Chemical detection of Z-DNA within the maize Adh1 promoter

Z-DNA is a left-handed helix which can form within tracts of alternating purines and pyrimidines. Tracts of potential Z-DNA identified by sequence inspection are often noted within regulatory portions of genes, but evidence that these tracts of sequence actually exist as Z-DNA is very limited, and not available for any plant gene. In this study, the chemical probes osmium tetroxide, diethylpyrocarbonate and hydroxylamine were used to show that a tract of alternating purines and pyrimidines in the Adh1 promoter (from -311 to -325) actually assumes a Z-DNA conformation under superhelical stress in vitro.     

Regulation of DNA replication by homopurine/homopyrimidine sequences

The simple repeating homopurine/homopyrimidine sequences dispersed throughout many eukaryotic genomes are known to form triple helical structures comprising three-stranded and single-stranded DNA. Several lines of evidence suggest that these structures influence DNA replication in cells. Homopurine/homopyrimidine sequences cloned into simian virus 40 (SV40) or SV40 origin-containing plasmids caused a reduced rate of DNA synthesis due to the pausing of replication forks. More prominent arrests were observed in in vitro experiments using single-stranded and double-stranded DNA with triplex-forming sequences. Nucleotides unable to form triplexes when present in the template DNA or when incorporated into the nascent strand prevented termination. Similarly, mutations destroying the triplex potential did not cause arrest while compensatory mutations restoring triplex potential restored it. These and other observations from a number of laboratories indicating that homopurine/homopyrimidine sequences act as arrest signals in vitro and as pause sites in vivo during replication fork movement suggest that these naturally occurring sequences play a regulatory role in DNA replication and gene amplification.     

In vivo footprinting identifies an activating element of the maize Adh2 promoter specific for root and vascular tissues

In vivo footprinting identifies four putative cis elements of Adh2 that interact with protein factors within the DNase I hypersensitive domains of the 5′ flanking region. The power of in vivo footprinting to identify functionally significant sites within a gene promoter was tested by biochemical and transgenic analyses of the putative element at position −160. Biochemical analyses show that proteins isolated from maize cell suspensions will bind to the Adh2 promoter in vitro to generate a footprint at −160 identical to that seen in vivo. The partially purified factor bound to the promoter in vitro can be specifically competed with fragments of DNA containing the element sequence, further demonstrating that a specific protein generates the footprint over that sequence. Transgenic analyses indicate that the −160 element is a functional element of the maize Adh2 promoter that acts as an activator in the meristem and vascular tissue of roots and in the vascular tissue of stems and leaves.     

Unusual Protonated Structure in the Homopurine · Homopyrimidine Tract of Supercoiled and Linearized Plasmids Recognized by Chemical Probes

Abstract

Plasmid pEJ4, which is a derivative of pUC19 containing an insert with 60-bp-long · homopurine homopyrimidine tract from sea urchin P. miliaris histone gene spacer, was studied by chemical probes of the DNA structure osmium tetroxide and glyoxal. The former probe reacts with pyrimidine bases, while the latter forms a stable product only with guanine residues. These probes can thus be applied as specific probes for the homopyrimidine and homopurine strands, respectively.

At pH 6.0 the site-specific modification of the homopurine · homopyrimidine tract by both probes was observed at native superhelical density of the plasmid. In the linear plasmid under the same conditions this modification was absent; it appeared, however, at more acid pH values. In supercoiled DNA the hypersensitivity of the homopurine homopyrimidine tract to osmium tetroxide did not substantially change when pH was decreased from 6.0 to 4.0. Changes in NaCl concentration at pH 4.5 did not influence the hypersensitivity to osmium tetroxide; at pH 6.0 this hypersensitivity decreased with increasing NaCl concentration. These results thus show that the chemical probes recognize an unusual protonated structure containing unpaired bases or non-Watson-Crick base pairs. At pH 5.6 the site-specific modification occurred at or near to the middle of the homopurine · homopyrimidine tract, suggesting that a hairpin may be involved in the unusual structure under the given conditions. From the models suggested so far for the unusual structure of homopurine · homopyrimidine tracts our results fit best the protonated triplex H form suggest by V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 3, 667 (1986).     

Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny.

Summary In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1–2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carring the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.     

Functional properties of the anaerobic responsive element of the maize Adh1 gene

The functional properties of the anaerobic responsive element (ARE) of the maize Adh1 gene have been analysed using a transient expression assay in electroporated maize protoplasts. The ARE functions in both orientations although inversion of the ARE sequence relative to the TATA box element produces slightly weaker promoter activity under anaerobic conditions and elevated expression under aerobic conditions. Promoter activity under anaerobic conditions is proportional to the number of complete ARE sequences in the Adh1 promotor. The ARE contains two sub-regions and dimers of sub-region II are as efficient as the wild-type sequence in activating gene expression under anaerobic conditions. However, sub-region I dimers do not appear capable of inducing gene expression in response to anaerobic stress. We conclude that sub-region II is essential for anaerobic induction of gene expression. Reporter gene expression remains constant when the spacing between sub-regions of the ARE is increased up to at least 64 bp, but increased spacing of 136 bp or greater abolishes expression in both aerobic and anaerobic conditions, indicating that a close association of the two sub-regions is required both for anaerobic responsiveness and for maximal levels of aerobic gene expression. When the ARE is placed upstream of position –90 of the CaMV 35S promoter, the ARE produces a high level of expression in both aerobic and anaerobic conditions. The general enhancement of gene expression driven by the hybrid ARE/35S promoter in aerobic conditions requires an intact sub-region II motif since mutation or deletion of sub-region II from the hybrid promoter reduces the level of expression to that observed for the truncated 35S promoter alone. In addition, mutation of the sub-region I sequences in the ARE/35S hybrid promoter does not significantly reduce expression in aerobic conditions, relative to pARE/35S(-90), suggesting that sub-region I does not contribute to this general enhancer function.     

Sequence-specific recognition of the major groove of DNA by oligodeoxynucleotides via triple helix formation. Footprinting studies.

Homopyrimidine oligodeoxynucleotides recognize the major groove of the DNA double helix at homopurine.homopyrimidine sequences by forming local triple helices. The oligonucleotide is bound parallel to the homopurine strand of the duplex. This binding can be revealed by a footprinting technique using copper-phenanthroline as a cleaving reagent. Oligonucleotide binding in the major groove prevents cleavage by copper-phenanthroline. The cleavage patterns on opposite strands of the duplex at the boundaries of the triple helix are asymmetric. They are shifted to the 3'-side, indicating that the copper-phenanthroline chelate binds in the minor groove of the duplex structure. Binding of the chelate at the junction between the triple and the double helix is not perturbed on the 5'-side of the bound homopyrimidine oligonucleotide. In contrast, a strong enhancement of cleavage is observed on the purine-containing strand at the triplex-duplex junction on the 3'-side of the homopyrimidine oligonucleotide.     

Triple Helix Structures: Sequence Dependence,Flexibility and Mismatch Effects

Abstract

By means of molecular modelling, electrostatic interactions are shown to play an important role in the sequence-dependent structure of triple helices formed by a homopyrimidine oligonucleotide bound to a homopurine, homopyrimidine sequence on DNA. This is caused by the presence of positive charges due to the protonation of cytosines in the Hoogsteen-bonded strand, required in order to form C.GxC+ triplets. Energetic and conformational characteristics of triple helices with different sequences are analyzed and discussed. The effects of duplex mismatches on the triple helix stability are investigated via thermal dissociation using UV absorption.     

Expression of maize Adh1 intron mutants in tobacco nuclei

In vivo and in vitro gene transfer experiments have suggested that the elements mediating intron recognition differ in mammalian, yeast and plant nuclei. Differences in the sequence dependencies, which also exist between dicotyledonous and monocotyledonous nuclei, have prevented some monocot introns from being spliced in dicot nuclei. To locate elements which modulate efficient recognition of introns in dicot nuclei, the maize Adh1 gene has been expressed in full-length and single intron constructs in Nicotiana benthamiana nuclei using an autonomously replicating plant expression vector. Quantitative PCR-Southern analyses indicate that the inefficient splicing of the maize Adh1 intron 1 (57% AU) in these dicot nuclei can be dramatically enhanced by increasing the degree of U1 snRNA complementarity at the 5′ splice site. This indicates that the 5′ splice site plays a significant role in defining the splicing efficiency of an intron in dicot nuclei and that, most importantly, the remainder of this monocot intron contains no elements which inhibit its accurate recognition in dicot nuclei. Deletions in intron 3 (66% AU) which effectively move the 3′ boundary between AU-rich intron and GC-rich exon sequences strongly activate a cryptic upstream splice site; those which do not reposition this boundary activate a downstream cryptic splice site. This suggests that 3′ splice site selection in dicot nuclei is extremely flexible and not dependent on strict sequence requirements but rather on the transition points between introns and exons. Our results are consistent with a model in which potential splice sites are selected if they are located upstream (5′ splice site) or downstream (3′ splice site) of AU transition points and not if they are embedded within AU-rich sequences.     

Isolation of a 6.2 kb genomic fragment carrying the Adh1 gene of tomato and its expression in transgenic tobacco

An 11 kb Eco RI genomic fragment containing the alcohol dehydrogenase (Adh1) gene was cloned. Cross-hybridization with three Adh2 cDNA clones suggested that the entire coding region of the Adh1 gene was contained on a 6.2 kb Xba I/Hind III subfragment. Using RFLP linkage analysis, the genomic clone was mapped on chromosome 4 between the markers TG 182 and TG 65 in a position corresponding to the Adh1 locus. To further confirm the Adh1 origin of the genomic clone, tobacco plants were transformed with the 6.2 kb Xba I/Hinb III genomic subfragment. Isozyme analysis demonstrated that in transgenic tobacco plants functional tomato specific ADH-1 homodimers were synthesized as well as heterodimers composed of tobacco and tomato subunits.     

Functional Analysis of Two Matrix Attachment Region (MAR) Elements in Transgenic Maize Plants

Matrix attachment regions (MARs) are binding sites for nuclear scaffold proteins in vitro, and are proposed to mediate the attachment of chromatin to the nuclear scaffold in vivo. Previous reports suggest that MAR elements may stabilize transgene expression. Here, we tested the effects of two maize MAR elements (P-MAR from the P1-rr gene, and Adh1-MAR from the adh1 gene) on the expression of a gusA reporter gene driven by three different promoters: the maize p1 gene promoter, a wheat peroxidase (WP) gene promoter, or a synthetic promoter (Rsyn7). The inclusion of P-MAR or Adh1-MAR on P::GUS transgene constructs did not reduce variation in the levels of GUS activity among independent transformation events, nor among the progeny derived from each event. The Adh1-MAR element did not affect GUS expression driven by the WP promoter, but did modify the spatial pattern of expression of the Rsyn7::GUS transgene. These results indicate that, in transgenic maize plants, the effects of MAR elements can vary significantly depending upon the promoter used to drive the transgene.     

In vivo detection of regulatory factor binding sites of Arabidopsis thaliana Adh

In vivo footprinting experiments have been used to analyze the binding of trans-acting regulatory factors in the 5 flanking region upstream of the alcohol dehydrogenase (Adh) gene from Arabidopsis thaliana. Protein-DNA interactions were detected by dimethyl sulfate footprinting and genomic sequencing, using an A. thaliana cell suspension culture that constitutively expressed the Adh gene. Several distinct footprinting domains have been characterized, and the potential effects of the corresponding trans-acting factors have been inferred from a comparison with data from the maize alcohol dehydrogenase-1 (Adh1) gene. One binding site is similar in sequence to one of the anaerobic response elements (ARE) of the maize gene, which has also been shown to bind to a trans-acting factor. Several of the remaining binding sites apparently represent a class of elements sharing the sequence 5-GTGG-3 within their footprint.Comparisons with maize Adh1 in vivo protein interactions reveal that the elements of Adh promoter structure are highly conserved, but the relative and absolute positions of the elements are variable.     

Strand orientation of [alpha]-oligodeoxynucleotides in triple helix structures: dependence on nucleotide sequence.

The aims of the present theoretical study of the conformations of [alpha]-oligodeoxynucleotides forming triple helices with DNA duplexes are to understand the structural and energetic factors involved in [alpha]-triple helix formation by means of energy minimization, and to explain the experimentally observed dependence of strand orientation on the nucleotide sequence. It is found that the energetically preferred orientation of the [alpha]-oligonucleotide with respect to the homopurine strand depends on the sequence of the homopurine.homopyrimidine tracts. This is a consequence of the structural heteromorphism of base triplets in the intrinsically more stable reverse Hoogsteen hydrogen bonding configuration. Practical rules are proposed for determining the orientation of the nuclease-resistant [alpha]-oligodeoxynucleotide strand which will form the most stable triple helix.     

Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants

A genomic clone encoding the potato homolog of the yeast ubiquitin-ribosomal protein fusion gene ubi3 was isolated and characterized. Chimeric genes containing the ubi3 promoter (920 bp of 5 to the ubiquitin start codon) were constructed in which the reporter gene -glucuronidase (GUS) was either fused directly to the promoter, or introduced as a translational fusion to the ubiquitin-coding region. After introduction into the potato by Agrobacterium-mediated transformation, GUS activities were measured in leaves and in tubers of transgenic clones. GUS activity was 5- to 10-fold higher in clones expressing the ubiquitin-GUS translational fusion than in clones containing GUS fused directly to the ubi3 promoter. For both types of constructs, GUS activity was highest in meristematic leaves and declined during leaf expansion, then rose again to near the meristematic levels during senescence. GUS activity in tubers was similar to that in young leaves. In contrast to the native ubi3 genes, the chimeric ubi3-GUS transgenes were not activated in the tuber by wounding.     

A repeated chromosomal DNA sequence is amplified as a circular extrachromosomal molecule in rice (Oryza sativa L.)

Summary The regulation in tobacco of the rolB and rolC promoters of Agrobacterium rhizogenes pRi 1855 T_L-DNA was studied by using the -glucuronidase (GUS) reporter system in transgenic plants. A 20- to 100-fold increase of GUS activity was selectively induced by auxin in rolB-GUS transformed mesophyll protoplasts, whereas this auxin-dependent increase was only 5-fold in rolC-GUS protoplasts. Moreover, both gene fusions exhibited similar tissue-specific expression in aerial parts but different patterns in roots. The spatial pattern of rolBGUS expression could be strongly modified by the addition of exogenous auxin, further suggesting that auxin plays a central role in the regulation of the rolB promoter in tobacco. The tissue-specific and auxin-dependent regulation of the rolB promoter is discussed in relation to the effects of the rolB gene on rhizogenesis and on cellular responses to auxin.Abbreviations BA benzoic acid - 6-BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - 2,4,5-T 2,4,5,-trichlorophenoxyacetic acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid - IAA indoleacetic acid - NAA naphthaleneacetic acid - MU 4-methyl umbelliferone - 35S CaMV cauliflower mosaic virus 35S (promoter) - TCA trichloroacetic acid - X-Glu 5-bromo-4chloro-3-indolyl -d-glucuronic acid     

Molecular Differences between Alleles Adh1-F and Adh1-Sin Sugar Beet Beta vulgaris L.

We compared nucleotide sequences of exon 4 and part of exon 5 of alleles F and S of the Adh1 locus controlling alcohol dehydrogenase in sugar beet. The Adh1-F and Adh1-S sequences of the examined fragment were shown to differ by two nucleotides. Adenine (A) and cytosine (C) of Adh1-F were substituted by respectively thymine (T) and adenine (A) in Adh1-S. Consequently, glutamine and asparagine from the F subunit of ADH1 are replaced by valine and lysine, respectively. Because of differences in the amino acid content, the F subunit is by two elementary charges more negatively charged electrically than the S subunit, which correlates with differences in their electrophoretic mobility. Comparison of the examined Adh1 fragment of sugar beet with its counterparts in other plants showed that the sites bearing substitutions in the former species are classed as variable.