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PubMed1992

A novel myoblast enhancer element mediates MyoD transcription.

The MyoD gene can orchestrate the expression of the skeletal muscle differentiation program. We have identified the regions of the gene necessary to reproduce transcription specific to skeletal myoblasts and myotubes. A proximal regulatory region (PRR) contains a conserved TATA box, a CCAAT box, and a GC-rich region that includes a consensus SP1 binding site. The PRR is sufficient for high levels of skeletal muscle-specific activity in avian muscle cells. In murine cells the PRR alone has only low levels of activity and requires an additional distal regulatory region to achieve high levels of muscle-specific activity. The distal regulatory region differs from a conventional enhancer in that chromosomal integration appears necessary for productive interactions with the PRR. While the Moloney leukemia virus long terminal repeat can enhance transcription from the MyoD PRR in both transient and stable assays, the simian virus 40 enhancer cannot, suggesting that specific enhancer-promoter interactions are necessary for PRR function.

Animals↗
PubMed1991

The expression of cathepsin B and other lysosomal proteinases in normal tissues and in tumors.

The mRNA for the lysosomal proteinases cathepsins B, D, H, L, and S are broadly distributed in normal rodent tissues. Although total cathepsin mRNA levels generally parallel the protein catabolic activity of the tissues, the expressions of the individual enzymes do not appear to be linked. Thus, the relative proportions of the individual messages are found to vary from tissue to tissue. Further evidence for the independent regulation of lysosomal proteinase expression is derived from observations of selective increases in mRNA levels for individual proteinases in rodent tumors. Only cathepsin B mRNA is elevated in a highly metastatic murine B16a melanoma and in a Walker-256 rat carcinosarcoma, while Moloney murine sarcoma virus-transformed fibroblasts express increased mRNA for cathepsins B, D, and L and normal levels for H and S. To address the regulation of cathepsin B expression, the mouse cathepsin B gene and its 5'-upstream region were cloned. The gene has 10 exons and 9 introns spanning about 20 kilobases. The 5'-upstream region and exon 1 are GC-rich with several potential Sp1 binding sites. TATA and CAAT motifs adjacent to the transcription start site are not evident. These properties are characteristic of mammalian "housekeeping" genes. B16 melanoma cells contain three cathepsin B transcripts of 2.2, 4.0 and 5.0 kilobases. The two larger messages, which were not found in normal tissues, contain unusually long 3'-untranslated regions resulting from the alternative cleavage and polyadenylation of the 3' end of the cathepsin B pre-mRNA in B16 melanomas. As all three messages encoded normal preprocathepsin B, cathepsin B secretion by melanoma cells is probably due to posttranslational mechanisms and not to alternative splicing or gene mutation.

Animals↗
PubMed1991

The oct3 gene, a gene for an embryonic transcription factor, is controlled by a retinoic acid repressible enhancer.

Oct3 is an embryonic octamer-binding transcription factor, whose expression is rapidly repressed by retinoic acid (RA). In this report, we have determined the transcriptional control region of the oct3 gene and studied the mechanism of the RA-mediated repression. The chromosomal oct3 gene consists of five exons. Three subdomains of the POU region and transactivating domain are located in separate exons. Transcription initiates at multiple sites in the GC-rich region lacking a typical TATA box. The upstream 2 kb region can confer the cell type-specific expression and RA-mediated repression. Analysis of the upstream region by deletion mutagenesis locates a cis element (RARE1) which functions as a stem cell-specific, yet RA-repressible, enhancer. Footprint and gel-retardation assays show that RARE1 is composed of two domains, each of which is recognized by distinct factors. Microinjection of oct3-lacZ constructs into fertilized eggs indicates that RARE1 can function in early embryos. We suggest that RARE1 is a critical cis element for oct3 gene expression in embryonic stem cells and for the RA-mediated repression.

Amino Acid Sequence↗
PubMed1992

Molecular and functional analysis of the XPBC/ERCC-3 promoter: transcription activity is dependent on the integrity of an Sp1-binding site.

The human XPBC/ERCC-3 gene, which corrects the excision-repair defect in xeroderma pigmentosum group B cells and the UV-sensitive CHO mutant 27-1 cells, appears to be expressed constitutively in various cell types and tissues. We have analysed the structure and functionality of the XPBC/ERCC-3 promoter. Transcription of the XPBC/ERCC-3 gene is initiated from heterogeneous sites, with a major startpoint mapped at position -54 (relative to the translation start codon ATG). The promoter region does not possess classical TATA and CAAT elements, but it is GC-rich and contains three putative Sp1-binding sites. In addition, there are two elements related to the cyclic AMP (cAMP)-response element (CRE) and the 12-O-tetradecanoyl phorbol-13-acetate-response element (TRE) in the 5'-flanking region. Transient expression analysis of XPBC/ERCC-3 promoter-CAT chimeric plasmids revealed that a 127-bp fragment, spanning position -129 to -3, is minimally required for the promoter activity. Transcription of the XPBC/ERCC-3 promoter depends on the integrity of a putative Sp1-binding site in close proximity to the major cap site. Band shift assays showed that this putative Sp1-binding site can interact specifically with a nuclear factor, most likely transcription factor Sp1 (or an Sp1-like factor) in vitro.

Animals↗
PubMed2008

TAp73beta and DNp73beta activate the expression of the pro-survival caspase-2S.

p73, the p53 homologue, exists as a transactivation-domain-proficient TAp73 or deficient deltaN(DN)p73 form. Expectedly, the oncogenic DNp73 that is capable of inactivating both TAp73 and p53 function, is over-expressed in cancers. However, the role of TAp73, which exhibits tumour-suppressive properties in gain or loss of function models, in human cancers where it is hyper-expressed is unclear. We demonstrate here that both TAp73 and DNp73 are able to specifically transactivate the expression of the anti-apoptotic member of the caspase family, caspase-2(S). Neither p53 nor TAp63 has this property, and only the p73beta form, but not the p73alpha form, has this competency. Caspase-2 promoter analysis revealed that a non-canonical, 18 bp GC-rich Sp-1-binding site-containing region is essential for p73beta-mediated activation. However, mutating the Sp-1-binding site or silencing Sp-1 expression did not affect p73beta's transactivation ability. In vitro DNA binding and in vivo chromatin immunoprecipitation assays indicated that p73beta is capable of directly binding to this region, and consistently, DNA binding p73 mutant was unable to transactivate caspase-2(S). Finally, DNp73beta over-expression in neuroblastoma cells led to resistance to cell death, and concomitantly to elevated levels of caspase-2(S.) Silencing p73 expression in these cells led to reduction of caspase-2(S) expression and increased cell death. Together, the data identifies caspase-2(S) as a novel transcriptional target common to both TAp73 and DNp73, and raises the possibility that TAp73 may be over-expressed in cancers to promote survival.

Binding Sites↗
PubMed1992

A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins.

The KAR2 gene of Saccharomyces cerevisiae codes for an essential chaperone protein (BiP) that is localized in the lumen of the endoplasmic reticulum (ER). The high basal rate of transcription of KAR2 is increased transiently by heat shock: prolonged induction occurs when unfolded proteins accumulate in the ER. Three cis-acting elements in the KAR2 promoter control expression of KAR2: (i) a GC-rich region that contributes to the high level of constitutive expression, (ii) a functional heat shock element (HSE) and (iii) an element (UPR) that is involved in the induction of BiP mRNA by unfolded proteins. By analyzing internal deletion mutants of the KAR2 promoter, we demonstrate here that these three elements regulate transcription of KAR2 independently. Furthermore, the 22 bp UPR element causes a heterologous (CYC1) promoter to respond to the presence of unfolded proteins in the ER. Extracts of both stressed and unstressed yeast cells contain proteins that bind specifically to synthetic HSE and UPR elements and retard their migration through gels. Binding proteins specific for the UPR element can be fractionated by ammonium sulfate precipitation. Two of the proteins UPRF-1 and UPRF-2 (which is apparently a proteolytic degradation product of UPRF-1) bind inefficiently to mutant versions of the UPR that are unable to confer responsiveness to unfolded proteins to the (CYC1) promoter. UPRF-1 therefore displays the properties expected of a transcription factor that is involved in the sustained response of the KAR2 promoter to unfolded proteins in the ER. These experiments show that yeast cells can activate a transcription factor that stimulates expression of a nuclear gene in response to the accumulation of unfolded proteins in another cellular compartment.

Base Sequence↗
PubMed1990

Photoregulated gene expression may involve ubiquitous DNA binding proteins.

Several promoter elements have previously been shown to influence the expression of the cab-E gene in Nicotiana plumbaginifolia. Here we demonstrate, by electrophoretic mobility shift and methylation interference assays, that a complex pattern of protein-DNA interactions characterizes this promoter. Among the multiple proteins identified, we focused on five different factors which either occupied important regulatory elements and/or were present in relatively large amounts in nuclear extracts. All of these proteins were distinguished on the basis of their recognition sequence and other biochemical parameters. One, GBF, interacted with a single sequence within the cab-E promoter homologous to the G-box found in many photoregulated and other plant promoters. A second factor, GA-1, bound to the GATA element which is located between the CAAT and TATA boxes of the cab-E and all other LHCII Type I CAB promoters. GA-1 also interacted in vitro with the I-boxes of the Arabidopsis rbcS-1A promoter and the as-2 site of the CaMV 35S promoter. Two other factors, GC-1 and AT-1, bound to multiple recognition sites localized within the GC-rich and AT-rich elements, respectively. GT-1, a protein which interacts with promoters of other light-regulated genes, bound to seven distinct sites distributed throughout the cab-E promoter.

Base Sequence↗
PubMed1991

Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants.

We have found previously that the sequences important for recognition of pre-mRNA introns in dicot plants differ from those in the introns of vertebrates and yeast. Neither a conserved branch point nor a polypyrimidine tract, found in yeast and vertebrate introns respectively, are required. Instead, AU-rich sequences, a characteristic feature of dicot plant introns, are essential. Here we show that splicing in protoplasts of maize, a monocot, differs significantly from splicing in a dicot, Nicotiana plumbaginifolia. As in the case of dicots, a conserved branch point and a polypyrimidine tract are not required for intron processing in maize. However, unlike in dicots, AU-rich sequences are not essential, although their presence facilitates splicing if the splice site sequences are not optimal. The lack of an absolute requirement for AU-rich stretches in monocot introns in reflected in the occurrence of GC-rich introns in monocots but not in dicots. We also show that maize protoplasts are able to process a mammalian intron and short introns containing stem--loops, neither of which are spliced in N.plumbaginifolia protoplasts. The ability of maize, but not of N.plumbaginifolia to process stem--loop-containing or GC-rich introns suggests that one of the functions of AU-rich sequences during splicing of dicot plant pre-mRNAs may be to minimize secondary structure within the intron.

Base Sequence↗
PubMed1979

Variable substructure in the secondary constriction of the human chromosome 1.

The secondary constriction in human chromosome 1 consists of a proximal segment stained by the GC-specific fluorochrome mithramycin and a distal segment stained by such fluorochromes as DAPI or DIPI, which show enhanced fluorescence intensities in AT-rich regions of the chromosomes. A study involving 21 individuals revealed that both parts are independently involved in length variability. In two cases, two GC-rich regions separated by an AT-rich segment and an additional distal AT-rich part were found.

Base Sequence↗
PubMed1990

Compositional patterns in the nuclear genome of cold-blooded vertebrates.

DNA preparations obtained from 122 species of fishes, 5 species of amphibians, and 13 species of reptiles were investigated in their compositional properties by analytical equilibrium centrifugation in CsCl density gradients. These species represented 21 orders of Osteichthyes, 3 orders of Chondrichthyes, 2 orders of amphibians, and 3 orders of reptiles. Modal buoyant densities of fish DNAs ranged from 1.696 to 1.707 g/cm3, the vast majority of values falling, however, between 1.699 and 1.704 g/cm3, which is the range covered by the DNAs of amphibians and reptiles. In all cases, DNA bands in CsCl were only weakly asymmetrical and only very rarely were accompanied by separate satellite bands (mostly on the GC-rich side). Intermolecular compositional heterogeneities were low in the vast majority of cases, and, like CsCl band asymmetries, at least partially due to cryptic or poorly resolved satellites. The present findings indicate, therefore, that DNAs from cold-blooded vertebrates are characterized by a number of common properties, namely a very wide spectrum of modal buoyant densities, low intermolecular compositional heterogeneities, low CsCl band asymmetries, and, in most cases, small amounts of satellite DNAs. In the case of fish DNAs a negative correlation was found between the GC level and the haploid size (c value) of the genome. If polyploidization is neglected, this phenomenon appears to be mainly due to the fact that increases and decreases in GC are associated with contraction and expansion phenomena, respectively, of intergenic noncoding sequences, which are GC poor relative to coding sequences.

Amphibians↗
PubMed1991

The distribution of genes in the human genome.

Previous investigations on the human genome determined: (i) the base compositions (GC levels) and the relative amounts of its isochore families; (ii) the compositional correlations (i.e., the correlations between GC levels) between third codon positions of a set of genes and the DNA fractions in which the genes were localized; and (iii) the compositional correlations between (a) third and first + second codon positions, as well as that between (b) introns and exons from the set of 'localized genes' and from all the coding sequences and genes (genomic sequences of exons + introns) available in gene banks. Here, we have shown that the correlations (iii, a and b) for 'localized genes' and genes from the bank are in full agreement, indicating that the former set is representative of the latter. We have then used the data (i) and the correlation (ii) to estimate the distribution of genes in isochore families. We have found that 34% of the genes are located in the GC-poor isochores (which represent 62% of the genome), 38% in the GC-rich isochores (31% of the genome) and 28% in the GC-richest isochores (3% of the genome). There is, therefore, a compositional gradient of gene concentration in the human genome. The gene density in the GC-richest 3% of the genome is about eight times higher than in the GC-rich 31%, and about 16 times higher than in the GC-poorest 62%.

Base Composition↗
PubMed1992

Compositional bimodality and evolution of retroviral genomes.

The compositional distributions of genomes, genes (and their third codon positions) and long terminal repeats from retroviruses of warm-blooded vertebrates are characterized by a striking bimodality which is accompanied by a remarkable compositional homogeneity within each retroviral genome. A first, major class of retroviral genomes is GC-rich, whereas a second, minor class is GC-poor. Representative expressed viral genomes from the two classes integrate in GC-rich and GC-poor isochores, respectively, of host genomes. The first class comprises all oncoviruses (except B-types and some D-types), the second, lentiviruses, spumaviruses, as well as B-type and some D-type oncoviruses (e.g., mouse mammary tumor virus and simian retroviruses type D, respectively). The compositional bimodal distribution of retroviral genomes and the accompanying compositional homogeneity within each retroviral genome appear to be the result of the compositional evolution of retroviral genomes in their integrated form.

Animals↗
PubMed1990

Interaction of berenil with the tyrT DNA sequence studied by footprinting and molecular modelling. Implications for the design of sequence-specific DNA recognition agents.

We have developed a technique of partially-restrained molecular mechanics enthalpy minimisation which enables the sequence-dependence of the DNA binding of a non-intercalating ligand to be studied for arbitrary sequences of considerable length (greater than = 60 base-pairs). The technique has been applied to analyse the binding of berenil to the minor groove of a 60 base-pair sequence derived from the tyrT promoter; the results are compared with those obtained by DNAse I and hydroxyl radical footprinting on the same sequence. The calculated and experimentally observed patterns of binding are in good agreement. Analysis of the modelling data highlights the importance of DNA flexibility in ligand binding. Further, the electrostatic component of the interaction tends to favour binding to AT-rich regions, whilst the van der Waals interaction energy term favours GC-rich ones. The results also suggest that an important contribution to the observed preference for binding in AT-rich regions arises from lower DNA perturbation energies and is not accompanied by reduced DNA structural perturbations in such sequences. It is therefore concluded that those modes of DNA distortion favourable to binding are probably more flexible in AT-rich regions. The structure of the modelled DNA sequence has also been analysed in terms of helical parameters. For the DNA energy-minimised in the absence of berenil, certain helical parameters show marked sequence-dependence. For example, purine-pyrimidine (R-Y) base pairs show a consistent positive buckle whereas this feature is consistently negative for Y-R pairs. Further, CG steps show lower than average values of slide while GC steps show lower than average values of rise. Similar analysis of the modelling data from the calculations including berenil highlights the importance of DNA flexibility in ligand binding. We observe that the binding of berenil induces characteristic responses in different helical parameters for the base-pairs around the binding site. For example, buckle and tilt tend to become more negative to the 5'-side of the binding site and more positive to the 3'-side, while the base steps at either side of the centre of the site show increased twist and decreased roll.

Amidines↗
PubMed1991

The structure of the gene coding for the mouse cell adhesion molecule uvomorulin.

We have recently shown that the Ca2+ dependent cell adhesion molecule uvomorulin is encoded by a single gene, localized on mouse chromosome 8. Here we describe the organization of the uvomorulin gene and give an initial characterization of the uvomorulin promoter. Uvomorulin is encoded by 16 exons, which are distributed over a region of more than 40 kb genomic DNA. The exon structure of the genes for uvomorulin and its chicken homologue L-CAM are nearly identical and thus highly conserved. The relationship between the exon structure and the structure of the uvomorulin protein is analysed. The initiation site of transcription of the uvomorulin gene is located 127 bp upstream of the translation start site in a GC-rich region with no TATA-box, but with a GC-box in position -48 and a CCAAT-box starting at position -65 with respect to the transcription start site. 1.6 kb of the uvomorulin promoter (-1492 to + 92) confer cell type specific promoter activity to the CAT reporter gene. Homologies to known cis acting elements of other promoters are discussed.

3T3 Cells↗
PubMed1991

An essential iteron-binding protein required for plasmid R1162 replication induces localized melting within the origin at a specific site in AT-rich DNA.

The R1162-encoded protein RepIB is essential for replication of the plasmid and binds specifically to iterons within the replicative origin. The protein causes the localized melting of DNA (determined by sensitivity to P1 nuclease) at a site within the AT-rich region of the origin, about 60 bp from the iteron binding sites and separated from them by a GC-rich tract. Point mutations have been isolated in the AT-rich DNA. These mutations interfere with origin activity and also prevent the protein-induced sensitivity to P1. A second-site suppressor of one of these mutations maps in the repIb gene and restores both origin function and sensitivity to P1. The results suggest a specific interaction between RepIB and origin DNA at a position distant from its primary binding site.

Adenine↗
PubMed1975

Denaturation mapping studies on the circular chloroplast deoxyribonucleic acid from pea leaves.

The structure of circular pea chloroplast DNA (ctDNA) has been analyzed by denaturation mapping. All of the pea ctDNA molecules that were examined had identical gross base sequences. Denaturation maps were constructed at denaturation levels of 2.5%, 22%, and 44%. These denaturation maps showed that the circular pea ctDNA contained six small AT-rich regions on one-half of the DNA molecule, and two small GC-rich regions on the other half of the DNA molecule. The structure of pea ctDNA circular dimers was also examined. The results showed that the pea ctDNA circular dimers consisted of two monomer length units integrated in tandem repeat.

Chloroplasts↗
PubMed1992

Structure and developmental regulation of the B-lymphoid tyrosine kinase gene blk.

The murine blk gene, which encodes a B-lymphoid-specific tyrosine kinase of the Src family (p55blk), contains 13 exons that span more than 30 kilobases of DNA on chromosome 14. In the first three exons, which encode the 5'-untranslated region and N-terminal amino acid sequence unique to p55blk, the blk gene differs from other members of the src family; in the last 10 exons, the organization of the blk gene is similar to that of other src genes. By primer extension and S1 nuclease protection analyses, we show that blk transcripts initiate from four major sites at the 5'-flank of blk; two sites predominate. The resulting transcripts differ only in the lengths of their 5'-untranslated sequences and encode identical proteins. None of the transcriptional start sites are preceded by consensus TATA elements, AT-rich elements, or extensive GC-rich regions. Expression of blk is regulated during B-cell development: blk RNA is expressed in all pro-B-, pre-B-, and mature B-cell lines examined, but is absent from plasma cell lines. Immunolocalization of p55blk in normal mouse spleen supports these observations: staining is restricted to lymphocytes and is concentrated in regions rich in B-cells; plasma cells and stromal cells are not stained with anti-Blk antibodies. Assays for RNA synthesis in isolated nuclei indicate that the lineage and developmental stage specificities of blk expression are regulated at least in part by changes in its rate of transcription.

Amino Acid Sequence↗
PubMed1992

Chromosome bands, their chromatin flavors, and their functional features.

To show that the input pattern of chromosomal mutations is highly organized relative to the band patterns along human chromosomes, a new term, "metaphase chromatin flavor," is introduced. Five different flavors of euchromatic metaphase bands are cytologically identified along a human ideogram. These are G-bands and, based upon combinations of extreme Alu richness and GC richness, four different R-band flavors. The two flavors with extremely GC-rich components, traditionally called "T-bands," represent only 15% of all bands. However, they contain 65% of mapped genes, 19 of 25 mapped oncogenes, most cancer-associated rearrangements, evolutionary rearrangements, meiotic chiasmata, and X-ray-induced breaks. Flavors with extremely Alu-rich flavors are also involved in melphalan-induced rearrangements, pachytene stretching, and mitotic chiasmata. Frequencies of CpG islands, CCGCCC boxes, retroposon families, and genes are characteristic to each chromatin flavor and will facilitate alignment of genome sequences onto ideograms of chromatin flavor. The influence of chromatin flavor on the evolution of a gene's sequence is so strong that one can infer the flavor of the band in which a gene resides from the sequence of the gene itself. Correlation coefficients for many pairs of mapped genetic variables, while globally high, are quite low within bands of one flavor, implicating a concerted mode of evolution for bands of one chromatin flavor.

Biological Evolution↗