Faithful Transcription Research Articles

Recruitment of Human TBP Selectively Activates RNA Polymerase II TATA-dependent Promoters

An increasing body of evidence suggests that eukaryotic activators stimulate polymerase II transcription by facilitating the assembly of the functional basal machinery at the promoter. Here we describe experiments that provide added support for the idea that recruitment of TATA-binding protein (TBP) is a rate-limiting step for transcription activation in mammalian cells. We found that, in human cell lines, recruitment of TBP to a promoter, as a GAL4-TBP fusion protein, can provide a substantial activation of transcription. Activation mediated by the hTBP, tethered to promoter DNA, is strictly dependent upon the presence of a functional TATA element, and it directs faithful transcription initiation. Interestingly, GAL4-hTBP activation was not observed from initiator (Inr) -dependent TATA-less promoters. These results suggest that TBP binding to DNA is not a rate-limiting step for the initial stages of TFIID recruitment to initiator-dependent TATA-less promoters. Finally, we provide evidence that synergy between GAL4-hTBP and defined transcription domains is restricted to activators, such as VP16 and Tat, which are likely to function at steps subsequent to the TFIID recruitment. These findings strengthen the idea that recruitment of TBP represents an important mechanism of activation of TATA-dependent promoters, and on the other hand, they suggest that TBP-DNA interactions are largely dispensable for specific transcription of initiator dependent TATA-less promoters.

Hutchinson–Gilford progeria: faithful DNA maintenance, inheritance and allelic transcription of β(1-4) galactosyltransferase

Hutchinson–Gilford progeria syndrome (HGPS) is a fatal segmental aging disorder affecting children. There is a paucity of prior data at the nucleotide level on DNA maintenance in HGPS. We have examined the specific nucleotide sequences and production of allelic transcripts from the locus GGTB2 encoding β(1-4) galactosyltransferase. Quantitative Northern blots of mRNA from HGPS and control fibroblasts indicated identical mature β(1-4) galactosyltransferase transcript sizes and amounts, regardless of their altered glycosylation status. DNA sequencing of cDNA derived from HGPS β(1-4) galactosyltransferase mRNA populations confirmed the encoded amino acid sequence was unaffected. Population studies of 41 unrelated individuals provided allelic frequency estimates for a novel FokI polymorphism, which was identified in two of six progeria cell strains. The polymorphism was faithfully inherited in a progeria pedigree in a Mendelian manner. Furthermore, the polymorphism provided direct evidence through sequencing of reverse transcription polymerase chain reaction products that both alleles were transcribed and generated mature mRNA. Any defects in transcripts were below detectable levels over the lengths of coding sequences examined, despite multiple replication events from conception leading to the production and maintenance of patient-derived cells. These results indicate faithful transcription in HGPS.

Targeted inactivation of a tobacco intron-containing open reading frame reveals a novel chloroplast-encoded photosystem I-related gene.

The chloroplast genome of all higher plants encodes, in its large single-copy region, a conserved open reading frame of unknown function (ycf3), which is split by two group II introns and undergoes RNA editing in monocotyledonous plants. To elucidate the function of ycf3 we have deleted the reading frame from the tobacco plastid genome by biolistic transformation. We show here that homoplasmic Deltaycf3 plants display a photosynthetically incompetent phenotype. Molecular analyses indicate that this phenotype is not due to a defect in any of the general functions of the plastid genetic apparatus. Instead, the mutant plants specifically lack detectable amounts of all photosystem I (PSI) subunits analyzed. In contrast, at least under low light conditions, photosystem II subunits are still present and assemble into a physiologically active complex. Faithful transcription of photosystem I genes as well as correct mRNA processing and efficient transcript loading with ribosomes in the Deltaycf3 plants suggest a posttranslational cause of the PSI-defective phenotype. We therefore propose that ycf3 encodes an essential protein for the assembly and/or stability of functional PSI units. This study provides a first example for the suitability of reverse genetics approaches to complete our picture of the coding capacity of higher plant chloroplast genomes.

Bent DNA in the Human Adenovirus Type 2 E1A Enhancer Is an Architectural Element for Transcription Stimulation

Bent DNA in the Human Adenovirus Type 2 E1A Enhancer Is an Architectural Element for Transcription Stimulation

Use of Lysolecithin-Permeabilized Infected-Cell Extracts to Investigate thein VitroBiochemical Phenotypes of Poxvirus ts Mutations Altered in Viral Transcription Activity

Lysolecithin permeabilization of vaccinia virus-infected cells was employed to prepare extracts that support faithful transcription initiationin vitroon plasmids possessing early, intermediate, and late viral gene promoters. Conditions which optimize transcription from each promoter were defined. Thein vitrosystem was used to investigate the multifunctional viral mRNA capping enzyme, which also functions as the viral early gene transcription termination factor (VTF) and a viral intermediate gene transcription initiation factor. A low level of signal-dependent termination of early gene transcription was observedin vitrowhich could be elevated by the addition of pure mRNA capping enzyme. VTF-dependent transcription termination was found to be restricted to templates that possessed an early promoter. This restriction mimics that observedin vivoand demonstrates that transcription termination is limited to RNA polymerase molecules that recognize early rather than intermediate or late gene promoters. Extracts prepared from cells infected at the nonpermissive temperature with a virus containing a ts mutation in gene D12L, which encodes the small subunit of VTF, are incapable of supporting both early gene transcription termination and intermediate gene transcription initiation. Both activities are restored upon addition of the purified wild-type mRNA capping enzyme.

Self-coded 3′-Extension of Run-off Transcripts Produces Aberrant Products during in Vitro Transcription with T7 RNA Polymerase

More than 70% of the RNA synthesized by T7 RNA polymerase during run-off transcription in vitro can be incorrect products, up to twice as long as the expected transcripts. Transcriptions with model templates indicate that false transcription is mainly observed when the correct product cannot form stable secondary structures at the 3'-end. Therefore, the following hypothesis is tested: after leaving the DNA template, the polymerase can bind a transcript to the template site and the 3'-end of the transcript to the product site and extend it, if the 3'-end is not part of a stable secondary structure. Indeed, incubation of purified transcripts with the polymerase in transcription conditions triggers a 3'-end prolongation of the RNA. When two RNAs of different lengths are added to the transcription mix, both generate distinct and specific patterns of prolonged RNA products without any interference, demonstrating the self-coding nature of the prolongation process. Furthermore, sequencing of the high molecular weight transcripts demonstrates that their 5'-ends are precisely defined in sequence, whereas the 3'-ends contain size-variable extensions which show complementarity to the correct transcript. Surprisingly, a reduction of the UTP concentration to 0.2-1.0 mM in the presence of 3.5-4.0 mM of the other NTPs leads to faithful transcription and good yields, irrespective of the nucleotide composition of the template.

Mutational Analysis of the Human Endogenous ERV9 Proviruses Promoter Region

ERV9 is a low repeated family of human endogenous retrovital elements whose expression is mainly detectable in undifferentiated embryonal carcinoma NT2/D1 cells. To define all the elements required for the correct transcription activity of the ERV9 promoter and to establish a precise correlation between the elements important for basal transcription, we have systematically analyzed the in vivo and in vitro transcriptional activity of many different ERV9 promoter mutants, including a series of linker-scanning mutations across the promoter region. We report here that the ERV9 promoter contains two elements controlling the selection of the correct start sites, a TATA box and an Inr-like region; the concerted action of both elements is necessary for faithful transcription. Finally, using a series of GAL4 protein fusion constructs in contransfection experiments, we demonstrated that various transcription factors can synergistically induce a high level of transcription when bound to an ERV9 DNA promoter.

Initiator-dependent transcription in vitro by a wheat germ chromatin extract

The development of plant in vitro transcription systems transcribing faithfully and efficiently from a broad range of plant nuclear promoters has remained a challenge. We examined the nucleotide sequence requirements for faithful and efficient transcription in a wheat germ chromatin extract (Yamazaki et al., Plant Mol Biol Rep 8: 114-123). The wheat germ chromatin extract was tested with a series of chimeric promoter constructs containing plant promoter sequences upstream from the TATA box, TATA boxes, and cap-site sequences (from -10 to +14, relative to the major in vivo initiation site) in different combinations. The plant extract transcribed faithfully from several chimeric promoters containing the capsite sequence of the parsley chalcone synthase promoter. The transcription was sensitive to the RNA polymerase II-specific inhibitor alpha-amanitin and was only dependent on the chalcone synthase cap-site sequence which therefore fulfils the operational criteria for a plant initiator element. Mutations of the putative chalcone synthase initiator element defined a core sequence '5'TAACAAC' around the initiation site that was necessary for efficient transcription in vitro. In contrast to the extract, purified wheat germ RNA polymerase II showed no preference for transcription from the major chalcone synthase in vivo initiation site.

Copurification of RNA polymerase I and the glucocorticoid-regulated transcription factor IC

A simple and rapid method to copurify RNA polymerase I and the glucocorticoid-regulated transcription factor, TFIC, is described. This protocol results in 1262-fold purification and 15% total recovery of the enzyme and factors needed to support faithful transcription in vitro from cloned mouse rRNA gene (rDNA). Using this method, proteins involved in rDNA transcription were purified from exponentially growing lymphosarcoma P1798 cells as well as cells treated with 0.1 μ m dexamethasone. A combination of transcription and reconstitution assays using G-free cassette-containing constructs and polyacrylamide gel electrophoretic analysis upon silver staining were used to detect TFIC activity as well as the characteristic TFIC polypeptides in control and dexamethasone-treated cell extracts. Treatment of P1798 cells with 0.1 gm dexamethasone for 24 h results in an over 95% reduction of TFIC activity, but no significant differences in the amount of TFIC polypeptides in the final product purified from control and glucocorticoid-treated cells could be detected. Our data indicate that glucocorticoid regulation of transcription of rDNA is mediated via post-translational modulation of the activity of TFIC.

In vitro transcription in isolated nucleoli of Tetrahymena pyriformis.

An in vitro transcription system was established using extrachromosomal nucleoli from Tetrahymena pyriformis macronuclei as a template. Ribosomal precursor RNA (pre-rRNA) and nascent pre-rRNA chains were removed from isolated nucleoli by treatment with RNase T1 and Sarkosyl. Nucleoli were then incubated in a RNA synthetic cocktail containing a cellular extract from Tetrahymena thermophila. The transcription product was examined for the presence of transcripts from T. pyriformis ribosomal DNA (rDNA) by P1 nuclease protection mapping using a DNA probe from a T. pyriformis rDNA clone. A sequence difference between T. pyriformis and T. thermophila in the 5' region of their 35S pre-rRNAs permitted exclusive detection of T. pyriformis transcripts. The results showed that faithful transcription initiation occurred in vitro from the in vivo initiation site of the nucleolar template and that the nucleolar template had a much higher efficiency of transcription than that of the purified rDNA clone. This system may offer unique advantages for future studies of transcriptional control during development and differentiation.

Activation of the Murine Thyrotropin β-Subunit Promoter by GH4Rat Pituitary Cell-Free Extracts

Expression of the TSH beta subunit gene is restricted to the thyrotroph cells of the anterior pituitary. Previously we identified several AT-rich DNA elements within the murine (m) TSH beta 5'-flanking region, denoted as D1 (-253 to -227), P4 (-142 to -131), P3 (-126 to -112), P2 (-106 to -98), and P1 (-76 to -68) which bind thyrotroph-specific factor(s). These sites are related to, but distinct from GHF-1 and LSF-1 binding sites, which restrict GH and PRL gene expression to pituitary somatotrophs and lactotrophs, respectively. To determine whether different pituitary cell types contain related factors capable of activating the mTSH beta promoter, cell-free transcription studies were performed using extracts from GH4 rat pituitary somatomammotroph cells. AI-through the endogenous mTSH beta gene is not expressed in GH4 cells, in vitro transcription of the mTSH beta promoter, normalized to the Rous sarcoma virus internal control, revealed faithful transcription initiation from the authentic mTSH beta CAP sites in GH4 but not in HeLa cell extracts. Cell-free transcription analysis of mTSH beta 5'-deletion mutants revealed consistent promoter activity with deletion to position -46 but complete loss of activity when deleted to position -9. To better define the specific factors in pituitary somatomammotrophs which interact with and activate the mTSH beta promoter, DNase I protection and gel-shift studies were performed using extracts from GC rat pituitary somatomammotroph cells and DNA affinity-purified lactotroph-specific transcription factor, LSF-1, required for rat PRL promoter activity, and purified from GC cells. These cells contain a factor(s) which binds to thyrotroph-specific elements of the mTSH beta promoter. These studies also show that LSF-1 binds the D1 and proximal thyrotroph-specific elements of the mTSH beta promoter and is capable of reconstituting the trans-activation of the mTSH beta promoter in HeLa nonpituitary cell extracts in vitro. Conversely, nuclear factors present in TtT-97 murine thyrotrophs bind the proximal lactotroph-specific elements on the rPRL promoter. This in vitro transcription assay provides a means to biochemically dissect the trans-activation of the mTSH beta promoter and to determine the functional overlap of distinct pituitary cell-specific factors in regulating GH, PRL, and TSH beta gene expression.

Glucocorticoid Inhibition of Transcription from Adenovirus Major Late Promoter

Glucocorticoids inhibit proliferation of lymphosarcoma P1798 cells in culture. This is accompanied by inhibition of expression of class I as well as some class II genes. We have used the technique of transcription in vitro and the adenovirus major late promoter (AdMLP) to investigate molecular mechanisms of glucocorticoid inhibition of transcription of class II genes. Nuclear extracts from exponentially growing P1798 cells support faithful transcription from AdMLP. However, treatment of P1798 cells with 10(-7) M dexamethasone (a synthetic glucocorticoid) for 24 h impairs the ability of nuclear extracts prepared from such cells to support faithful transcription from AdMLP. Transcription from the human histone H4 gene promoter is unaffected by dexamethasone treatment. Gel mobility shift assays using synthetic oligonucleotide probe indicate no difference in the CAAT box binding activity of control and dexamethasone-treated cell extracts. Similarly, dexamethasone treatment does not affect the upstream stimulatory factor activity of P1798 cells. Furthermore, up-stream stimulatory factor purified from control cell extracts is unable to reconstitute the glucocorticoid-treated cell extracts for transcription from AdMLP. Fractionation of the control cell extracts on Sepharose S yields two protein fractions, neither of which supports transcription from AdMLP. However, one of these fractions, S-II, confers upon glucocorticoid-treated cell extracts the ability to support faithful transcription from AdMLP. We conclude that glucocorticoids regulate the transcription from AdMLP by regulating the activity of a trans-acting transcription factor which copurifies with fraction S-II.

Differential Transcription of the Fibroin and Sericin-1 Genes in Cell-Free Extracts1 : (fibroin gene/sericin gene/differential transcription/nuclear extracts/Bombyx mori).

Devices of the extraction procedues and a recovery of the final supernatant as tiers accompanied with a transcription ability assessment of the individual tiers have enabled us to develop a variety of cell-free transcription systems. The latter step revealed that the factors necessary for faithful transcription as well as enhanced transcription form aggregates or complexes indicating an intrinsic nature of these factors to interact each other. Altogether 14 cell-free transcription systems have been developed from Bombyx mori embryos and tissues of various developmental stages and a cultured cell line, and screened for activities enhancing the basal promoter levels of the silk genes. Using these extracts a differential transcription of plural tissue-specific genes was tried. The fibroin gene was preferentially transcribed than the sericin-1 gene in the extracts from the posterior silk glands where the fibroin gene is specifically transcribed in vivo, while the sericin-1 gene was dominant to the fibroin gene in the extracts from the middle silk glands where the sericin-1 gene is specifically transcribed. Thus, these nuclear extracts offer us a biochemical clue assaying factors responsible for the differential transcription.

Sequence and factor requirements for faithful in vitro transcription of human 7SL DNA

We have analysed the transcription of a functional 7SL gene by RNA polymerase III (RNAPIII) in S100 extracts in vitro. Accurate and efficient synthesis of 7S L RNA depends on the presence of ( i) an upstream sequence and ( ii) an internal promoter element located within the first 22 bp of the gene. These findings were substantiated by DNase I footprinting. Mutations of the internal promoter identified the doublet CG [nucleotide (nt) +15/+16] outside the A-box homologue (nt +15/ to +14) as being essential for both proper promoter function in the vitro transcription assay and competition in the template-exclusion assay. Fractionation of S100 extracts identified two fractions required in addition to RNAPIII for faithful transcription of the gene. Each of these two fractions gave rise to one of two footprints observed in DNase I protection experiments, indicating that at least two DNA-binding factors are involved.

Infectious Recombinant Vectored Virus Vaccines

Publisher Summary This chapter presents the development and the potential of infectious recombinant vectored virus vaccines. Experimental immunizations with infectious virus vectors carrying one or more heterologous genes for immunogenic proteins have been remarkably successful in experimental animals systems. Such immunizations have enabled the presentation of immunogenic proteins to the host immune system in an authentic way, mimicking closely the way antigens are presented in natural infections. Member viruses of the three families— Poxviridae, Herpesviridae, and Adenoviridae — described in the chapter as having potential for use as infectious vectored virus vaccines, have characteristics that lead to a complex judgment as to the overall promise for the practical use of end products in the field. Feasibility seems clear even though, in most cases, questions of stability of recombinants and the level of expression of inserted gene products have not been fully answered. At present, virus vectors under development have the ability to carry enough heterologous DNA to code for a single or multiple genes for immunogenic proteins. Vector systems allow remarkably faithful transcription and translation from inserted genes, as well as proper posttranslational processing and transport.

Selected Letters of Eugene O’Neill ed. by Travis Bogard, Jackson R. Bryer

178 Comparative Drama major narrative incidents. Notable accretions do occur in other variants of "Little Musgrave"-a threat or bnoe is held against the page to secure his silence; the lord and his enemy are blood relations; Musgrave is himself married; Lord Barnard either kills himself, is hanged, or does penance; or the lady's blood is caught in a basin of silver (Child, II, 243)-yet clearly none of these touches derives from Heywood's play. More decisively, such secondary treatments are commonly marked by a faithful transcription of significant character names from their sources. None of Child's or Bronson's versions refer remotely to appellations like "Frankford," "Acton," "Wendoll ," or "Anne," but contain instead recognizable offshoots of "Barnard" (Barnet, Burnett, Barnaby, Barnabas, Barhoas) and "Musgrave" (Mousgrove, Mossgrey, Messgrove , Sir Grove, Matty Groves, Mathew Grew). 13 Allan Holaday, "Thomas Heywood and the Puritans," lournal of English and Germanic Philology, 49 (1950), 197-98. 14 McNeir, "Heywood's Sources," pp. 199-200. 15 Keith Sturgess, ed., Three Elizabethan Domestic Tragedies (Hannondsworth: Penguin, 1985), p. 30. 16 McNeir, "Heywood's Sources," p. 206; Clark, Thomas Heywood, p. 236. 17 Cromwell, Thomas Heywood, p. 105. REVIEWS Travis Bogard and Jackson R. Bryer, eds. Selected Letters of Eugene O'Neill. New Haven and London: Yale University Press, 1988. pp. xi + 602. $35.00. That the personal and professional letters of Eugene O'Neill are deeply fascinating and of immense use to O'Neill scholars goes without saying. That the work done by Travis Bogard and Jackson R. Bryer in selecting, editing, annotating, and providing introductory statements for the present volume is a careful and thoughtful scholarly effort needs saying. One can comment little on the selecting, of course, without detailed knowledge of the body of material selected from (which includes far more letters than the number published here), but those in the volume cover every period of the playwright's life and include correspondence with family, acquaintances, colleagues, critics, theatrical producers , legal advisors, and public inquirers. There seems little chance that anything important has been omitted, and the notes are clear and informative. Especially notable is the fact that in the introductions to the different sections of the book, Bogard and Bryer speculate sensitively and usefully on the possible significances of important correspondence, especially in treating the later periods of the playwright's creative life when many of his letters, like his plays, appear to deepen in understanding and self-awareness. The letters are divided into sections representing chronologically eight different periods of the playwright's life. They progress in the earlier sections from his voluminous love letters to Beatrice Ashe (in 1914-15) through the numerous letters to people associated with his developing career as a playwright-prospective producers like George C. Tyler and professional friends like Kenneth McGowan. He shows himself in these years to be as tough and stubborn in defense of what he perceived to be his integrity as an artist as he was maudlin, verbose, and self-pitying as a lover and young husband. With his burgeoning success of the mid-20's came the breakdown of his marriage and an outpouring of letters to both his new lover (Carlotta Monterey) and to the wife (Agnes Boulton) he at first sought to placate and later vilified viciously for her delay of their divorce-these accompanied by many letters to his ever-faithful attorney Harry Weinberger. So many of the letters written before 1932 have to do with the many diffuse elements of O'Neill's life-his love life, his "divorce" life, his business life (including his byzantine tax problems), not to mention his theatrical life-that the focus of reader interest is drawn away from his artistic development and ideas. This period was also marked by his surprisingly prolific correspondence with his son and nameSake by his first, hastily ended marriage (of 1909). His belief in the wisdom and stability of Eugene O'Neill, Jr., apparently persisted right up to the moment of that classicist's suicide 179 180 Comparative Drama in 1950. The letters show him repeatedly throwing Eugene Jro's apparent success up to the indecisive and ill-fated Shane (whose suicide followed Eugene...

Conditional mutants of RPC160, the gene encoding the largest subunit of RNA polymerase C in Saccharomyces cerevisiae.

A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA polymerase C (RPC160) was cloned by hybridization to a previously isolated fragment of that gene. RPC160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740. Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSO3 and substituted for the wild-type allele on the chromosome. Four of them were unambiguously identified as rpc160 mutants by failure to complement a fully defective mutation rpc160::URA3. The faithful transcription of a yeast tRNA gene by mutant cell-free extracts is strongly reduced as compared to wild-type. In vivo, the rpc160 mutations specifically affect the synthesis of tRNA in a temperature sensitive way, with comparatively little effect on the synthesis of 5S rRNA and no effect on 5.8S rRNA. An unlinked mutation (pcil-3) suppresses the temperature sensitive phenotype of the rpc160-41 mutation.

Cyclosporin A inhibits rDNA transcription in lymphosarcoma P1798 cells.

Effects of cyclosporin A (CsA) on rRNA synthesis in vivo and in vitro were studied using lymphosarcoma P1798 in culture. Pulse labeling with [3H]uridine indicated that treatment of P1798 cells with 1 microgram/ml of CsA for 24-h reduced rRNA levels by 50-60%, whereas rRNA levels of cells rescued from CsA and grown for 24 h were similar to those of controls. Transcription experiments using nuclei from control, treated, and rescued cells indicated that the reduction in rRNA synthesis in treated cells was due to reversible inhibition of transcription of rDNA. Transcription studies in vitro indicated that S100 extracts from CsA-treated cells were unable to carry out faithful transcription of cloned mouse rDNA, even though RNA polymerase I levels of control and treated cell extracts were similar. Mixing experiments indicated that the inability of the CsA-treated cell extract to transcribe cloned rDNA in vitro was not due to the presence of inhibitor(s) or nuclease(s) in such extracts. Supplementation of CsA-treated cell extract with partially or highly purified preparations of a transcription initiation factor for RNA polymerase I, obtained from control cell extracts, conferred transcriptional ability on the CsA-treated cell extract. Extracts from cells treated with cyclosporin H, an inactive analogue of CsA, faithfully transcribed rDNA, indicating the specificity of CsA action. These data indicate that CsA-treated cells lack the ability to initiate rDNA transcription in vivo and in vitro, due to specific, reversible reduction in the amount or activity of transcription factor IC. Significance of these results in understanding the mechanisms of the lymphostatic activity of CsA is discussed.

Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.

The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription.

Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.

The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription.