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Biomedical subjects

E S Dennis

Publications and source records attributed to E S Dennis.

At least 19 recordsLinked to original sources
PubMed1995

Effective ribozyme delivery in plant cells.

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.

Base Sequence↗
PubMed1995

Symbiotic and nonsymbiotic hemoglobin genes of Casuarina glauca.

Casuarina glauca has a gene encoding hemoglobin (cashb-nonsym). This gene is expressed in a number of plant tissues. Casuarina also has a second family of hemoglobin genes (cashb-sym) expressed at a high level in the nodules that Casuarina forms in a nitrogen-fixing symbiosis with the actinomycete Frankia. Both the nonsymbiotic and symbiotic genes retained their specific patterns of expression when introduced into the legume Lotus corniculatus. We interpret this finding to mean that the controls of expression of the symbiotic gene in Casuarina must be similar to the controls of expression of the leghemoglobin genes that operate in nodules formed during the interaction between rhizobia and legumes. Deletion analyses of the promoters of the Casuarina symbiotic genes delineated a region that contains nodulin motifs identified in legumes; this region is critical for the controlled expression of the Casuarina gene. The finding that the nonsymbiotic Casuarina gene is also correctly expressed in L. corniculatus suggests to us that a comparable non-symbiotic hemoglobin gene will be found in legume species.

Actinomycetales↗
PubMed1994

The expression and anaerobic induction of alcohol dehydrogenase in cotton.

The alcohol dehydrogenase (ADH) system in cotton is characterized, with an emphasis on the cultivated allotetraploid species Gossypium hirsutum cv. Siokra. A high level of ADH activity is present in seed of Siokra but quickly declines during germination. When exposed to anaerobic stress the level of ADH activity can be induced several fold in both roots and shoots of seedlings. Unlike maize and Arabidopsis, ADH activity can be anaerobically induced in mature green leaves. Three major ADH isozymes were resolved in Siokra, and it is proposed that two genes, Adh1 and Adh2, are coding for these three isozymes. The genes are differentially expressed. ADH1 is predominant in seed and aerobically grown roots, while ADH2 is prominent in roots only after anaerobic stress. Biochemical analysis demonstrated that the ADH enzyme has a native molecular weight of approximately 81 kD and a subunit molecular weight of approximately 42 kD, thus establishing that ADH in cotton is able to form and is active as dimers. Comparisons of ADH activity levels and isozyme patterns between Siokra and other allotetraploid cottons showed that the ADH system is highly conserved among these varieties. In contrast, the diploid species of cotton all had unique isozyme patterns.

Alcohol Dehydrogenase↗
PubMed1994

Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene.

The Adh (alcohol dehydrogenase, EC 1.1.1.1.) gene from Arabidopsis thaliana (L.) Heynh. can be induced by dehydration and cold, as well as by hypoxia. A 1-kb promoter fragment (CADH: -964 to +53) is sufficient to confer the stress induction and tissue-specific developmental expression characteristics of the Adh gene to a beta-glucuronidase reporter gene. Deletion mapping of the 5' end and site-specific mutagenesis identified four regions of the promoter essential for expression under the three stress conditions. Some sequence elements are important for response to all three stress treatments, whereas others are stress specific. The most critical region essential for expression of the Arabidopsis Adh promoter under all three environmental stresses (region IV: -172 to -141) contains sequences homologous to the GT motif (-160 to -152) and the GC motif (-147 to -144) of the maize Adh1 anaerobic responsive element. Region III (-235 to -172) contains two regions shown by R.J. Ferl and B.H. Laughner ([1989] Plant Mol Biol 12: 357-366) to bind regulatory proteins; mutation of the G-box-1 region (5'-CCACGTGG-3', -216 to -209) does not affect expression under uninduced or hypoxic conditions, but significantly reduces induction by cold stress and, to a lesser extent, by dehydration stress. Mutation of the other G-box-like sequence (G-box-2: 5'-CCAAGTGG-3', -193 to -182) does not change hypoxic response and affects cold and dehydration stress only slightly. G-box-2 mutations also promote high levels of expression under uninduced conditions. Deletion of region I (-964 to -510) results in increased expression under uninduced and all stress conditions, suggesting that this region contains a repressor binding site. Region II (-510 to -384) contains a positive regulatory element and is necessary for high expression levels under all treatments.

Alcohol Dehydrogenase↗
PubMed1994

Promoter elements required for developmental expression of the maize Adh1 gene in transgenic rice.

To define the regions of the maize alcohol dehydrogenase 1 (Adh1) promoter that confer tissue-specific expression, a series of 5' promoter deletions and substitution mutations were linked to the Escherichia coli beta-glucuronidase A (uidA) reporter gene and introduced into rice plants. A region between -140 and -99 not only conferred anaerobically inducible expression in the roots of transgenic plants but was also required for expression in the root cap, embryo, and in endosperm under aerobic conditions. GC-rich (GC-1, GC-2, and GC-3) or GT-rich (GT-1 and GT-2) sequence motifs in this region were necessary for expression in these tissues, as they were in anaerobic expression. Expression in the root cap under aerobic conditions required all the GC- and GT-rich motifs. The GT-1, GC-1, GC-2, and GC-3 motifs, and to a lesser extent the GT-2 motif, were also required for anaerobic responsiveness in rice roots. All elements except the GC-3 motif were needed for endosperm-specific expression. The GC-2 motif and perhaps the GT-1 motif appeared to be the only elements required for high-level expression in the embryos of rice seeds. Promoter regions important for shoot-, embryo-, and pollen-specific expression were proximal to -99, and nucleotides required for shoot-specific expression occurred between positions -72 and -43. Pollen-specific expression required a sequence element outside the promoter region, between +54 and +106 of the untranslated leader, as well as a silencer element in the promoter between -72 and -43.

Alcohol Dehydrogenase↗
PubMed1991

Reactivation of a silent Ac following tissue culture is associated with heritable alterations in its methylation pattern.

Tissue cultures were initiated from embryos with an inactive form of Ac in the wx-m9 Ds-cy allele. Plants regenerated from the cultures showed a high frequency of activation of Ac. That activation was shown to be associated with reduced methylation of cytosine residues at the 5' end of the transposable element. An examination of Ac activity and methylation status of the Ac element in progenies of the regenerant plants demonstrated transmission of the altered epigenotype through two sexual generations. In these progenies no evidence for trans activation of inactive, partially methylated, Ac elements was obtained. These results confirm that in certain instances altered methylation patterns can be inherited through the germ line.

Alleles↗
PubMed1991

The anaerobic responsive element contains two GC-rich sequences essential for binding a nuclear protein and hypoxic activation of the maize Adh1 promoter.

We have identified a protein (GCBP-1) in nuclear extracts from maize suspension cell cultures that binds to specific sequences within the Anaerobic Responsive Element (ARE) of the maize Adh1 promoter. Competition analyses show that the GCBP-1 binding activity distinguishes ARE sequence motifs from other enhancer elements or pUC19 sequences. The binding activities of several mutant ARE sequences define two regions of the ARE important for GCBP-1 binding in vitro, between nucleotides -135 to -131 and nucleotides -120 to -112 of the maize Adh1 promoter. Both regions are required for efficient GCBP-1 binding to occur in vitro. The minimum consensus binding site for GCBP-1 is 5'-GC(G/C)CC-3'. This sequence is similar to a part of the binding site of the human transcription factor Sp1 (1). We demonstrate that maize GCBP-1 and human Sp1 have similar recognition properties. Using ARE mutants in a transient assay in maize protoplasts we have shown that mutation of the GCBP-1 binding sites prevents significant hypoxic activation of the maize Adh1 promoter. These results suggest a direct role for GCBP-1 in the hypoxic activation of Adh1 gene expression. GCBP-1 is present in both uninduced and induced nuclei, indicating that inducible gene expression is not dependent upon synthesis of GCBP-1 and suggesting that post-translational modification of bound GCBP-1 may be important for enhanced transcription to occur.

Alcohol Dehydrogenase↗
PubMed1990

DNA methylation of maize transposable elements is correlated with activity.

Transposition of the maize transposable elements Ac, Spm and Mu is correlated with a lack of methylation. For both Ac (Schwartz & Dennis 1986) and Spm (Fedoroff et al. 1988) methylation of a region upstream and downstream of the site of initiation of transcription results in a loss both of mRNA encoding for the transposase and the ability to excise. We have studied the activation of an inactive, methylated derivative of Ac by using a cycle of tissue culture. A passage through tissue culture results in activation of the element and is correlated with demethylation of the 5' end of the transposase gene. Demethylation and activation may occur as a multistep process. We propose a model in which binding of the Ac transposase to the 5' region of the Ac element prevents methylation following replication. Consequently, demethylation and reactivation occurs.

Alleles↗
PubMed1990

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 promoter. 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/delta 35S(-90), suggesting that sub-region I does not contribute to this general enhancer function.

Alcohol Dehydrogenase↗
PubMed1990

A DNA-binding protein factor recognizes two binding domains within the octopine synthase enhancer element.

A protein that binds to the enhancing element of the octopine synthase gene has been identified in nuclear extracts from maize cell suspension cultures. Two protein-DNA complexes are distinguishable by electrophoretic mobility in gel retardation assays. Footprint analyses of these low and high molecular weight complexes show, respectively, half and complete protection of the ocs-element DNA from cleavage by methidiumpropyl-EDTA.FE(II). Two lines of evidence indicate that the element has two recognition sites, each of which can bind identical protein units. Elements that are mutated in one or the other half and form only the low molecular weight complex interfere with the formation of both the low and high molecular weight complexes by the wild-type element. Protein isolated from a complex with only one binding site occupied can bind to the wild-type ocs-element and generate complexes with protein occupying one or both binding sites. Occupation of both sites of the ocs-element is a prerequisite for transcriptional enhancement.

Amino Acid Oxidoreductases↗
PubMed1990

Nonlegume hemoglobin genes retain organ-specific expression in heterologous transgenic plants.

Hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa have been isolated [Landsmann et al. (1986). Nature 324, 166-168; Bogusz et al. (1988). Nature 331, 178-180]. The promoters of these genes have been linked to a beta-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus. Both promoters directed root-specific expression in transgenic tobacco. When transgenic Lotus plants were nodulated by Rhizobium loti, both promoter constructs showed a high level of nodule-specific expression confined to the central bacteroid-containing portion of the nodule corresponding to the expression seen for the endogenous Lotus leghemoglobin gene. The T. tomentosa promoter was also expressed at a low level in the vascular tissue of the Lotus roots. The hemoglobin promoters from both nonlegumes, including the non-nodulating species, must contain conserved cis-acting DNA signals that are responsible for nodule-specific expression in legumes. We have identified sequence motifs postulated previously as the nodule-specific regulatory elements of the soybean leghemoglobin genes [Stougaard et al. (1987). EMBO J. 6, 3565-3569].

Base Sequence↗
PubMed1990

Chitinase, beta-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation.

Sequence analysis of five gene families that were isolated from tobacco thin cell layer explants initiating floral development [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35] showed that two encode the pathogenesis-related proteins basic chitinase and basic beta-1,3-glucanase, while a third encodes the cell wall protein extensin, which also accumulates during pathogen attack. Another sequence family encodes the water stress-induced protein osmotin [Singh et al. (1989). Plant Physiol. 90, 1096-1101]. We found that osmotin was also induced by viral infection and wounding and, hence, could be considered a pathogenesis-related protein. These genes, which were highly expressed in explants during de novo flower formation but not in explants forming vegetative shoots [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35], were also regulated developmentally in day-neutral and photoresponsive tobacco plants with high expression levels in the roots and moderate- to low-level expression in other plant organs including flowers. An unidentified gene family, FB7-4, had its highest level of expression in the basal internodes. Our findings indicate that these genes, some of which are conventionally considered to encode pathogen-related proteins, also have a complex association with normal developmental processes, including the floral response, in healthy plants.

Amino Acid Sequence↗
PubMed1990

OCSBF-1, a maize ocs enhancer binding factor: isolation and expression during development.

The ocs-elements comprise a family of related 20-base pair DNA sequences with dyad symmetry that are functional components of the promoters of several genes introduced into the plant nucleus by Agrobacterium transformation or infection by DNA viruses. We describe the isolation and characterization of a maize cDNA that encodes a protein, OCSBF-1, that binds specifically to ocs-element sequences. The 21-kilodalton OCSBF-1 protein was encoded by a single copy, intron-less gene. The gene was differentially expressed in maize plants. Developing leaves had a gradient of OCSBF-1 mRNA with the basal portion of the leaves, which contain dividing and differentiating cells, having 40-fold to 50-fold higher levels of OCSBF-1 transcripts than the apical portion of the leaves, where the cells are fully differentiated. Roots and shoots of young plants had levels of OCSBF-1 mRNA similar to the basal portions of developing leaves. OCSBF-1 contained a small basic amino acid region and a potential leucine zipper motif homologous to the DNA-binding domains of the basic region-leucine zipper family of transcription factors such as Jun and GCN4. A truncated protein with the amino-terminal 76 amino acids of OCSBF-1, encompassing the basic domain and leucine zipper motif, still bound to ocs-element sequences in vitro. OCSBF-1 was able to bind to a site within each half of the ocs-element as well as to animal AP-1 and CREB sites.

Amino Acid Sequence↗
PubMed1989

The ocs-element is a component of the promoters of several T-DNA and plant viral genes.

The ocs-element is an enhancer element first identified in the promoter of the octopine synthase gene (OCS) where it occurs as a 16 bp palindromic sequence. The transcriptional enhancing activity of the ocs-element correlated with in vitro binding of a transcription factor. We have now identified ocs-elements in the promoter regions of six other T-DNA genes involved in opine synthesis and three plant viral promoters including the 35S promoter of cauliflower mosaic virus. These elements bind the ocs transcription factor in vitro and enhance transcription in plant cells. Comparison of the sequences of these 10 elements has defined a 20 bp consensus sequence, TGACG(T/C)AAG(C/G)(G/A)(A/C)T(G/T)ACG(T/C)(A/C)(A/C), which includes the 16 bp palindrome in its central region. We propose the name ocs-element for this class of promoter elements of similar sequence and function.

Base Sequence↗
PubMed1989

Molecular analysis of the ADH1-Cm allele of maize.

The Adh1-Cm allele and each gene in the Adh1-FCm duplication have been cloned and restriction-mapped. Of the Cm allele 6 kb was sequenced. A single amino acid substitution of aspartate for tyrosine at residue 52 accounts for the altered enzymatic properties of the Cm protein. Comparison of the nucleotide sequence to that of Adh1-1F and Adh1-1S shows structural and restriction site polymorphisms in the 3' flanking DNA. Cm lacks the insertion sequence present in 1F and 1S and contains a complex sequence composed of two direct repeats and an inverted repeat. The two genes of the duplication allele have similar restriction maps to Cm and each other.

Alcohol Dehydrogenase↗