Eucaryotic Genes Research Articles

The Mof2/Sui1 protein is a general monitor of translational accuracy.

Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed -1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene. mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed -1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.

Analysis of expression of the gene encoding for the nuclear autoantigen La/SS-B using reportergene constructs

In earlier studies mRNA isoforms encoding for the nuclear autoantigen La were identified. In an alternative La mRNA form the exon 1 was replaced with the exon 1′. Moreover, exon 1′ La mRNAs were found to start at different 5′-regions. In dependence on the 5′-start the exon 1′ La mRNAs encoded for up to three open reading frames upstream of the La frame, which starts in the exon 2. The exon 1′ was located in the intron about 70 nts downstream of the exon 1. The exon 1′ La mRNA was proposed to be the result of a promoter switch in combination with an alternative splicing mechanism. The commonly used technique to study the expression of a eucaryotic gene is to fuse a reportergene immediately downstream of the proposed regulatory elements. Due to (i) the short distance between exon 1 and exon 1′, (ii) the varying 5′-starts of the exon 1′ La mRNAs, and (iii) the upstream open reading frames in the exon 1′ La mRNAs this technique appeared to be difficult to apply to the La gene. In order to overcome these problems a luciferase reportergene construct was cloned which started about 2500 nts upstream of the exon 1 and contained the exon 1, the intron including the exon 1′, and a portion of the exon 2. Luciferase was fused into the exon 2. This construct was used to prepare 5′-deletion mutants. The constructs were transiently transfected into HeLa cells. RNAs were isolated from the transiently transfected cells and analyzed using the 5′-Rapid Amplification of cDNA End technique. The PCR products were subcloned and sequenced. This analysis showed that exon 1 and exon 1′ transcripts were correctly transcribed and spliced from the La luciferase fusion construct. Moreover, the 5′-start of the respective transcript allowed to identify those genomic regions in the La gene that were most likely being involved in determining the respective transcription initiation site. In parallel to the estimation of the 5′-start of the transcripts, the luciferase activity was measured. Thereby we detected a cryptic promoter element in the intron between the exon 1 and exon 2.

The NOT proteins are part of the CCR4 transcriptional complex and affect gene expression both positively and negatively.

The CCR4 transcriptional regulatory complex consisting of CCR4, CAF1, DBF2 and other unidentified factors is one of several groups of proteins that affect gene expression. Using mass spectrometry, we have identified the 195, 185 and 116 kDa species which are part of the CCR4 complex. The 195 and 185 kDa proteins were found to be NOT1 and the 116 kDa species was identical to NOT3. NOT1, 2, 3 and 4 proteins are part of a regulatory complex that negatively affects transcription. All four NOT proteins were found to co-immunoprecipitate with CCR4 and CAF1, and NOT1 co-purified with CCR4 and CAF1 through three chromatographic steps in a complex estimated to be 1.2x10(6) Da in size. Mutations in the NOT genes affected many of the same genes and processes that are affected by defects in the CCR4 complex components, including reduction in ADH2 derepression, defective cell wall integrity and increased sensitivity to monoand divalent ions. Similarly, ccr4, caf1 and dbf2 alleles negatively regulated FUS1-lacZ expression, as do defects in the NOT genes. These results indicate that the NOT proteins are physically and functionally part of the CCR4 complex which forms a unique and novel complex that affects transcription both positively and negatively.

The rnhB gene encoding RNase HII of Streptococcus pneumoniae and evidence of conserved motifs in eucaryotic genes.

A single RNase H enzyme was detected in extracts of Streptococcus pneumoniae. The gene encoding this enzyme was cloned and expressed in Escherichia coli, as demonstrated by its ability to complement a double-mutant rnhA recC strain. Sequence analysis of the cloned DNA revealed an open reading frame of 290 codons that encodes a polypeptide of 31.9 kDa. The predicted protein exhibits a low level of homology (19% identity of amino acid residues) to RNase HII encoded by rnhB of E. coli. Identification of the S. pneumoniae RNase HII translation start site by amino-terminal sequencing of the protein and of mRNA start sites by primer extension with reverse transcriptase showed that the major transcript encoding rnhB begins at the protein start site. Comparison of the S. pneumoniae and E. coli RNase HII sequences and sequences of other, putative bacterial rnhB gene products surmised from sequencing data revealed three conserved motifs. Use of these motifs to search for homologous genes in eucaryotes demonstrated the presence of rnhB genes in a yeast and a roundworm. Partial rnhB gene sequences were detected among expressed sequences of mouse and human cells. From these data, it appears that RNase HII is universally present in living cells.

Microsatellite deletions in the c-myb transcriptional attenuator region associated with over-expression in colon tumour cell lines.

In the hemopoietic system c-myb expression is required for proliferation of immature cells and its down-regulation is required for differentiation. In colonic mucosa c-myb expression occurs at levels comparable to immature hemopoietic cells. Inhibition of c-myb expression in colon cell lines, using anti-sense oligonucleotides, indicates that c-myb expression is required for proliferation. However, the mechanism of c-myb regulation during colon cell differentiation has not been explored. Using the LIM1215 and CaCo-2 colon carcinoma cell lines induced to differentiate with sodium butyrate, we demonstrate that c-myb mRNA is down-regulated as an early event in differentiation by a mechanism involving transcriptional attenuation in intron 1. By analogy with procaryotic and eucaryotic genes, transcriptional attenuation probably occurs in a region containing nineteen consecutive thymidine residues. Computer prediction of the secondary structure of the nascent mRNA chain encoded by this region suggests a strong potential for stem-loop formation. Sequence analysis of several colon tumour cell lines reveals mutations in this region that may disrupt transcriptional attenuation and result in the increased c-myb expression observed in colon tumours and tumour cell lines.

Homologous and heterologous expression of a ribosomal protein gene in Podospora anserina requires an intron.

Although the role of introns in eucaryotic nuclear genes has been much debated, it remains underinvestigated in fungi. The AS1 gene of Podospora anserina contains three introns and encodes a ribosomal protein (S12) belonging to the well-conserved bacterial S19 family. We attempted to complement the highly pleiotropic mutation AS1-4 with a cDNA encoding the homologous human (S15) protein (rig gene) under the control of the AS1 promoter. In a control experiment, the AS1+ cDNA was unable to complement fully the AS1-4 mutation. It was assumed that the AS1 cDNA was not well expressed and that the AS1 gene needed intron(s) to be efficiently expressed. Addition of the first intron of the AS1 gene to the AS1 and rig cDNAs did indeed allow complementation of all the phenotypic defects of the AS1-4 mutation. These data lead to two main conclusions. First, the human S15 ribosomal protein is functional in Podospora. Second, full expression of the Podospora AS1 gene requires at least one intron.

Immediate-Early Baculovirus Vectors for Foreign Gene Expression in Transformed or Infected Insect Cells

Baculovirus expression vectors are used routinely for foreign gene expression and are under intense development as improved biological pesticides. Conventional baculovirus expression vectors are recombinant viruses that can express a foreign gene in insect cells under the control of thepolyhedrinpromoter, which provides high-level transcription during the very late phase of infection. For some applications, including foreign glycoprotein production and insect pest control, it might be advantageous to have baculovirus vectors that could express foreign gene products in uninfected cells or earlier after infection. To fulfill this need, we have constructed a new set of plasmids that can be used to clone and express foreign genes under the control of a baculovirusie1promoter, which is active in uninfected insect cells and throughout infection. We used a subset of these new plasmids to isolate recombinant baculoviruses containing various foreign genes and compared expression of these genes by the resulting immediate-early baculovirus vectors and by conventional baculovirus vectors. As expected, the immediate-early vectors began to express each foreign gene earlier in infection but, by 36–48 h postinfection, the conventional vectors had produced more of each foreign protein. Conventional baculovirus vectors also produced more enzymatic activity from two different procaryotic genes than the immediate–early baculovirus vectors. However, immediate-early vectors produced as much or more enzymatic activity from two different eucaryotic genes encoding secretory pathway proteins than the conventional vectors, even at 48 h postinfection. Hence, this report describes a new set of plasmids that can be used to clone and express foreign genes under the control of the baculovirusie1promoter and suggests that immediate-early baculovirus vectors might be as useful as conventional baculovirus expression vectors for producing biologically active eucaryotic secretory pathway proteins.

Selective cloning of a defined number of tandem DNA repeats in Escherichia coli.

Repetitive DNA sequences play an important role in biology, especially at the level of eucaryotic gene transcription control. Their construction, assembly and cloning by conventional means or by PCR derived methods is usually very cumbersome. This method is based on the association of a small DNA tag with the sequence to be polymerised. The multimeric sequence is constructed in a stepwise manner, the tag allowing for simple in vivo selection of the (n+1) polymer containing vector at each step. Thus, n rounds of cloning generate a family of vectors, each harbouring a defined number of ordered tandem copies of the sequence of interest, ranging from 1 to n. Contrary to the multimerization methods based on PCR, this method is independent of the size of the sequence to be polymerised. It is, in fact, especially well suited for the moderate polymerisation of short DNA sequences such as regulatory proteins binding sites, or the preparation of small size DNA ladders. The outline of the method is as follows (Fig. 1). The sequence to be polymerised is first cloned between two compatible ends restriction sites in a cloning vector (sites A and B), generating vector 1X. Then, a small cassette encompassing an amber suppressor tRNA gene with its promoter and transcription terminator is cloned next to it (between sites C and D), to obtain vector 1X+S which is used to transform an amber mutated strain, such as, for example, XAC-1 (1). The 2X+S vector is obtained by cutting out the 1X+S fragment with restriction enzymes A and D, and cloning it between sites B and D of vector 1X. Growth of the transformants on minimal medium ensures that only the correct construct is selected for. In the same manner, the 3X+S vector is obtained by cloning the 2X+S A/D fragment into the 1X vector between the B and the D sites and selecting the transformants for the presence of the suppressor gene. The 3X+S vector will thus contain three adjacent copies of the sequence of interest, in the same orientation, between two unique restriction sites A and B, as the sites between the copies are destroyed by the cloning procedure and the asymmetrical cloning ensures that only one orientation of the sequence is inserted. Each time the cloning step is repeated, one unit of the sequence is added to the polymerised sequence. With a recombination deficient amber mutated strain as a host for the successive transformations, the limit to the number of copies of the sequence of interest is only given by the size of the plasmid. If the nX+S containing vector is destroyed by additional restriction cuts when preparing the nX+S fragment, then no purification of this fragment is required before cloning, and the successive rounds of cloning are very simple. Figure 1. Outline of the method. A and B are compatible ends restriction sites such as SalI and XhoI for example.

L'effet de position : influence de la conformation chromatinienne sur l'expresson des gènes eucaryotes

It is now well established that the eucaryotic nucleus is highly organized in euchromatic versus heterochromatic domains. Heterochromatin is mostly found in centrometric and telomeric regions of chromosomal arms are essentially euchromatic. The proper expression of a gene strongly depends on its chromatin structure. For instance, when delocalized to a heterochromatic area, a euchromatic gene displays a mosaic inactivation. Silencing of gene expression by heterochromatin is randomly distributed in some cells and is permanent. This phenomenon is named the position-effect variegation and has been extensively studied in Drosophila and yeast. Furthermore, similar silencing effects canalso be observed in other organisms including mammals. In some cases, the epigenetic extinction of an allele can be transmitted to its homologous conterpart as shown by dominant variegation in Drosophila or paramutation in plants. Similar effects involving cis and trans-inactivating chromatin structure might also be responsible for certain human dominant diseases of the nervous system associated with an abnormal expansion of repeated sequences typically silencing gene expression in Drosophila. Changes in chromatin conformation are widely observed in the epigenetic control of eucaryotic gene expression. The position-effect variegation provides a useful model to better understand chromatin modification during development or during the cell cycle and how it is inherited by daughter cells leading to the permanent silencing of euchromatic genes.

Domains in human interferon alpha-1 gene containing tandems of arginine codons AGG play the role of translational initiators in E. coli

The AGG and AGA are the least used arginine codons in E. coli but they are the most preferable ones in eukaryotes. The low expression of some eucaryotic genes (such as human alpha-1 interferon gene) which contain clusters of AGG codons is explained either by the limited pool of the tRNA AGG (Varenne and Lazdunski, 1986) or by the competition of these clusters with the Shine-Dalgarno (SD) sequence (Ivanov et al., 1992). The aim of the present study is to demonstrate the in vivo capacity of AGG tandems to bind to bacterial ribosomes. The two tandems of AGG codons (Arg 12 Arg 13 and Arg 163 Arg 164) of hIFα 1 with their surrounding nucleotides were cloned in a bacterial expression plasmid containing a strong promoter and a reporter gene (chloramphenicol acetyltransferase, CAT) devoid of a ribosome binding site. The results obtained showed that both AGG tandems initiated translation of the CAT mRNA with an efficiency equal to that of the consensus SD sequence and several fold higher than the native SD sequence of the CAT gene.

Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation.

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) stimulates the transcription of many eucaryotic genes by catalyzing the phosphorylation of the cAMP-regulatory element binding protein (CREB). Conversely, the attenuation or inhibition of cAMP-stimulated gene transcription would require the dephosphorylation of CREB by a nuclear protein phosphatase. In HepG2 cells treated with the protein serine/threonine (Ser/Thr) phosphatase inhibitor okadaic acid, dibutyryl-cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter was enhanced over the level of PEPCK gene transcription observed in cells treated with dibutyryl-cAMP alone. This process was mediated, at least in part, by a region of the PEPCK promoter that binds CREB. Likewise, okadaic acid prevents the dephosphorylation of PKA-phosphorylated CREB in rat liver nuclear extracts and enhances the ability of PKA to stimulate transcription from the PEPCK promoter in cell-free reactions. The ability of okadaic acid to enhance PKA-stimulated transcription in vitro was entirely dependent on the presence of CREB in the reactions. The phospho-CREB (P-CREB) phosphatase activity present in nuclear extracts coelutes with protein Ser/Thr phosphatase type 2A (PP2A) on Mono Q, amino-hexyl Sepharose, and heparin agarose columns and was chromatographically resolved from nuclear protein Ser/Thr-phosphatase type 1 (PP1). Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by the heat-stable protein inhibitor-2, which is a potent and selective inhibitor of PP1. Nuclear PP2A dephosphorylated P-CREB 30-fold more efficiently than did nuclear PP1. Finally, when PKA-phosphorylated CREB was treated with immunopurified PP2A and PP1, the PP2A-treated CREB did not stimulate transcription from the PEPCK promoter in vitro, whereas the PP1-treated CREB retained the ability to stimulate transcription. Nuclear PP2A appears to be the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.

Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation.

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) stimulates the transcription of many eucaryotic genes by catalyzing the phosphorylation of the cAMP-regulatory element binding protein (CREB). Conversely, the attenuation or inhibition of cAMP-stimulated gene transcription would require the dephosphorylation of CREB by a nuclear protein phosphatase. In HepG2 cells treated with the protein serine/threonine (Ser/Thr) phosphatase inhibitor okadaic acid, dibutyryl-cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter was enhanced over the level of PEPCK gene transcription observed in cells treated with dibutyryl-cAMP alone. This process was mediated, at least in part, by a region of the PEPCK promoter that binds CREB. Likewise, okadaic acid prevents the dephosphorylation of PKA-phosphorylated CREB in rat liver nuclear extracts and enhances the ability of PKA to stimulate transcription from the PEPCK promoter in cell-free reactions. The ability of okadaic acid to enhance PKA-stimulated transcription in vitro was entirely dependent on the presence of CREB in the reactions. The phospho-CREB (P-CREB) phosphatase activity present in nuclear extracts coelutes with protein Ser/Thr phosphatase type 2A (PP2A) on Mono Q, amino-hexyl Sepharose, and heparin agarose columns and was chromatographically resolved from nuclear protein Ser/Thr-phosphatase type 1 (PP1). Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by the heat-stable protein inhibitor-2, which is a potent and selective inhibitor of PP1. Nuclear PP2A dephosphorylated P-CREB 30-fold more efficiently than did nuclear PP1. Finally, when PKA-phosphorylated CREB was treated with immunopurified PP2A and PP1, the PP2A-treated CREB did not stimulate transcription from the PEPCK promoter in vitro, whereas the PP1-treated CREB retained the ability to stimulate transcription. Nuclear PP2A appears to be the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.

Analysis of the promoter sequence and the transcription initiation site of the mouse 5-HT 1C serotonin receptor gene

The serotonin 1c (5-HT 1C) receptor is found in many brain regions, but is particularly enriched on the epithelial cells of the choroid plexus. A major challenge in neurobiology is to delineate the molecular processes that regulate the specific pattern of neuronal gene expression in the brain. As an initial step towards cis-acting DNA sequences that control the expression of the 5-HT 1C receptor, we have isolated the promoter sequence of its gene. Sequence analysis of a 1.8 kb fragment indicated that the 3′ end of this fragment overlaps with the 5′ untranslated region of the 5-HT 1C receptor mRNA, and primer extension using mouse brain poly(A) + RNA mapped the transcription initiation site within this fragment. There are a number of sequence elements upstream from the transcription initiation site that are homologous to regulatory elements found in other eucaryotic genes. To determine the promoter activity, a plasmid was constructed that contains this fragment as promoter region and the cDNA for the 5-HT 1C receptor as the promoter. When injected into the nucleus of Xenopus oocytes, this construct resulted in functional expression of the reporter gene. Primer extension using the RNA extracted from the injected oocytes indicated a single transcription initiation site of the reporter mRNA. These results suggest that the 5-HT 1C receptor was functionally expressed under the promoter activity of the 1.8 kb 5′ sequence of its gene. The system will be useful for further analysis of the cis-acting elements in the promotor region of the 5-HT 1C receptor gene and the trans-acting factors that regulate tissue-specific expression of the receptor.

Regulation of hepatitis B virus gene expression

The hepatitis B virus has a compact genome, yet codes for at least seven primary translation products. Not surprisingly, the cis-acting elements that regulate gene expression overlap each other and the open reading frames extensively, rendering dissection of each element difficult. Nevertheless, various groups have identified and characterized four promoters and two enhancers, whose combined actions lead to preferential gene expression in hepatocytes, thereby partially explaining the hepatotropism of the virus. In addition, the X gene product trans-activates transcription from a wide variety of promoters, both cellular and viral, by as yet unclear mechanisms, while a core gene product may negatively regulate gene expression. The HBV genome thus provides a well-defined system for studying complex cis- and trans-regulation of eucaryotic gene expression.

Structure and expression of the Kas12 gene encoding a beta-ketoacyl-acyl carrier protein synthase I isozyme from barley.

The beta-ketoacyl-acyl carrier protein (ACP) synthase I in the plant fatty acid synthetase catalyzes the condensations of acetate units to a growing acyl-ACP leading to the synthesis of palmitoyl-ACP. Barley chloroplasts contain three cerulenin sensitive beta-ketoacyl-ACP synthase I isoforms, alpha 2, alpha beta, and beta 2. The Kas12 gene encoding the beta 2 isozyme has been isolated and sequenced. The gene spans 3.8 kilobases and contains seven exons separated by six intervening sequences varying from 75 to 1008 base pairs in length. The mosaic gene structure is different compared with that of the beta-ketoacyl synthase in the multifunctional rat and goose fatty acid synthetases. Southern blot analyses of genomic DNA from barley, wheat, and the barley-wheat chromosome addition lines indicate that Kas12 is a single copy gene located on chromosome 2. Primer extension analyses identified four transcription start sites located 168-171 nucleotides upstream from the translation initiation codon. The Kas12 promoter lacks an appropriately positioned TATA box and contains a GC-rich region including two GC elements similar to the Sp1 transcription factor-binding site. In this regard Kas12 closely resembles a set of ubiquitously expressed eucaryotic genes. In accord with this deduction, polymerase chain reaction analysis showed that the Kas12 transcript is present in barley roots, germinating embryos, developing kernels, and leaves.

Isolation and sequence analysis of the genomic DNA fragment encoding an aspartic proteinase inhibitor homologue from potato (Solanum tuberosum L.).

A genomic DNA clone encoding an aspartic proteinase inhibitor of potato was isolated from a lambda EMBL3 phage library using the aspartic proteinase inhibitor cDNA as a hybridization probe. The gene has all characteristic sequences normally found in eucaryotic genes. Typical CAAT and TATA box sequences were found in the 5'-upstream region. In this part are also two putative regulatory AGGA box sequences located. In the genomic sequence there are no intron sequences interrupting the coding region. An open reading frame of the gene encodes a precursor protein of 217 amino acids which shows high percent identity with the aspartic proteinase inhibitor cDNA.

Processing-Independent Analysis (Pia) -A New Diagnostic Tool

Posttranslational processing is an important phase of the expression of most eucaryotic genes in terms of functional proteins. Among these, secretory proteins and peptides are of particular interest for clinical chemists, since diagnostic measurements of circulating proteins and peptides constitute a major discipline in clinical chemistry. The posttranslational covalent maturation of secretory proteins and peptides involves multiple enzymatic modifications of the corresponding proproteins along the intracellular secretory pathway. During the eighties, an increasing amount of evidence has indicated that sick secretory cells fail to process their secretory products normally. The diseased cells therefore fail to process their secretory products normally. The diseased cells therefore release also incompletely processed precursors and processing-intermediates. In order to measure the degree of disease, assays that measure proteins and peptides independent of the degree of processing are therefore desirable. We have now designed a new analytical principle, according to which secretory proteins, peptides and their precursors can be accurately quantitated irrespective of the degree of processing. This principle, named processing-independent analysis (PIA), is generally applicable to all cellular synthesized substances. The principle has been applied to and developed first for a well-defined secretory peptide system, progastrin and its products. Using this model, the results obtained so far confirm the diagnostic superiority of processing-independent analysis in comparison with conventional assays for bioactive peptides.

Transcriptional Control of the Invariant Chain Gene Involves Promoter and Enhancer Elements Common to and Distinct from Major Histocompatibility Complex Class II Genes

The invariant chain (Ii) is a glycoprotein coexpressed with the major histocompatibility complex (MHC) class II antigens. Although Ii is encoded by a single gene unlinked to the MHC gene complex, Ii and MHC class II appear to have similar patterns of tissue specific expression and generally are coordinately regulated by cytokines. Here we present evidence that transcription of the murine Ii gene is controlled by multiple cis-acting elements. The 5' regulatory region of the Ii gene appears to be combined of conserved class II regulatory elements with promoter elements commonly found in other eucaryotic genes. A region containing characteristic class II promoter elements (H box, X box, and a modified Y box) serves as an upstream enhancer in the Ii gene and might contribute to the coexpression of MHC class II and Ii genes. A series of positive control elements, the kappa B element, Sp1-binding site, and CCAAT box, are present in the Ii promoter and apparently serve distinct regulatory functions. The kappa B site in the Ii gene is a cell type-specific element, contributing to expression in a B-cell line but not in a fibroblast cell line, and the Sp1 site is required by the H-X-Y' enhancer element to stimulate promoter activity. In addition, an Ii enhancer in the first intron that specifically stimulates its own promoter has been identified. Our results suggest that a sequence match between enhancers and certain promoter elements is critical.

Transcriptional control of the invariant chain gene involves promoter and enhancer elements common to and distinct from major histocompatibility complex class II genes.

The invariant chain (Ii) is a glycoprotein coexpressed with the major histocompatibility complex (MHC) class II antigens. Although Ii is encoded by a single gene unlinked to the MHC gene complex, Ii and MHC class II appear to have similar patterns of tissue specific expression and generally are coordinately regulated by cytokines. Here we present evidence that transcription of the murine Ii gene is controlled by multiple cis-acting elements. The 5' regulatory region of the Ii gene appears to be combined of conserved class II regulatory elements with promoter elements commonly found in other eucaryotic genes. A region containing characteristic class II promoter elements (H box, X box, and a modified Y box) serves as an upstream enhancer in the Ii gene and might contribute to the coexpression of MHC class II and Ii genes. A series of positive control elements, the kappa B element, Sp1-binding site, and CCAAT box, are present in the Ii promoter and apparently serve distinct regulatory functions. The kappa B site in the Ii gene is a cell type-specific element, contributing to expression in a B-cell line but not in a fibroblast cell line, and the Sp1 site is required by the H-X-Y' enhancer element to stimulate promoter activity. In addition, an Ii enhancer in the first intron that specifically stimulates its own promoter has been identified. Our results suggest that a sequence match between enhancers and certain promoter elements is critical.

The adenovirus EII early promoter has multiple EIA-sensitive elements, two of which function cooperatively in basal and virus-induced transcription

The mechanism by which the adenovirus-encoded nuclear oncogene EIA activates transcription of several viral and host promoters is an important issue in the regulation of eucaryotic gene expression and virus-host cell interactions. Identification of cis-acting elements of the promoters and the cognate host transcription factors that are targets for EIA action is crucial for our understanding of the EIA-mediated control of coordinately regulated genes. The adenovirus EII early promoter has a complex architecture and contains two overlapping promoters with start sites at +1 (major promoter) and -26 (minor promoter). The major promoter responds strongly to virus-encoded trans activators EIA and EIV and contains four elements: a TAGA motif analogous to the TATA box, two EIIF sites present in an inverted orientation, and an ATF/CREB site. To determine precisely the roles played by these cis-acting elements in both basal and virus-induced transcription when the promoter is situated in its natural context, we investigated the phenotype of a series of linker scan promoter substitution mutants inserted into the viral chromosome. Promoter constructs harboring linker scan mutations in each element were rebuilt into a novel EIA- adenovirus vector, and transcriptional activity was monitored in virus-infected cells. In the absence of virus-encoded trans activators, basal activity in vivo was dependent on all four cis-acting elements. Surprisingly, a promoter mutant with only one of the two EIIF sites intact could not promote transcription in vivo, suggesting that the two EIIF sites function cooperatively even in basal transcription. Promoters harboring mutations in either of these two EIIF sites also failed to bind to an infection-specific form of EIIF in gel shift assays and competed only very weakly for EIIF binding with the wild-type promoter fragment. The dramatic cooperativity shown by the two inverted EIIF sites of the EII promoter both in vivo and in vitro could reflect simultaneous contact of both sites by the transcription factor EIIF. Furthermore, promoter mutants with mutations in the TAGA motif, the two EIIF sites, and the single ATF site all failed to respond to virus-encoded trans activators. Whereas recent results demonstrate that EIIF activity can be modulated independently by EIV, leading to transactivation of this promoter, our results and those published previously strongly indicate that the three different transcription factors that bind to TAGA, EIIF, and ATF motifs of the EII early promoter are all targets for EIA regulation in vivo. Thus, strong transactivation of the EII early promoter through these multiple EIA-sensitive elements and independently by the recently discovered EIV pathway suggests that the EII early promoter is stringently regulated in virus-infected cells.(ABSTRACT TRUNCATED AT 400 WORDS)