DNA Sequence Elements Research Articles

Repetitive DNA Elements as a Major Component of Plant Genomes

A major part of the nuclear genome of most plants is composed of different repetitive DNA elements. Studying these sequence elements is essential for our understanding of the nature and consequences of genome size variation between different species, and for studying the large-scale organization and evolution of plant genomes. Sugar beet ( Beta vulgarisL.) is an important crop and a suitable model for such investigations: with a genome size of 0.8 pg 1C (760 Mbp) it contains significant amounts of all major groups of repetitive sequences among its nine chromosome pairs, but analysis is not complicated by polyploidy or the huge size of some genomes, and there are valuable genetic data, recombinant DNA libraries and wild relatives to complement studies of sequence contribution to genome size in sugar beet. A sophisticated understanding of the structure of the genome will provide valuable data about the major factors responsible for genome size variation, useful aids in the development of a molecular understanding of genome evolution, and perhaps indicate strategies for crop improvement. Using molecular and cytological approaches, we have characterized a range of differentially organized repetitive DNA sequence elements from the genomes of cultivated and wild beet species, leading to an extensive model of the repetitive DNA, its organization and evolution.

Sequence-specific Binding of Ku Autoantigen to Single-stranded DNA

Glucocorticoid-induced transcription of mouse mammary tumor virus is repressed by Ku antigen/DNA-dependent protein kinase (DNA-PK) through a DNA sequence element (NRE1) in the viral long terminal repeat. Nuclear factors binding to the separated single strands of NRE1 have been identified that may also be important for transcriptional regulation through this element. We report the separation of the upper-stranded NRE1 binding activity in Jurkat T cell nuclear extracts into two components. One component was identified as Ku antigen. The DNA sequence preference for Ku binding to single-stranded DNA closely paralleled the sequence requirements of Ku for double-stranded DNA. Recombinant Ku bound the single, upper strand of NRE1 with an affinity that was 3-4-fold lower than its affinity for double-stranded NRE1. Sequence-specific single-stranded Ku binding occurred rapidly (t1/2 on = 2.0 min) and was exceptionally stable, with an off rate of t1/2= 68 min. While Ku70 cross-linked to the upper strand of NRE1 when Ku was bound to double-stranded and single-stranded DNAs, the Ku80 subunit only cross-linked to single-stranded NRE1. Intriguingly, addition of Mg2+ and ATP, the cofactors required for Ku helicase activity, induced the cross-linking of Ku80 to a double-stranded NRE1-containing oligonucleotide, without completely unwinding the two strands.

Differential Transcriptional Regulation of the Human Squalene Synthase Gene by Sterol Regulatory Element-binding Proteins (SREBP) 1a and 2 and Involvement of 5′ DNA Sequence Elements in the Regulation

Transcription of the human squalene synthase (HSS) gene is regulated by variations in the level of cellular cholesterol. Three regulatory elements in the HSS promoter region are known to be involved in the regulation: 1) a modified sterol regulatory element (SRE) 1 (HSS-SRE-1), 2) an inverted SRE-3 (Inv-SRE-3), 3) an inverted Y box (Inv-Y-Box). We report here the regulatory role of distinct cis-elements in the HSS promoter by using mutants of an HSS-luciferase promoter reporter. The activity of a wild-type promoter reporter transiently transfected into HepG-2 cells is increased by sterol depletion of the cells or by coexpression of mature forms of the SRE-binding proteins (SREBP) 1a and SREBP-2. Differential activation by SREBP-1a and SREBP-2 of the reporter gene mutated at various regions of the promoter is observed. Mutation of either the HSS-SRE-1 or the Inv-SRE-3 sequence diminished the activation by SREBP-1a and by sterol depletion but did not affect the activation by SREBP-2. Simultaneous mutations of both of these sequences almost completely abolished activation of the promoter by SREBP-1a or by sterol depletion, but activation by SREBP-2 was retained at 70%. Mutation of the Inv-Y-Box sequence element decreased the activity of the promoter by 50% or more, and if mutated together with both SREs, the activation was almost completely abolished. Mutation of any single GC box of the two located at -40 to -57 did not affect activity, whereas simultaneous mutation of the two decreased activation by SREBP-2 by 60%, by lipid depletion by 20%, and had no effect on the activation by SREBP-1a. A Y box motif at -159 to -166 and an SRE-like sequence element (SRE-1(8/10)) at position -101 to -108 are also involved in the sterol regulation. These results indicate that the complex sterol-mediated transcriptional regulation of the HSS gene is due to the presence of multiple copies of diverse cis elements in the HSS promoter. The differential activation of the HSS promoter may point to specific role of the SREBPs in cholesterogenesis.

Spacing requirements for interactions between the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase and the cAMP receptor protein.

During transcription initiation at bacterial promoters, the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) can interact with DNA-sequence elements (known as UP elements) and with activator proteins. We have constructed a series of semi-synthetic promoters carrying both an UP element and a consensus DNA-binding site for the Escherichia coli cAMP receptor protein (CRP; a factor that activates transcription by making direct contacts with alphaCTD). At these promoters, the UP element was located at a variety of distances upstream of the CRP-binding site, which was fixed at position -41.5 bp upstream of the transcript start. At some positions, the UP element caused enhanced promoter activity whereas, at other positions, it had very little effect. In no case was the CRP-dependence of the promoter relieved. DNase I and hydroxyl-radical footprinting were used to study ternary RNA polymerase-CRP-promoter complexes formed at two of the most active of these promoters, and co-operativity between the binding of CRP and purified alpha subunits was studied. The footprints show that alphaCTD binds to the UP element as it is displaced upstream but that this displacement does not prevent alphaCTD from being contacted by CRP. Models to account for this are discussed.

Regulation of transcription initiation at the Escherichia coli nir operon promoter: a new mechanism to account for co-dependence on two transcription factors.

Expression from the Escherichia coli nir promoter is co-dependent on Fnr (a transcription factor triggered by oxygen starvation) and on NarL or NarP (transcription factors triggered by nitrite and nitrate ions). Fnr binds to a single DNA site centred between basepairs 41 and 42 upstream from the nir transcript start, whereas NarL and NarP bind to a site upstream, centred between basepairs 69 and 70. A novel mechanism to account for co-dependence on Fnr and NarL/NarP is suggested from experiments in which the spacing between the DNA sites for Fnr and NarL/NarP was altered. DNA sequence elements located upstream of the NarL/NarP-binding site are the targets for two or more proteins that act to repress Fnr-dependent activation of the nir promoter. This inhibition is counteracted by NarL or NarP. The model has been corroborated by the effects of several deletions and single base substitutions in the nir promoter upstream sequences: these deletions and substitutions prevent the binding of the repressor proteins. One of these repressors has been identified as the Fis protein, that binds to a site located 135-149bp upstream of the nir transcript start: the binding of Fis is suppressed by a single base substitution at position -146. The other repressor protein(s) have yet to be identified, but appear to bind downstream of the DNA site for Fis: binding is suppressed by a single base substitution at position -99.

The human plasminogen activator inhibitor type I gene promoter targets to kidney.

The plasminogen activator inhibitor type 1 (PAI-1) gene encodes the physiological inhibitor of tissue-type and urokinase-type plasminogen activators and is induced by cytokines such as transforming growth factor-beta (TGF-beta). Studies have identified DNA sequence elements within the first 1.3 kb of the 5'-upstream DNA that mediate cytokine responsiveness in transfected cells in vitro. However, the DNA sequences that mediate PAI-1 expression in vivo have not yet been delineated. To define these regulatory sequences, we generated transgenic mice that expressed a hybrid gene comprising sequences between -1,272 and +75 of the human PAI-1 gene ligated to a LacZ reporter gene. Transgene expression detected in two independent lines was observed only in kidney from embryonic day 13 to adult and was seen primarily in proximal tubule cells of the outer medulla. Transgene expression and activity were unchanged in response to TGF-beta and remained restricted to kidney. Thus we have identified a promoter region within the PAI-1 gene that targets transgene expression to kidney but, unlike the native promoter, is unresponsive to TGF-beta in the experimental protocol used.

Structure and polymorphism of ribosomal RNA genes in the marine shrimp Penaeus vannamei

We have examined the structure and polymorphism of the genes that encode ribosomal RNA in the Pacific whiteleg shrimp, Penaeus vannamei. We have found that the rRNA genes are polymorphic within and between individual shrimp and between selectively bred families. The polymorphism suggests the presence of repeated DNA sequence elements in the intergenic spacer region upstream from the promoter for transcription of the rRNA genes. In other organisms, such repeated elements have been shown to serve as alternative promoters or enhancers for transcription. If the same is true in P. vannamei, the observed polymorphism might be linked to traits important for shrimp aquaculture.

Regulation of RNA Polymerase I Transcription in Yeast and Vertebrates

This article focuses on what is currently known about the regulation of transcription by RNA polymerase I (pol I) in eukaryotic organisms at opposite ends of the evolutionary spectrum--a yeast, Saccharomyces cerevisiae, and vertebrates, including mice, frogs, and man. Contemporary studies that have defined the DNA sequence elements are described, as well as the majority of the basal transcription factors essential for pol I transcription. Situations in which pol I transcription is known to be regulated are reviewed and possible regulatory mechanisms are critically discussed. Some aspects of basal pol I transcription machinery appear to have been conserved from fungi to vertebrates, but other aspects have evolved, perhaps to meet the needs of a metazoan organism. Different parts of the pol I transcription machinery are regulatory targets depending on different physiological stimuli. This suggests that multiple signaling pathways may also be involved. The involvement of ribosomal genes and their transcripts in events such as mitosis, cancer, and aging is discussed.

Telomere variation in Xenopus laevis.

Eukaryotic telomeres are variable at several levels, from the length of the simple sequence telomeric repeat tract in different cell types to the presence or number of telomere-adjacent DNA sequence elements in different strains or individuals. We have investigated the sequence organization of Xenopus laevis telomeres by use of the vertebrate telomeric repeat (TTAGGG)n and blot hybridization analysis. The (TTAGGG)n-hybridizing fragments, which ranged from less than 10 to over 50 kb with frequently cutting enzymes, defined a pattern that was polymorphic between individuals. BAL 31 exonuclease treatment confirmed that these fragments were telomeric. The polymorphic fragments analyzed did not hybridize to 5S RNA sequences, which are telomeric according to in situ hybridization. When telomeric fragments from offspring (whole embryos) were compared to those from the spleens of the parents, the inheritance pattern of some bands was found to be unusual. Furthermore, in one cross, the telomeres of the embryo were shorter than the telomeres of the parents' spleen, and in another, the male's testis telomeres were shorter than those of the male's spleen. Our data are consistent with a model for chromosome behavior that involves a significant amount of DNA rearrangement at telomeres and suggest that length regulation of Xenopus telomeres is different from that observed for Mus spretus and human telomeres.

Retrofitting YACs for direct DNA transfer into plant cells.

The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant-selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6- to 12-fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures.

Human bZIP Transcription Factor GeneNRL:Structure, Genomic Sequence, and Fine Linkage Mapping at 14q11.2 and Negative Mutation Analysis in Patients with Retinal Degeneration

TheNRLgene encodes an evolutionarily conserved basic motif-leucine zipper transcription factor that is implicated in regulating the expression of the photoreceptor-specific gene rhodopsin.NRLis expressed in postmitotic neuronal cells and in lens during embryonic development, but exhibits a retina-specific pattern of expression in the adult. To understand regulation ofNRLexpression and to investigate its possible involvement in retinopathies, we have determined the complete sequence of the humanNRLgene, identified a polymorphic (CA)nrepeat (identical to D14S64) within theNRL-containing cosmid, and refined its location by linkage analysis. Since a locus for autosomal recessive retinitis pigmentosa (arRP) has been linked to markers at 14q11 and since mutations in rhodopsin can lead to RP, we sequenced genomic PCR products of theNRLgene and of the rhodopsin-Nrl response element from a panel of patients representing independent families with inherited retinal degeneration. The analysis did not reveal any causative mutations in this group of patients. These investigations provide the basis for delineating the DNA sequence elements that regulateNRLexpression in distinct neuronal cell types and should assist in the analysis ofNRLas a candidate gene for inherited diseases/syndromes affecting visual function.

Distinct cis-acting sequences are required for the germination and sugar responses of the cucumber isocitrate lyase gene

Recent data have shown that distinct DNA sequence elements direct the germination and sugar responses of the cucumber ( Cucumis sativus, L.) malate synthase ( Ms) gene ( Sarah et al. (1996)Mol. Gen. Genet., 250, 153–161). Such information is, however, lacking for the isocitrate lyase ( Icl) gene which is coordinately regulated with Ms. Deletions from the 5′ end of the Icl promoter were therefore created specifically to address this question. Analysis of expression in seeds of transgenic Nicotiana plumbaginifolia plants showed that whereas a promoter sequence of 2.9 kilobases (kb) produced a normal germination response, deletion to −1568 base pairs (bp) dramatically reduced this response. Examination of the sugar response employed a transgenic cucumber root system. In this case, the 2900 bp and 1568 bp promoters both gave a strong sugar response, but further deletion to −1367 bp eliminated the response. Therefore, the germination and sugar responses of the Icl gene require distinct cis-acting elements, located respectively upstream and downstream of −1568 bp. This observation is consistent with distinct signal transduction systems regulating gene expression in each case.

Structural flexibility in transcription complex formation revealed by protein-DNA photocrosslinking.

The Oct-1 POU domain binds diverse DNA-sequence elements and forms a higher-order regulatory complex with the herpes simplex virus coregulator VP16. The POU domain contains two separate DNA-binding domains joined by a flexible linker. By protein-DNA photocrosslinking we show that the relative positioning of the two POU DNA-binding domains on DNA varies depending on the nature of the DNA target. On a single VP16-responsive element, the POU domain adopts multiple conformations. To determine the structure of the Oct-1 POU domain in a multiprotein complex with VP16, we allowed VP16 to interact with previously crosslinked POU-domain-DNA complexes and found that VP16 can associate with multiple POU-domain conformations. These results reveal the dynamic potential of a DNA-binding domain in directing transcriptional regulatory complex formation.

Activation of RNA polymerase II by topologically linked DNA-tracking proteins.

Almost all proteins mediating transcriptional activation from promoter-distal sites attach themselves, directly or indirectly, to specific DNA sequence elements. Nevertheless, a single instance of activation by a prokaryotic topologically linked DNA-tracking protein has also been demonstrated. The scope of the latter class of transcriptional activators is broadened in this work. Heterologous fusion proteins linking the transcriptional activation domain of herpes simplex virus VP16 protein to the sliding clamp protein beta of the Escherichia coli DNA polymerase III holoenzyme are shown to function as topologically DNA-linked activators of yeast and Drosophila RNA polymerase II. The beta:VP16 fusion proteins must be loaded onto DNA by the clamp-loading E. coli gamma complex to be transcriptionally active, but they do not occupy fixed sites on the DNA. The DNA-loading sites of these activators have all the properties of enhancers: they can be inverted and their locations relative to the transcriptional start site are freely adjustable.

Accessory Subunit of Mitochondrial DNA Polymerase fromDrosophila Embryos: CLONING, MOLECULAR ANALYSIS, AND ASSOCIATION IN THE NATIVE ENZYME

A full-length cDNA of the accessory (beta) subunit of mitochondrial DNA polymerase from Drosophila embryos has been obtained, and its nucleotide sequence was determined. The cDNA clone encodes a polypeptide with a deduced amino acid sequence of 361 residues and a predicted molecular mass of 41 kDa. The gene encoding the beta subunit lies within 4 kilobase pairs of that for the catalytic subunit in the Drosophila genome, on the left arm of chromosome 2. The two genes have similar structural features and share several common DNA sequence elements in their upstream regions, suggesting the possibility of coordinate regulation. A human cDNA homolog of the accessory subunit was identified, and its nucleotide sequence was determined. The human sequence encodes a polypeptide with a predicted molecular mass of 43 kDa that shows a high degree of amino acid sequence similarity to the Drosophila beta subunit. Subunit-specific rabbit antisera, directed against the recombinant catalytic and accessory subunit polypeptides overexpressed and purified from Escherichia coli, recognize specifically and immunoprecipitate the native enzyme from Drosophila embryos. Demonstration of the physical association of the two subunits in the Drosophila enzyme and identification of a human accessory subunit homolog provide evidence for a common heterodimeric structure for animal mitochondrial DNA polymerases.

A transcriptional insulator element, the su(Hw) binding site, protects a chromosomal DNA replication origin from position effects.

Eukaryotic chromosomes are organized into domains of activity for both transcription and DNA replication. Transcriptional "border," or "insulator," elements have been implicated in mediating the organization of transcriptional domains. However, the DNA sequence elements which might demarcate domains of DNA replication activity are unknown. su(Hw) protein binding sites [su(Hw)BSs] are potent transcriptional insulator elements which can block enhancer action, as well as positive and negative chromosomal position effects. Here we report that flanking su(Hw)BSs can also create a chromosomal domain permissible for activity of the chorion gene DNA replication origin. During Drosophila oogenesis the chorion (eggshell) gene loci are amplified approximately 80-fold through repeated initiation of DNA replication. The cis-acting amplification control element, on the third chromosome (ACE3), is required for high levels of amplification initiating at the nearby major origin of replication, Ori-beta. A transgenic chorion locus construct containing ACE3 and Ori-beta was able to amplify but was extremely sensitive to position effects: only 7 of 21 independent insertions amplified >10-fold. The inclusion of flanking su(Hw)BSs in the construct dramatically protected DNA replication from position effects: 31 of 31 insertions now amplified >10-fold, and this protection was reduced in a su(Hw) mutant background. Amplification was equal on both sides of the su(Hw)BS, demonstrating that replication fork passage is not significantly impeded by these sites. Inclusion of only a single su(Hw)BS in the construct did not detectably protect the chorion gene DNA replication origin from position effects.

Sequence-specific DNA Binding and Transcription Factor Phosphorylation by Ku Autoantigen/DNA-dependent Protein Kinase: PHOSPHORYLATION OF Ser-527 OF THE RAT GLUCOCORTICOID RECEPTOR

NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (Kd = 0.84 +/- 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.

Positive and negative DNA sequence elements are required to establish the pattern of Pax3 expression.

The transcription start site and DNA sequence elements required for the induction of Pax3 expression in differentiating P19 embryonal carcinoma cells have been localized. These elements consist of a promoter and additional elements located within 1.6 kbp 5' to the transcription start site. Sequence elements within this 1.6 kbp region are also sufficient to mediate the induction and dorsal restriction of Pax3 in the neural tube and somites of transgenic mice throughout the hindbrain and trunk. Additional elements required for expression anterior to the hindbrain and in migrating myoblasts are located within 14 kbp 5' to the transcription start site. This region also contains element(s) that repress Pax3 expression in the ventral body wall mesoderm of the tail bud.

Regulation of the major detoxication functions by phenobarbital and 3-methylcholanthrene in co-cultures of rat hepatocytes and liver epithelial cells.

In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CYP), including CYP1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase alpha (GST alpha), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O-deethylation activity was well maintained and ethoxyresorufin O-deethylation activity gradually decreased during co-culture time. In addition, we showed that phenobarbital and 3-methylcholanthrene treatments resulted in a significant increase of these activities. Two general patterns of accumulation of liver-specific mRNAs were observed. CYP1A1/2, CYP2B1/2, CYP3A1/2, GST alpha, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYP1A1/2 (13.6-fold) and GST alpha (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (11.2-fold), GST alpha (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treated co-cultures. Furthermore, we demonstrated that liver-specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1 which were expressed in both cell types during the co-culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells. Finally, to investigate whether co-cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163-bp DNA sequence element reported to confer phenobarbital responsiveness. A 2-3-fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital-treated co-cultures transfected with the phenobarbital-responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments. Our results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.

Characterization of the chicken beta-globin insulator.

Insulators, first identified in Drosophila, are DNA sequence elements that shield a promoter from nearby regulatory elements. We have previously reported that a DNA sequence at the 5' end of the chicken beta-globin locus can function as an insulator. It is capable of shielding a reporter gene from the activating effects of a nearby mouse beta-globin locus control region element in the human erythroleukemic cell line K562. In this report, we show that most of the insulating activity lies in a 250-bp CpG island (core element), which contains the constitutive DNase I-hypersensitive site (5'HS4). DNA binding assays with the core sequence reveal a complex protein binding pattern. The insulating activity of the core element is multiplied when tandem copies are used. Although CpG islands are often associated with promoters of housekeeping genes, we find little evidence that the core element is a promoter. Furthermore, the insulator differs from a promoter in its ability to block the locus control region effect directionally.