and transcription in other systems, between the 3' end of the atpB gene, encoding the beta-subunit of the chloroplast ATP synthase, and a downstream chimeric gene, aadA, encoding antibiotic resistance. Run-on transcription and RNA analyses revealed that in cells containing (dA)40 and (dAAAGGG)8, aadA was transcribed at a higher rate, and its RNA accumulated to a relatively high level. It is concluded that poly(dA/dT) can function in the chloroplast as a transcription enhancer element. Therefore, the insertion of poly(dA/dT) sequence into the intergenic region of a multicistronic transcription unit may modulate gene expression at the transcriptional level.

High and Low Levels of Cottontail Rabbit Papillomavirus E2 Protein Generate Opposite Effects on Gene Expression

The papillomavirus E2 protein plays an important role in viral transcriptional regulation and replication. We chose to study the cottontail rabbit papillomavirus (CRPV) E2 protein as a transcriptional regulator because of the availability of an animal model for papilloma formation, which may be relevant for human papillomavirus (HPV) infection and replication. We studied the effect of expression levels of E2 on the long control region, which contains transcriptional promoter and enhancer elements, and synthetic E2-dependent artificial promoters in which the E2 was the dominant factor in the transcriptional activation. These experiments indicated that high levels of E2 were inhibitory and low levels were stimulatory for transactivation. In addition, we showed that the complex formed between CRPV E2 and the cognate binding site was less stable than the complex formed between HPV E2 and the same cognate binding site. Furthermore, we showed that CRPV E2 binding to its transcriptional regulatory sequence was stabilized by other proteins such as E1, which produced increments in transcriptional activation of E2-dependent genes. The data may be used to define conditions in which the rabbit model can be used for the screening of drugs which are inhibitory to the HPV and CRPV replication and gene expression.

A CCAAT/Enhancer-binding Protein Site within Antioxidant/Electrophile Response Element Along with CREB-binding Protein Participate in the Negative Regulation of RatGST-Ya Gene in Vascular Smooth Muscle Cells

A CCAAT/Enhancer-binding Protein Site within Antioxidant/Electrophile Response Element Along with CREB-binding Protein Participate in the Negative Regulation of RatGST-Ya Gene in Vascular Smooth Muscle Cells

A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity.

This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5' untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Gtx 5' UTR that is 100% complementary to the 18S rRNA at nucleotides 1132-1124. In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits. Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Gtx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements.

Discrete Enhancer Elements Mediate Selective Responsiveness of Enhancer of split Complex Genes to Common Transcriptional Activators

In Drosophila, genes of the Enhancer of split Complex [E(spl)-C] are important components of the Notch (N) cell–cell signaling pathway, which is utilized in imaginal discs to effect a series of cell fate decisions during adult peripheral nervous system development. Seven genes in the complex encode basic helix-loop-helix (bHLH) transcriptional repressors, while 4 others encode members of the Bearded family of small proteins. A striking diversity is observed in the imaginal disc expression patterns of the various E(spl)-C genes, suggestive of a diversity of function, but the mechanistic basis of this variety has not been elucidated. Here we present strong evidence from promoter–reporter transgene experiments that regulation at the transcriptional level is primarily responsible. Certain E(spl)-C genes were known previously to be direct targets of transcriptional activation both by the N-signal-dependent activator Suppressor of Hairless [Su(H)] and by the proneural bHLH proteins achaete and scute. Our extensive sequence analysis of the promoter-proximal upstream regions of 12 transcription units in the E(spl)-C reveals that such dual transcriptional activation is likely to be the rule for at least 10 of the 12 genes. We next show that the very different wing imaginal disc expression patterns of E(spl)m4 and E(spl)mγ are a property of small (200–300 bp), evolutionarily conserved transcriptional enhancer elements, which can confer these distinct patterns on a heterologous promoter despite their considerable structural similarity [each having three Su(H) and two proneural protein binding sites]. We also demonstrate that the characteristic inactivity of the E(spl)mγ enhancer in the notum and margin territories of the wing disc can be overcome by elevated activity of the N receptor. We conclude that the distinctive expression patterns of E(spl)-C genes in imaginal tissues depend to a significant degree on the capacity of their transcriptional cis-regulatory apparatus to respond selectively to direct proneural- and Su(H)-mediated activation, often in only a subset of the territories and cells in which these modes of regulation are operative.

Analysis of human sodium/iodide symporter, thyroid transcription factor-1, and paired-box-protein-8 gene expression in benign thyroid diseases.

The ability to concentrate iodide, a fundamental property of normally functioning thyroid tissue, is altered in various thyroid diseases. Given the critical role of the Na+/I- symporter (NIS) in controlling iodide access to the thyroid gland, altered expression of NIS may be responsible, at least in part, for an enhanced or diminished capacity to concentrate iodide. In this study, we used Northern blot analysis, a newly established quantitative polymerase chain reaction (PCR) assay and in addition hNIS-directed immunohistochemical analysis to assess the levels of hNIS mRNA and protein expression in various localized and diffuse benign thyroid abnormalities, including Graves' disease (GD), scintigraphically cold solitary benign thyroid nodule (CBTN), nontoxic multinodular goiter (NMNG), solitary autonomously functioning thyroid nodule (AFTN), and mild diffuse iodine deficiency goiter (IDG). In addition, in view of the recent identification of putative binding sites for the transcription factors thyroid transcription factor-1 (TTF-1) and human paired-box-protein-8 (Pax-8) in the human NIS gene promoter, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to assess in these same samples the levels of TTF-1 and Pax-8 gene expression. Northern blot analysis revealed high levels of hNIS gene expression in thyroid specimens derived from patients with GD and AFTN. In contrast, levels of hNIS mRNA expression were moderate in NMNG, low in diffuse IDG, and very low in CBTN. Quantitative RT-PCR analysis of hNIS mRNA transcripts revealed variable but generally low levels of hNIS gene expression in IDG and NMNG, and undetectable or very low levels of hNIS mRNA in all scintigraphically CBTN studied. In contrast, markedly elevated levels of hNIS mRNA transcripts were detected in active GD (up to 17-fold) and AFTN (up to 25-fold). Immunohistochemical analysis revealed abundant hNIS protein expression by thyroid follicular cells in GD, moderate and heterogeneous levels in NMNG, and very low levels in CBTN. hNIS mRNA levels were correlated with TTF-1 and Pax-8 gene expression in GD and, to a lesser degree, in AFTN, NMNG, and IDG, but not in CBTN. In general, hNIS gene expression was more closely correlated with TTF-1 as compared to Pax-8 gene expression. In conclusion, the abundance of hNIS mRNA and protein expression in a broad range of benign thyroid pathologies correlated well with their functional state as assessed by thyroid scintigraphy. In addition to TTF-1 and Pax-8, other transcription factors and enhancer elements may contribute to regulation of NIS gene promoter activity.

Structural and Functional Analysis of the Chick Chondroitin Sulfate Proteoglycan (Aggrecan) Promoter and Enhancer Region

Aggrecan is a large chondroitin sulfate proteoglycan, the expression of which is both tissue-specific and developmentally regulated. Here we report the cloning and sequencing of the 1.8-kilobase genomic 5' flanking sequence of the chick aggrecan gene and provide a functional and structural characterization of its promoter and enhancer region. Sequence analysis reveals potential Sp1, AP2, and NF-I related sites, as well as several putative transcription factor binding sites, including the cartilage-associated silencers CIIS1 and CIIS2. A number of these transcription factor binding motifs are embedded in a sequence flanked by prominent inverted repeats. Although lacking a classic TATA box, there are two instances in the 1.8-kb genomic fragment of TATA-like TCTAA sequences, as have been defined previously in other promoter regions. Primer extension and S1 protection analyses reveal three major transcription start sites, also located between the inverted repeats. Transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively reduced portions of the aggrecan promoter region allowed mapping of chondrocyte-specific transcription enhancer and silencer elements that are consistent with the sequence analysis. These findings suggest the importance of this regulatory region in the tissue-specific expression of the chick aggrecan gene.

Cooperative regulation of the human aromatase cytochrome P450 gene transcription by placenta-specific cis-acting elements

Aromatase cytochrome P450 catalyses the reaction to convert androgens to estrogens by coupling with NADPH-cytochrome P450 reductase in the endoplasmic reticulum. The human aromatase cytochrome P450 gene (CYP19) is expressed in a variety of tissues under regulation of tissue-specific promoters. Previously, we localized a cell-type specific transcriptional enhancer element between −242 and −166 relative to the major cap site of the gene, by transient expression analysis in human BeWo choriocarcinoma cells. In the present study, we demonstrate that the enhancer element consists of two subelements, element I (located between −238 and −200), and element II (located between −196 and −176) as analysed by DNase I footprinting using the nuclear extracts of BeWo cells. The gel mobility shift assay shows that each of these subelements binds specific nuclear factor(s). The transient expression of the bacterial chloramphenicol acetyltransferase gene constructs involving the subelements in BeWo cells reveals that the elements activate reporter gene expression synergistically when present together, nevertheless each of the elements by itself also has an enhancer activity. The transient expression analysis further shows that element I is responsible for the transcriptional synergism with the binding site of a nuclear factor-interleukin-6 (NF-IL-6) (also known as CCAAT enhancer/binding protein β), which is located between −2141 and −2115 relative to the major cap site of the gene. These results suggest that the enhancer element plays important roles in sustaining the high levels of CYP19 expression in placental cells in cooperation with other cis-acting transcritional regulatory elements.

Cooperative Regulation of the Human Aromatase Cytochrome P450 Gene Transcription by Placenta-specific cis-acting Elements

Aromatase cytochrome P450 catalyses the reaction to convert androgens to estrogens by coupling with NADPH-cytochrome P450 reductase in the endoplasmic reticulum. The human aromatase cytochrome P450 gene (CYP19) is expressed in a variety of tissues under regulation of tissue-specific promoters. Previously, we localized a cell-type specific transcriptional enhancer element between −242 and −166 relative to the major cap site of the gene, by transient expression analysis in human BeWo choriocarcinoma cells. In the present study, we demonstrate that the enhancer element consists of two subelements, element I (located between −238 and −200), and element II (located between −196 and −176) as analysed by DNase I footprinting using the nuclear extracts of BeWo cells. The gel mobility shift assay shows that each of these subelements binds specific nuclear factor(s). The transient expression of the bacterial chloramphenicol acetyltransferase gene constructs involving the subelements in BeWo cells reveals that the elements activate reporter gene expression synergistically when present together, nevertheless each of the elements by itself also has an enhancer activity. The transient expression analysis further shows that element I is responsible for the transcriptional synergism with the binding site of a nuclear factor-interleukin-6 (NF-IL-6) (also known as CCAAT enhancer/binding protein β), which is located between −2141 and −2115 relative to the major cap site of the gene. These results suggest that the enhancer element plays important roles in sustaining the high levels of CYP19 expression in placental cells in cooperation with other cis-acting transcritional regulatory elements.

Interactions of a Transcriptional Activator in the env Gene of the Mouse Mammary Tumor Virus with Activation-dependent, T Cell-specific Transacting Factors

The mouse mammary tumor virus env gene contains a transcriptional activator (META) that can control transcription of the adjacent long terminal repeat region. Transcriptional control by META parallels that of several lymphokine genes, being specific to T cells, dependent on their activation, and inhibited by the immunosuppressive drug cyclosporine (CsA). DNase I footprinting indicated that nuclear factors from activated T lymphocytes bound a promoter-proximal site, META(P), and a promoter-distal site, META(D+), within the 400-base pair META region. Nuclear factors from unstimulated, but not from activated cells, bound a site, META(D-), adjacent to META(D+). META(D+) directed transcription of a linked luciferase gene, and gel shift analysis revealed binding of inducible, CsA-sensitive T cell factors, in parallel with transfection results. Authentic NFAT and NF-kappaB targets did not compete for the META(D+) binding factor(s). The SV40 core sequence competed for META(D+) binding factors, but META(D+) failed to compete for the complexes obtained with the SV40 probe. Our results, taken together, indicate that META(D+) is a novel transcriptional enhancer element that is similar in its cell-type specificity, activation dependence, and CsA sensitivity to the NFAT element. It may be relevant to the role of MMTV in expression of Mls antigens or the induction of T cell lymphomas.

A (G+C)‐Rich Motif in the Aldolase C Promoter Functions as a Constitutive Transcriptional Enhancer Element

The enzyme fructose-1,6-bisphosphate aldolase consists of three isozymes that are expressed in a tissue-specific manner. Using antibodies against aldolase B and C, it is shown that aldolase C is expressed in virtually all neuronal cell lines derived from the central and peripheral nervous system. Recently, experiments with transgenic mice indicated that a (G+C)-rich region of the aldolase C promoter might function as a neuron-specific control element of the rat aldolase C gene [Thomas, M., Makeh, I., Briand, P., Kahn, A. & Skala, H. (1993) Eur. J. Biochem. 218, 143-151). To functionally analyse this element, a plasmid consisting of four copies of this (G+C)-rich sequence, a TATA box, and the rabbit beta-globin gene as reporter was constructed. This plasmid was transfected into neuronal and nonneuronal cell lines and transcription was monitored by RNase protection mapping of the beta-globin mRNA. It is shown that the (G+C)-rich element of the aldolase C promoter directs transcription in neuronal as well as in nonneuronal cells. In contrast, the synapsin I promoter, used as a control for neuron-specific gene expression, directed transcription only in neuronal cells. In gel-retardation assays, two major DNA-protein complexes were detected with the (G+C)-rich element of the aldolase C promoter used as a DNA probe and nuclear extracts from brain and liver as a source for DNA-binding proteins. These DNA-proteins interactions could be impaired by a DNA probe that contained an Sp1-binding site, indicating that Sp1 or an Sp1-related factor binds to the aldolase C promoter (G+C)-rich element. This was confirmed by supershift analysis with antibodies specific for Sp1. The zinc finger transcription factor zif268/egr-1, also known to recognize a (G+C)-rich consensus site, did not, however, bind to the (G+C)-rich motif of the aldolase C promoter, nor could it stimulate transcription in transactivation assays from this control region. From these data, we conclude that the (G+C)-rich element of the aldolase C promoter functions as a constitutive transcriptional response element mediated by Sp1 and Sp1-related transcription factors.

Promotion of V(D)J recombinational accessibility by the intronic E kappa element: role of the kappa B motif.

The accessibility of a chromosomally integrated TCR beta minilocus recombination substrate in a V(D)J recombinase-inducible cell line (HDR37) depends on incorporation of transcriptional enhancer elements such as the Ig kappa light chain intronic enhancer (E kappa). The E kappa element contains several functional motifs including the kappa B motif, which binds the NF-kappa B transcription factor. To assess molecular mechanisms by which E kappa promotes V(D)J recombinational accessibility, we compared the abilities of the wild-type E kappa, a corresponding E kappa sequence with a mutant kappa B motif (E kappa-kappa B-) and a kappa B motif dimer (kappa B2) to function in the context of the TCR beta minilocus/HDR37 system. The E kappa-containing minilocus underwent demethylation, transcription and V(D)J recombination, independently of copy number of integration site. Transfectants containing low copy numbers (one or two) of the E kappa-kappa B(-)-containing minilocus, like enhancerless or kappa B2-containing miniloci at any copy number, were inactive with respect to all three processes. In contrast, high-copy-number integrants of the E kappa-kappa B- substrates showed an integration-site dependent activation of all three processes. Together these data show that the kappa B motif plays a critical role in the ability of E kappa to confer V(D)J recombinational accessibility, but that it is not sufficient to mediate this process by itself.

Retinoic Acid Receptor β2 (RARβ2) Null Mutant Mice Appear Normal

Vertebrates are highly sensitive to both retinoic acid (RA) deficiency and excess. The RA signal is thought to be transduced by nuclear receptors (the RAR and RXR families) which activate the expression of target genes via cis -acting transcriptional enhancer elements. Each of the three RAR genes, RARα, RARβ, and RARγ, gives rise to several isoforms by differential usage of two promoters and alternative splicing. RARβ2 is the most abundant of the four RARβ isoforms, and its transcription is spatially and temporally restricted in developing embryos, suggesting that it might perform specific functions. Furthermore, RARβ2 expression can be induced via a retinoic acid response element located in its promoter region. This RA effect is particularly interesting since under conditions of RA excess, RARβ2 promoter activity and transcript accumulation are induced in regions of developing embryos in which malformations subsequently appear, such as the craniofacial region, the hindbrain, and the limbs. These findings have led to the suggestion that the RARβ2 isoform might mediate some of the teratogenic effects of RA. In this study, we have eliminated RARβ2 expression by targeted gene disruption. RARβ2 null mutants exhibit an apparently normal phenotype, indicating that other RARs must compensate for RARβ2 sufficiently well to allow normal prenatal and postnatal development to proceed. By challenging RARβ2 null embryos with teratogenic doses of RA, we have also directly addressed the question of whether RARβ2 is required for mediating RA-induced malformations.

Regulation and targeting of recombination in extrachromosomal substrates carrying immunoglobulin switch region sequences.

We have used extrachromosomal substrates carrying immunoglobulin heavy-chain S mu and S gamma 3 switch region sequences to study activation and targeting of recombination by a transcriptional enhancer element. Substrates are transiently introduced into activated primary murine B cells, in which recombination involving S-region sequences deletes a conditionally lethal marker, and recombination is measured by transformation of Escherichia coli in the second step of the assay. Previously we found that as many as 25% of replicated substrates recombined during 40-h transfection of lipopolysaccharide (LPS)-stimulated primary cells and that efficient recombination was dependent on the presence of S-region sequences as well as a transcriptional activator region in the constructs (H. Leung and N. Maizels, Proc. Natl. Acad. Sci. USA 89:4154-4158, 1992). Here we show that recombination of the switch substrates is threefold more efficient in LPS-cultured primary B cells than in the T-cell line EL4; the activities responsible for switch substrate recombination thus appear to be more abundant or more active in cells which can carry out chromosomal switch recombination. We test the role of the transcriptional activator region and show that the immunoglobulin heavy-chain intron enhancer (E mu) alone stimulates recombination as well as E mu combined with a heavy-chain promoter and that mutations that diminish enhancer-dependent transcription 500-fold diminish recombinational activation less than 2-fold. These observations suggest that the enhancer stimulates recombination by a mechanism that does not depend on transcript production or that is insensitive to the level of transcript production over a very broad range. Furthermore, we find that E mu stimulates recombination when located either upstream or downstream of S mu but that the position of the recombinational activator does affect the targeting of recombination junctions, suggesting that the relatively imprecise targeting of switch junctions in vivo may reflect the availability of many potential activator sites within each switch region.

Regulation and Targeting of Recombination in Extrachromosomal Substrates Carrying Immunoglobulin Switch Region Sequences

We have used extrachromosomal substrates carrying immunoglobulin heavy-chain S mu and S gamma 3 switch region sequences to study activation and targeting of recombination by a transcriptional enhancer element. Substrates are transiently introduced into activated primary murine B cells, in which recombination involving S-region sequences deletes a conditionally lethal marker, and recombination is measured by transformation of Escherichia coli in the second step of the assay. Previously we found that as many as 25% of replicated substrates recombined during 40-h transfection of lipopolysaccharide (LPS)-stimulated primary cells and that efficient recombination was dependent on the presence of S-region sequences as well as a transcriptional activator region in the constructs (H. Leung and N. Maizels, Proc. Natl. Acad. Sci. USA 89:4154-4158, 1992). Here we show that recombination of the switch substrates is threefold more efficient in LPS-cultured primary B cells than in the T-cell line EL4; the activities responsible for switch substrate recombination thus appear to be more abundant or more active in cells which can carry out chromosomal switch recombination. We test the role of the transcriptional activator region and show that the immunoglobulin heavy-chain intron enhancer (E mu) alone stimulates recombination as well as E mu combined with a heavy-chain promoter and that mutations that diminish enhancer-dependent transcription 500-fold diminish recombinational activation less than 2-fold. These observations suggest that the enhancer stimulates recombination by a mechanism that does not depend on transcript production or that is insensitive to the level of transcript production over a very broad range. Furthermore, we find that E mu stimulates recombination when located either upstream or downstream of S mu but that the position of the recombinational activator does affect the targeting of recombination junctions, suggesting that the relatively imprecise targeting of switch junctions in vivo may reflect the availability of many potential activator sites within each switch region.

Molecular analysis of transgenic plants generated by microprojectile bombardment: effect of petunia transformation booster sequence.

Supercoiled plasmid expression vectors containing the petunia transformation booster sequence (TBS) were introduced by microprojectile bombardment into dicotyledenous (tobacco) and monocotyledonous (maize) cells. TBS effected a 7.8- to 16-fold increase in transformation frequencies in tobacco, and a 1.7- to 2.4-fold increase in maize. Although TBS contains a well-defined transcription enhancer element, no increases in plasmid gene expression were observed. TBS did not alter integration patterns in transformants, and did not affect segregation of linkage in R1 progeny. Computer analyses of the TBS sequence revealed numerous modular elements previously shown to be associated with putative chromosomal replication origin regions in eukaryotes, including DNA unwinding elements, scaffold-associated regions and pyrimidine tracts.

Cell-type-specific activity of the human papillomavirus type 18 upstream regulatory region in transgenic mice and its modulation by tetradecanoyl phorbol acetate and glucocorticoids

The upstream regulatory region (URR) of human papillomavirus type 18 (HPV-18) harbors transcriptional promoter and enhancer elements which are thought to determine the cell-type specificity of the virus. In order to study the regulation of HPV-18 expression in vivo, we constructed transgenic mice carrying the bacterial lacZ gene under the control of the HPV-18 URR. Analysis of beta-galactosidase activity by histochemical staining of tissue sections of four independent transgenic mice showed that the viral promoter was specifically active in epithelial cells within a variety of organs (e.g., tongue, ovary, uterus, testis, and small intestine). Very strong staining was observed in newborn transgenic mice in contrast to a weak activity found during fetal life. Determination of beta-galactosidase activity in crude extracts from tissues of three lines of transgenic mice proved to be a useful tool for a quantitative analysis of transgene expression. In mice from two different transgenic lines treated with dexamethasone such measurements revealed a biphasic effect of the hormone on the activity of the enzyme in the stratified epithelium of the tongue (transient increase followed by a decrease). Northern (RNA) blot analysis showed similar changes in beta-galactosidase mRNA in that tissue. Treatment with tetradecanoyl phorbol acetate (TPA) led to a twofold increase in both enzymatic activity and mRNA levels. Finally, combined treatments with dexamethasone and TPA showed that both factors interfered with each other in their respective effects on transgene expression, suggesting that a cross-talk mechanism between transcription factors could be involved in the regulation of the HPV-18 URR.