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Quantitative footprinting analysis of drug-DNA interactions: Fe(III) methidium-propyl-EDTA as a probe.

Quantitative footprinting studies involving a 139-base pair restriction fragment from pBR322 DNA, a lexitropsin ligand and two different DNA cleavage agents, the enzyme DNase I and the footprinting reagent Fe(III) methidium-propyl-EDTA (Fe-MPE), are described. The autoradiographic data showed that the ligand, an analogue of netropsin possessing two N-methylimidazole groups, binds to four regions on the 139-mer which are rich in GC. Analysis of the data leading to individual binding constants for each of the four loading events on the 139-mer revealed that Fe-MPE and DNase I report the same binding constants for the lexitropsin bound to its interaction sequences. The fact that the data from both probes can be analyzed using a common model indicates that the DNA cleavage specificity of the probe and not its binding/cleavage mechanism is the important factor in reporting of site loading information in the footprinting experiment. The study also showed that under certain conditions it is possible to gain information on the density of ligand binding sites on carrier DNA by monitoring site loading events on the labeled fragment.

Base Sequence↗
PubMed1990

DNA sequencing in HydroLink matrices: extension of reading ability to greater than 600 nucleotides.

All the systems for optimizing DNA sequencing published so far have introduced modifications regarding: (i) linearization of band migration via ionic strength gradients or wedge-shaped gels; (ii) automatization of band reading via introduction of fluorescent probes; (iii) direct blotting analysis; (iv) pulsed electric fields and (v) discontinuous buffer systems. In all these systems, DNA sequence reading with an accuracy of ca. 98% rarely exceeds a length of 350 bases. We have chosen, in order to increase the reading ability of a single gel, to manipulate the characteristics of the gel matrix. The Seq-HydroLink gel formation here reported allows optimal reading, from a single gel run, of at least 600 bases. In order to guarantee this reading ability in a single run, the upper and lower ends of the ladder are time-resolved, i.e. the same sample is applied to the gel matrix at three different time intervals. The present system represents an increase of at least 30% in reading ability as compared with any type of polyacrylamide gel formulation so far reported.

Bacteriophages↗
PubMed1983

Structural organization of the rat gene for the arginine vasopressin-neurophysin precursor.

The rat arginine vasopressin-neurophysin precursor gene has been isolated from a genomic library cloned in lambda phage Charon 4A. Restriction mapping and nucleotide sequence analysis demonstrated that the gene is 1.85 kilobase pairs long and contains two intervening sequences located in the protein coding region. Exon A encodes a putative signal peptide, the hormone arginine vasopressin and the variable N terminus of the carrier protein neurophysin, exon B encodes the highly conserved middle part of neurophysin and exon C its variable C terminus together with glycoprotein. Thus, the three functional domains of the percursor - arginine, vasopressin, neurophysin, glycoprotein - are encoded on three distinct exons.

Animals↗
PubMed1983

Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.

Amino Acid Sequence↗
PubMed1987

A cellular transcription factor E4F1 interacts with an E1a-inducible enhancer and mediates constitutive enhancer function in vitro.

Efficient transcription of the adenovirus E1a-inducible E4 gene is mediated by an E1a-dependent enhancer in vivo. In vitro, the enhancer functions constitutively in the absence of E1a, conferring high levels of transcription on the E4 and heterologous promoters. Binding of a cellular transcription factor, E4F1, to two sites within the E4 enhancer and to one other functionally important site located directly upstream of the E4 TATA box is required for transcriptional activity. The relationship between the enhancer and the TATA box proximal site is further demonstrated by the fact that the E4 enhancer can be functionally substituted by two copies of the TATA box proximal site. These and other results suggest that the E4 promoter may be comprised solely of multiple E4F1 binding sites and a TATA box. In addition to the E4 promoter, E4F1 interacts with other adenovirus early promoters and thus may be involved in the co-ordinate expression of E1a-inducible early viral genes.

Adenovirus Early Proteins↗
PubMed1987

Construction of hybrid Tn501/Tn21 transposases in vivo: identification of a region of transposase conferring specificity of recognition of the 38-bp terminal inverted repeats.

In order to study the transposase enzymes of Class II prokaryotic transposable elements, we have constructed genes encoding hybrid transposase proteins. This was done by recombination in vivo between the tnpA genes of transposons Tn501 and Tn21. These hybrid genes can complement in trans a transposition-defective mutant of Tn501. The structures of the products of this complementation indicate whether the specificity of the hybrid transposase in recognising the 38 bp terminal inverted repeats is that of Tn501 or that of Tn21. The determinant of this specificity is in the N-terminal region of the transposase protein, between amino acids 28 and 216. The predicted amino acid sequences so far determined of transposases from the Class II family reveal an area of homology in this region.

Amino Acid Sequence↗
PubMed1987

Cloning and expression of the cDNA coding for a human lymphocyte IgE receptor.

Low-affinity receptors (Fc epsilon R) and secreted factors (IgE-BF) which bind to immunoglobulins of the IgE isotype play a key role in the regulation of human IgE synthesis. We report here the cloning of a cDNA coding for the Fc epsilon R of the human B-lymphoblast cell line RPMI 8866. The nucleotide sequence of this cDNA predicts a polypeptide with 321 amino acids and a mol. wt of 36,281 daltons. A functional Fc epsilon R capable of binding IgE was expressed in Chinese hamster ovary cells after stable transformation with the cDNA which had been cloned into a mammalian expression vector. Amino acid sequence analysis of IgE-BF purified from RPMI 8866 cells revealed an amino-terminal sequence of 19 residues which coincides with the predicted amino acid sequence of the Fc epsilon R, starting at residues 148 and 150. A computer search with the translated amino acid sequence of the Fc epsilon R revealed a domain of 120 amino acids having striking homology to the human asialoglycoprotein receptors.

Amino Acid Sequence↗
PubMed1987

Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes.

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.

Animals↗
PubMed1988

Expression of the G6PD locus on the human X chromosome is associated with demethylation of three CpG islands within 100 kb of DNA.

We have previously reported that expression of the G6PD locus is correlated with the methylation status of two islands of CpG dinucleotides which are 3' to the locus and in the 5' region of two adjacent genes of unknown function, P3 and GdX. We have now examined the methylation of a third CpG island in the promoter region of the G6PD gene itself in DNA from males, females and reactivants that express G6PD on the inactive X chromosome. Our results show that expression of the G6PD gene is associated with concordant demethylation of all three CpG islands in this 100-kb region of DNA.

Animals↗
PubMed1988

Alternative RNA products from the Ultrabithorax domain of the bithorax complex.

The homeotic gene, Ultrabithorax (Ubx) is involved in specifying the identities of several segments in the fly Drosophila melanogaster. The structures of over 60 independent Ubx cDNAs have been examined. There are two major species of transcripts, 3.2 and 4.3 kb in length, which are produced by alternate sites of polyadenylation. Differential splicing gives rise to at least five variant Ubx proteins. The variant forms share common 5' and 3' exons but differ in their small internal 'micro' exons. Additional variation is generated by two separate splice donor sites at the end of the common 5' exon, situated 27 bp apart. Northern hybridization and S1 nuclease protection studies of RNA from various developmental stages and tissue types reveal that the alternate splicing and the choice of polyadenylation site are each differentially regulated in both a temporal and a tissue specific manner. Additional transcripts were found just downstream of the Ubx transcription unit, which may be products of the lethal left of bithorax gene (llb).

Animals↗
PubMed1988

Cis-acting regions sufficient for spermatocyte-specific transcriptional and spermatid-specific translational control of the Drosophila melanogaster gene mst(3)gl-9.

In Drosophila spermatogenesis transcription occurs only premeiotically while translation can be detected also in postmeiotic spermatids. To analyse the underlying processes mst(3)gl-9, a gene specifically expressed in the male germ cells of Drosophila melanogaster, was studied. The putative protein encoded by mst(3)gl-9 is mostly composed of repetitive Cys-Gly-Pro motifs. The transcriptional and translational control of expression of mst(3)gl-9 has been investigated by P-mediated transformation. Only 102 bp of 5' upstream sequences and the first 201 bp of the gene are sufficient to maintain the gene specific characteristics of expression, namely premeiotic transcription and postmeiotic translation separated by 3 days of development.

Amino Acid Sequence↗
PubMed1988

Multiple RNAs expressed from the int-2 gene in mouse embryonal carcinoma cell lines encode a protein with homology to fibroblast growth factors.

Mouse embryonal carcinoma cell lines that differ in their patterns of expression of the potential oncogene int-2 have been exploited in a structural analysis of the multiple RNA transcripts characteristic of this gene. Ribonuclease protection experiments indicate that four major classes of int-2 RNA initiate at heterogeneous cap sites within two distinct promoter regions, P1 and P2, spanning approximately 50 and 150 bp respectively. The more downstream promoter P2 is located in a region of the DNA that constitutes an intron in RNA transcripts that initiate at the upstream promoter P1. Otherwise, all four RNA structures share the same splice donor and acceptor sites that define the boundaries of the second and third exons. Further complexity arises through usage of two distinct polyadenylation signals, both variants of the normal consensus, that are separated by 1100 bp. Despite these structural variations, the results suggest that all four major classes of RNA encode the same protein product which shows significant homology to the family of heparin-binding proteins typified by basic fibroblast growth factor (FGF).

Animals↗
PubMed1988

The genomic organization and transcription of the ubiquitin genes of Trypanosoma cruzi.

We describe here the organization of the ubiquitin genes of the parasitic protozoan Trypanosoma cruzi. T. cruzi contains greater than 100 ubiquitin coding sequences all of which are clustered into a 27 kb segment of the genome. Two types of ubiquitin coding sequences were found. There are five fusion genes (FUS1-5) consisting of a ubiquitin coding sequence fused to a basic non-ubiquitin sequence. The T. cruzi ubiquitin fusion protein is 84% homologous to the product of the UBI gene of Saccharomyces cerevisiae. The non-ubiquitin domains of the two proteins are 67% homologous. There are five polyubiquitin coding genes (PUB) each consisting of varying lengths of polyubiquitin coding sequence and terminating with a single copy of the larger fusion gene. Transcription of the ubiquitin genes results in the generation of six major poly(A)+ mRNAs. The pattern of transcription accurately reflects the genomic organization, in that the transcripts consist of either a single copy of the ubiquitin fusion coding sequence or varying lengths of polyubiquitin (up to 52 copies of the ubiquitin coding unit) each ending with a single copy of the ubiquitin fusion sequence. Finally, there are heat shock elements 5' to the PUB genes and transcription patterns are altered under conditions of stress.

Amino Acid Sequence↗
PubMed1988

The 11-1 gene of Plasmodium falciparum codes for distinct fast evolving repeats.

The 11-1 gene of Plasmodium falciparum has been investigated by DNA sequence analysis. It begins at the 5' end with a putative miniexon coding for a polypeptide which has the characteristics of a signal sequence. The miniexon is followed by a small intron. This again is followed by a large exon consisting of 9-, 18- and 27-bp repeats embedded in unique DNA. Specific antibodies isolated by affinity chromatography on a purified recombinant fusion protein expressing the three- and six-amino acid repeats were used to identify the product of the 11-1 gene. In exhibits size variations from 260 to 350 kd in different strains. Southern blot analysis with synthetic DNA as probe demonstrates that the 18-bp repeat is absent or drastically altered in two strains whereas the other repeats are present in all seven strains investigated. The unusual preference for G in the third position of some codons of the repeats but not in the unique sequences indicates rapid evolution of the repeats. Slippage during replication, unequal crossing over and selection are discussed as possible mechanisms leading rapidly to extreme diversity.

Amino Acid Sequence↗
PubMed1988

Interaction of a nodule specific, trans-acting factor with distinct DNA elements in the soybean leghaemoglobin Ibc(3) 5' upstream region.

Nuclear extracts from soybean nodules, leaves and roots were used to investigate protein-DNA interactions in the 5' upstream (promoter) region of the soybean leghaemoglobin lbc(3) gene. Two distinct regions were identified which strongly bind a nodule specific factor. A Bal31 deletion analysis delimited the DNA elements responsible for the binding of this factor, which map at nucleotides -223 to -246 (element 1) and -161 to -176 (element 2), relative to the start point of transcription. Competition experiments strongly suggest that both elements bind to the same nodule specific factor, but with different affinities. Elements 1 and 2 share a common motif, although their AT-rich DNA sequences differ. Element 2 is highly conserved at an analogous position in other soybean lb gene 5' upstream regions.

Journal Article↗
PubMed1988

Wheat germ splicing endonuclease is highly specific for plant pre-tRNAs.

Intron-containing pre-tRNAs from organisms as different as yeast, Nicotiana, Xenopus and man are efficiently spliced and processed in a HeLa cell extract. They are also correctly processed in a wheat germ extract; however, the intron is removed only from the tobacco pre-tRNA. To determine whether plant pre-tRNA introns have any specific structural and/or sequence feature we have cloned two intron-containing tRNATyr genes from the plant Arabidopsis. Comparison of these genes, of the Nicotiana tRNATyr gene and of a Glycine max tRNAMet gene reveals that plant introns from three different species have no sequence homology and are only 11 to 13 nucleotides long. Thus, short length may be one important feature of plant introns. Furthermore, the 5' and 3' splice sites are separated by 4 bp in the extended anticodon stems of these pre-tRNA structures. In contrast, yeast and vertebrate introns are rather variable in length and the splice sites are separated by 5 or 6 bp. These differences in distance and relative helical orientation of the splice sites in plant pre-tRNAs versus pre-tRNAs from other organisms are obviously tolerated by the vertebrate splicing endonuclease, but not at all by the plant enzyme.

Endoribonucleases↗
PubMed1989

Purification and characterization of the deoR repressor of Escherichia coli.

The deoR gene, which encodes the deor repressor protein in Escherichia coli, was fused to the strong Ptrc promoter in plasmid pKK233-2. The Ptrc promoter is kept repressed by lacI repressor to prevent cell killing. Induction of the Ptrc--deoR fusion plasmid resulted in the accumulation of 4% of the soluble protein as deoR protein. The deoR repressor protein was purified to 80% purity using conventional techniques; it has a mass of 28.5 kd and appears to exist as an octamer in solution. The deoR repressor is shown by DNase I footprinting to bind to the 16 bp palindromic sequence in the Pribnow box region of the deoP1 promoter. Also, the deoR repressor binds cooperatively in vitro to a DNA template with two deoR binding sites separated by 224 bp in keeping with the conclusion from genetic experiments that more than one operator is required for efficient repression of the deo operon.

Bacterial Proteins↗
PubMed1989

DNA replication origin and transcriptional enhancer in c-myc gene share the c-myc protein binding sequences.

We have previously reported that c-myc protein, or protein(s) complexed with c-myc protein, binds to the region upstream of the first exon of the c-myc gene and that this region contains an origin of cellular DNA replication (ori) and also a transcriptional enhancer. Here we show by Southwestern blotting that c-myc protein binds directly to a 7 bp sequence within the above region. Furthermore, we show that the c-myc protein binding sequences are indispensable for both ori and enhancer functions, but that additional sequences are required for maximal ori and enhancer activities. Thus, c-myc protein is a sequence specific factor which is apparently used both in initiation of DNA replication and in regulation of RNA transcription.

Animals↗