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PubMed1992

FGF-mediated aspects of skeletal muscle growth and differentiation are controlled by a high affinity receptor, FGFR1.

Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) play major roles in vertebrate embryogenesis, including control of skeletal muscle growth and differentiation. Understanding their roles requires delineating the specific FGF and FGFR isoforms involved. This study analyzes the FGFR transcripts found in a model mouse skeletal myoblast cell line (MM14) during growth and terminal differentiation. MM14 cells express transcripts for FGFR1 (flg) but not FGFR2 (bek). The predominate FGFR1 transcript contains three immunoglobulin (Ig)-like domains in the extracellular ligand binding region. Approximately one-fourth of the three Ig-like domain transcripts possess a 6-nt deletion between the first and second Ig-like domains which after translation would result in deletion of an Arg-Arg pair. Cloning of mouse genomic DNA surrounding the region of the FGFR1 6-nt deletion indicates that the deletion is derived by alternative splicing of FGFR1 transcripts. Transcripts containing two Ig-like domains account for less than 5% of total FGFR1 mRNA in MM14 cells. A survey of RNA from mouse tissues indicated that two Ig-like domain FGFR1 transcripts are rare in all tissues except in lung, in which the two Ig-like domain form accounts for roughly 70% of the lung FGFR1 mRNA. PCR RACE cloning studies disclosed 162 nt of additional FGFR1 5'-flanking RNA which was highly GC-rich. FGFR1 transcripts decline 8- to 10-fold during low serum, (-)FGF-mediated differentiation of MM14 cultures. The kinetics of the FGFR1 mRNA decline is similar to the previously described differentiation-dependent decrease in cell surface FGF receptors.

Amino Acid Sequence↗
PubMed1992

Screening for mutations in human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE).

Previously, we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2, the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.

Base Sequence↗
PubMed1992

Analysis of the regulation of the pelBC genes in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi secretes five major isoenzymes of pectate lyases encoded by the pelABCDE genes. The nucleotide sequence of the region surrounding the pelB gene of E. chrysanthemi 3937 was determined, including the regulatory regions involved in pelB and pelC expression. Analysis of the transcripts showed that transcription of pelB or pelC gave, in both cases, only one transcript. The transcription initiation sites of both pelB and pelC were precisely determined as well as the position of the transcription termination of pelB. The pelB and pelC promoters are very similar, showing a good homology with the -35 consensus region but low homology with the -10 consensus. In both cases a KdgR-box overlaps the -35 region. The pelC gene may have two KdgR operators. Moreover, the pelB and pelC genes are preceded by other sequences presenting the typical symmetry of operator sites that could be involved in more specific regulations. Comparison of E. chyrsanthemi pel regulatory regions revealed three classes of homology: pelA, pelB-pelC and pelD-pelE. The sole regulatory sequence conserved among the three classes corresponds to the KdgR-binding site. Moreover, all the pel regulatory regions are AT-rich in contrast to the coding regions which are GC-rich. Gel retardation experiments with fragments overlapping the pelB or pelC regulatory regions demonstrated that the KdgR protein specifically binds to these regions. Other proteins probably also interact with these DNA fragments. Transcription of pelB terminates in a region corresponding to a GC-rich inverted repeat followed by a run of T residues, typical of rho-independent transcription termination sites. Moreover, preliminary results imply that a region adjacent to pelC provoke, directly or indirectly, the repression of pelB and pelC expression.

Amino Acid Sequence↗
PubMed1992

Drug-DNA sequence-dependent interactions analysed by electric linear dichroism.

The interactions between 20 drugs and a variety of synthetic DNA polymers and natural DNAs were studied by electric linear dichroism (ELD). All compounds tested, including several clinically used antitumour agents, are thought to exert their biological activities mainly by virtue of their abilities to bind to DNA. The selected drugs include intercalating agents with fused and unfused aromatic structures and several groove binders. To examine the role of base composition and base sequence in the binding of these drugs to DNA, ELD experiments were carried out with natural DNAs of widely differing base composition as well as with polynucleotides containing defined alternating and non-alternating repeating sequences, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT),poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). Among intercalating agents, actinomycin D was found to be by far the most GC-selective. GC selectivity was also observed with an amsacrine-4-carboxamide derivative and to a lesser extent with methylene blue. In contrast, the binding of amsacrine and 9-aminoacridine was practically unaffected by varying the GC content of the DNAs. Ethidium bromide, proflavine, mitoxantrone, daunomycin and an ellipticine derivative were found to bind best to alternating purine-pyrimidine sequences regardless of their nature. ELD measurements provided evidence for non-specific intercalation of amiloride. A significant AT selectivity was observed with hycanthone and lucanthone. The triphenyl methane dye methyl green was found to exhibit positive and negative dichroism signals at AT and GC sites, respectively, showing that the mode of binding of a drug can change markedly with the DNA base composition. Among minor groove binders, the N-methylpyrrole carboxamide-containing antibiotics netropsin and distamycin bound to DNA with very pronounced AT specificity, as expected. More interestingly the dye Hoechst 33258, berenil and a thiazole-containing lexitropsin elicited negative reduced dichroism in the presence of GC-rich DNA which is totally inconsistent with a groove binding process. We postulate that these three drugs share with the trypanocide 4',6-diamidino-2-phenylindole (DAPI) the property of intercalating at GC-rich sites and binding to the minor groove of DNA at other sites. Replacement of guanines by inosines (i.e., removal of the protruding exocyclic C-2 amino group of guanine) restored minor groove binding of DAPI, Hoechst 33258 and berenil. Thus there are several cases where the mode of binding to DNA is directly dependent on the base composition of the polymer. Consequently the ELD technique appears uniquely valuable as a means of investigating the possibility of sequence-dependent recognition of DNA by drugs.

Base Sequence↗
PubMed1992

Construction of a chimeric ArsA-ArsB protein for overexpression of the oxyanion-translocating ATPase.

Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli is conferred by the conjugative R-factor R773, which encodes an ATP-driven anion extrusion pump. The ars operon is composed of three structural genes, arsA, arsB, and arsC. Although transcribed as a single unit, the three genes are differentially expressed as a result of translational differences, such that the ArsA and ArsC proteins are produced in high amounts relative to the amount of ArsB protein made. Consequently, biochemical characterization of the ArsB protein, which is an integral membrane protein containing the anion-conducting pathway, has been limited, precluding studies of the mechanism of this oxyanion pump. To overexpress the arsB gene, a series of changes were made. First, the second codon, an infrequently used leucine codon, was changed to a more frequently utilized codon. Second, a GC-rich stem-loop (delta G = -17 kcal/mol) between the third and twelfth codons was destabilized by changing several of the bases of the base-paired region. Third, the re-engineered arsB gene was fused 3' in frame to the first 1458 base pairs of the arsA gene to encode a 914-residue chimeric protein (486 residues of the ArsA protein plus 428 residues of the mutated ArsB protein) containing the entire re-engineered ArsB sequence except for the initiating methionine. The ArsA-ArsB chimera has been overexpressed at approximately 15-20% of the total membrane proteins. Cells producing the chimeric ArsA-ArsB protein with an arsA gene in trans excluded 73AsO2- from cells, demonstrating that the chimera can function as a component of the oxyanion-translocating ATPase.

Adenosine Triphosphatases↗
PubMed1992

Structure of the human DNA ligase I gene.

The gene encoding DNA ligase I, the major DNA ligase activity in proliferating mammalian cells, maps to human chromosome 19q13.2-13.3. We have determined the complete structure of the gene, which is composed of 28 exons spanning 53kb on this chromosome. The first exon is untranslated, and utilises a GC dinucleotide instead of the canonical GT splice donor. The 5' flanking region lacks a TATA box and is highly GC-rich, as is characteristic of a 'housekeeping' gene. In common with the promoters of genes encoding other DNA replication enzymes, such as DNA polymerase alpha, the 5' flanking region of the DNA ligase I gene contains recognition elements for several transcription factors which may mediate increased expression in quiescent cells in response to growth factors.

Base Sequence↗
PubMed1992

A nuclear protein is required for thyroid hormone receptor binding to an inhibitory half-site in the epidermal growth factor receptor promoter.

The epidermal growth factor (EGF) receptor (EGFR) promoter is negatively regulated by thyroid hormone and retinoic acid. This regulation can be mapped to a 36-basepair GC-rich region of the promoter (EGFR P/E) that functions autonomously as a promoter and an enhancer when placed in front of the thymidine kinase gene TATA element. Direct high affinity binding of the thyroid hormone receptor (T3R) to this element requires a nuclear protein. Through ion exchange chromatography and gel filtration of HeLa nuclear extract, this activity was identified as a protein of approximately 67 kilodaltons. This protein did not bind to DNA alone, but greatly augmented T3R binding to the EGFR P/E sequence in gel mobility shift and DNA precipitation assays. When combined with the T3R auxillary protein (TRAP), the T3R migrated as a larger complex on the DNA. Chemical cross-linking identified this complex as a heterodimer between T3R and TRAP. T3R-TRAP binds to a 7-basepair site in the EGFR P/E (GGGACTC) that has weak homology to a consensus thyroid response element half-site. Thus, on this element, T3R-TRAP heterodimers contact the DNA primarily on a single site that comprises an inhibitory thyroid response element.

Base Sequence↗
PubMed1992

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.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1992

[Chiasma distribution in the lampbrush chromosomes of the chicken Gallus gallus domesticus: hot spots of recombination and their possible role in proper dysjunction of homologous chromosomes at the first meiotic division].

Chiasma distribution in the lambrush chromosomes of the chicken Gallus gallus domesticus was studied. The data of the authors show that the general pattern of chiasmata in the interstitional region of chromosomes corresponds to the Poisson distribution. However, in the telomeric and subtelomeric regions of all chicken macrochromosomes one can see chiasma as a rule. In the half of 140 microchromosomes from 24 different oocytes, there are also the telomeric chiasmata. On the basis of this observation, it may be predicted that there are hot spots of recombination near or into the telomeric GC-rich heterochromatic bands of chicken chromosomes. We suggest that these hot spots of recombination near the telomeres are a necessary facility for not only macrochromosomes but all microchromosomes as well to have at least one chiasma. The constant presence of at least one chiasma in a bivalent in needed for correct disjunction of homologous chromosomes at the first meiotic division.

Animals↗
PubMed1992

The parathyroid hormone-related protein (PTHrP) gene preferentially utilizes a GC-rich promoter and the PTHrP 1-139 coding pathway in normal human amnion.

Parathyroid hormone-related protein (PTHrP) is an oncofetal protein that is widely expressed in mammalian tissues. The PTHrP gene is a complex with three transcriptional start-sites, two TATA boxes and a GC-rich region, and three predicted polypeptide products, PTHrP 1-141, PTHrP 1-139, and PTHrP 1-173. The originally discovered form of PTHrP, PTHrP 1-141, and the classical TATA box promoters are generally assumed to be the major pathways of PTHrP gene expression. We have used the polymerase chain reaction (PCR) to study PTHrP gene expression in the human amnion. Our studies demonstrate that the GC-rich promoter is preferentially used and that PTHrP 1-139 is the major PTHrP mRNA expressed in human amnion. PTHrP 1-139 lacks the carboxy-terminal arginine and histidine residues of PTHrP 1-141; these two basic amino acids could have significant effects on the biological activity of PTHrP. These preferential pathways for PTHrP gene expression are shared by malignant and normal human tissues.

Amnion↗
PubMed1992

Cloning and characterization of the 5'-flanking region of the human topoisomerase II alpha gene.

Topoisomerases are essential enzymes for DNA metabolism in prokaryotes and eukaryotes. In human cells, DNA topoisomerase II enzyme activity can be modulated by both viral transformation and changes in proliferation status. To identify elements important for regulation of topoisomerase II alpha gene expression, genomic DNA clones covering the 5'-end of the gene were isolated. The intron/exon structure of a 2.5-kilobase region encompassing the translation start site was determined. Transcription was found to initiate at multiple sites clustered around 90 base pairs 5' to the ATG initiation codon. Transient expression of chimeric topoisomerase II-reporter gene constructs in HeLa cells revealed that the 5'-flanking region exhibited promoter activity. The region -90 to -1 upstream of the major transcription start site was shown by deletion analysis to include a promoter. This minimal promoter lacks a TATA box, is moderately GC-rich, and contains a high frequency of CpG dinucleotides; characteristic of a "housekeeping" gene promoter. Maximal promoter activity was observed using a fragment extending to position -562. Putative regulatory elements are contained within and immediately upstream of the minimal promoter region. The regulatory region of the topoisomerase II alpha gene identified here is similar in basic structure to those of the human thymidine kinase and DNA polymerase alpha genes, which are also controlled by proliferation-specific factors.

Base Sequence↗
PubMedDate not supplied

Polymerase chain reaction analysis of fragile X mutations.

The mutation that underlies the fragile X syndrome is presumed to be a large expansion in the number of CGG repeats within the gene FMR-1. The unusually GC-rich composition of the expanded region has impeded attempts to amplify it by the polymerase chain reaction (PCR). We have developed a PCR protocol that successfully amplifies the (CGG)n region in normal, carrier and affected individuals. The PCR analysis of several large fragile X families is presented. The PCR results agree with those obtained by direct genomic Southern blot analyses. These favorable comparisons suggest that the PCR assay may be suitable for rapid testing for fragile X mutations and premutations and genetic screening of at-risk individuals.

Base Sequence↗
PubMed1992

The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II.

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

Animals↗
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↗
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↗
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↗
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↗
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↗