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E Lam

Publications and source records attributed to E Lam.

At least 73 records · Page 4Linked to original sources
PubMed1990

Plant nuclear factor ASF-1 binds to an essential region of the nopaline synthase promoter.

We have characterized a tobacco nuclear factor that binds to the -118 region of the nopaline synthase (nos) promoter from the Ti plasmid of Agrobacterium tumefaciens. The binding site for this factor, identified by DNase I footprinting, encompasses the region from -138 to -103 of the nos promoter. This region, which contains a potential Z-DNA-forming sequence, was previously shown to be essential for nos promoter activity in transgenic tobacco. A synthetic 21-base pair sequence from the protected region (from -131 to -111), designated as nos-1, was sufficient for factor recognition in vitro. In transgenic tobacco, a tetramer of nos-1 can confer leaf and root expression when fused upstream of a truncated 35 S promoter from the cauliflower mosaic virus. Mutations at the two TGACG-like motifs in nos-1 abolish factor binding while preserving the potential for Z-DNA formation. A tetramer of the nos-1 mutant sequence has no significant activity above background when tested in transgenic tobacco. Competition experiments with activation sequence factor (ASF)-1 binding sites from the 35 S promoter of cauliflower mosaic virus (as-1) and the wheat histone H3 promoter (hex-1) demonstrate that ASF-1 is the factor that binds to nos-1.

Amino Acid Oxidoreductases↗
PubMed1990

GT-1 binding site confers light responsive expression in transgenic tobacco.

Light-dependent expression of rbcS, the gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase, which is the key enzyme involved in carbon fixation in higher plants, is regulated at the transcriptional level. Sequence analysis of the gene has uncovered a conserved GT motif in the -150 to -100 region of many rbcS promoters. This motif serves as the binding site of a nuclear factor, designated GT-1. Analysis of site-specific mutants of pea rbcS-3A promoter demonstrated that GT-1 binding in vitro is correlated with light-responsive expression of the rbcS promoter in transgenic plants. However, it is not known whether factors other than GT-1 might also be required for activation of transcription by light. A synthetic tetramer of box II (TGTGTGGTTAATATG), the GT-1 binding site located between -152 to -138 of the rbcS-3A promoter, inserted upstream of a truncated cauliflower mosaic virus 35S promoter is sufficient to confer expression in leaves of transgenic tobacco. This expression occurs principally in chloroplast-containing cells, is induced by light, and is correlated with the ability of box II to bind GT-1 in vitro. The data show that the binding site for GT-1 is likely to be a part of the molecular light switch for rbcS activation.

Base Sequence↗
PubMed1990

A metal-dependent DNA-binding protein interacts with a constitutive element of a light-responsive promoter.

We have used DNase I footprinting to characterize nuclear factors that bind to the light-responsive promoter of pea rbcS-3A, one member of the gene family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. A sequence-specific binding activity, designated 3AF1, binds to an AT-rich sequence present at the -45 region of the rbcS-3A promoter. A tetramer of the 3AF1 binding site, designated as Box VI, can form multiple complexes with tobacco leaf and root nuclear extracts. Mutations of 3 base pairs in Box VI severely reduce DNA-protein complex formation in vitro. The wild-type Box VI tetramer, but not the mutant tetramer, is active in transgenic tobacco plants when placed upstream of the cauliflower mosaic virus 35S promoter truncated at -90. These results correlate binding of 3AF1 to the in vivo function of Box VI. The Box VI tetramer/35S chimeric construct confers expression in diverse cell types and organs and its activity is not dependent on light. By using the Box VI tetramer as a probe to screen a cDNA expression library, we have obtained a putative cDNA clone for the 3AF1 DNA-binding activity. Lysogen extracts of Escherichia coli expressing the cDNA clone give sequence-specific complexes with Box VI. The deduced amino acid sequence of the protein encoded by the cDNA contains two stretches of about 100 residues that are 80% homologous. Moreover, in each of the two repeats, there is an arrangement of histidines and cysteines, which may be related to the two known types of zinc-finger motifs found in many DNA-binding proteins. Consistent with the expectation that metal coordination plays an important role in DNA binding by this protein, we found that 1,10-phenanthroline can abolish the formation of DNA-protein complexes. Interestingly, we found that the same treatment did not abolish the DNA binding activity of 3AF1 in crude nuclear extracts of tobacco. These data indicate that the nuclear 3AF1 activity is likely due to multiple DNA-binding proteins all interacting with Box VI in vitro. RNA gel blot analysis shows that multiple transcripts homologous to this cDNA clone are expressed in different tobacco organs.

Amino Acid Sequence↗
PubMed1989

Light to dark transition modulates the phase of antenna chlorophyll protein gene expression.

The genes coding for the light-harvesting chlorophyll a/b-binding protein of photosystem II (cab) in wheat are regulated by phytochrome and an endogenous circadian clock. The regulation by these factors has been shown to be at the transcriptional level. Here we show that the phase of cab gene expression is reset by light to dark transition of the previous day. The time of light to dark transition is linearly related to the time of maximal cab gene expression under free running conditions in darkness. We propose that in nature the onset of darkness resets the phase of the circadian clock, which regulates the timing of cab gene expression, so as to anticipate the time of sunrise in the following day.

Chlorophyll↗
PubMed1989

Two tobacco DNA-binding proteins with homology to the nuclear factor CREB.

The 35S promoter of the cauliflower mosaic virus (CaMV) contains a tandem repeat of the sequence TGACG in the region -83 to -63. This 21-base pair (bp) sequence, called as-1, is involved in root expression of the 35S promoter. When inserted in a promoter of a gene expressed specifically in photosynthetic tissues, as-1 confers high level expression in roots. We have described a factor, ASF-1, that binds specifically to as-1 in vitro. There is a good correlation between ASF-1 binding affinity to as-1 related sequences in vitro and the function of these sequences in vivo. These results strongly suggest that ASF-1 is responsible for the function of as-1. Here we report the isolation of tobacco complementary DNA clones encoding two TGACG-sequence-specific binding-proteins (TGA1a and TGA1b). Sequence analysis of the cDNA clones shows that both proteins contain a basic region that shows high homology to a stretch of basic amino acids in the nuclear factors CREB, GCN4, and c-Jun to a 'leucine-zipper' region. On the basis of binding specificity we propose TGA1a to be a good candidate for ASF-1.

Activating Transcription Factors↗
PubMed1989

Phytochrome activation of two nuclear genes requires cytoplasmic protein synthesis.

We have investigated the effects of protein synthesis inhibitors on light-induced expression of two plant nuclear genes, Cab and rbcS, in wheat, pea and transgenic tobacco. Light activation of these two genes is very sensitive to cycloheximide, an inhibitor of cytoplasmic protein synthesis but not to chloramphenicol, an inhibitor of organellar protein synthesis. Studies with chimeric gene constructs in transgenic tobacco seedlings show that cycloheximide exerts its effect at the transcriptional level. As a control, we show that the expression of the cauliflower mosaic virus (CaMV) 35S promoter is enhanced by cycloheximide treatment, irrespective of the coding sequence used. Escape-time analyses with green wheat seedlings show that the cycloheximide block for Cab gene expression is after the primary signal transduction step linked to phytochrome photoconversion. Our results suggest that phytochrome activation of Cab and rbcS is mediated by a labile protein factor(s) synthesized on cytoplasmic ribosomes.

Blotting, Northern↗
PubMed1989

Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants.

The 35S promoter of cauliflower mosaic virus (CaMV) is able to confer high-level gene expression in most organs of transgenic plants. A cellular factor from pea and tobacco leaf tissue, which recognizes nucleotides in a tandemly repeated TGACG motif at the -75 region of this promoter, has been detected by DNase I footprinting and gel retardation assays. This factor is named activation sequence factor 1 (ASF-1). A cellular factor binding to the two TGACG motifs can also be detected in tobacco root extracts. Mutations at these motifs inhibit binding of ASF-1 to the 35S promoter in vitro. When examined in transgenic tobacco, these mutations cause a 50% drop in leaf expression of the 35S promoter. In addition, these same mutations attenuate stem and root expression of the 35S promoter about 5- to 10-fold when compared to the level of expression in leaf. In contrast, mutations at two adjacent CCAAT-box-like sequences have no dramatic effect on promoter activity in vivo. A 21-base-pair element containing the two TGACG motifs is sufficient for binding of ASF-1 in vitro when inserted in a green-tissue-specific promoter. In vivo, the insertion of an ASF-1 binding site caused high levels of expression in root. Thus, a single factor binding site that is defined by site-specific mutations is shown to be sufficient to alter the expression pattern of promoters in vivo.

Base Sequence↗
PubMed1989

ASF-2: a factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in Cab promoters.

We have used nuclear extracts prepared from tobacco leaf tissue to characterize a factor binding site, designated as-2 (activating sequence-2), at the -100 region of the cauliflower mosaic virus 35S promoter. The activity of this factor, called ASF-2 (activating sequence factor-2), is not detected in tobacco root extracts. as-2 includes two GT motifs with sequence homology to the SV40 enhancer core A element and the Box II element of pea rbcS. Nevertheless, oligomers of these sequence elements do not compete for ASF-2 binding in gel retardation assays, indicating that the GT motifs may not be involved. Methylation interference studies identify two guanines (G93 and G98) that are required for interaction with ASF-2. Sequences surrounding these two critical guanines display homologies to a GATA repeat conserved among several light-responsive promoters. One such sequence from a petunia Cab promoter is able to compete with as-2 for factor binding. In transgenic plants, a tetramer of as-2 is able to confer leaf expression when fused 5' to the -90 derivative of the 35S promoter. The expression is not dependent on light and, thus, the as-2 tetramer does not function as a light-responsive element in this context. Histochemical localization of the reporter gene product suggests that the as-2 tetramer directs expression in trichomes, vascular elements, and epidermal and mesophyll cells.

Base Sequence↗
PubMed1989

Characterization of phytochrome-regulated gene expression in a photoautotrophic cell suspension: possible role for calmodulin.

A photoautotrophic suspension culture of soybeans was found to exhibit light-dependent expression of the genes encoding the major chlorophyll a- and b-binding protein (CAB). The expression was mediated by phytochrome, since it was induced by red light and reversed by far-red light. The maximal level as well as the kinetics of the induction were comparable between the suspension culture and soybean seedlings. Using this cell culture, we addressed the question of whether a calcium- and/or calmodulin-mediated step is involved in the signal transduction process between phytochrome and CAB expression. We found that W-7, a potent calmodulin antagonist, severely attenuated the induction of CAB mRNA by light, whereas W-5, a weak calmodulin antagonist, had little effect. Control experiments demonstrated that W-7 treatment did not block the induction of hsp-75 by heat shock. The addition of ionomycin, a calcium ionophore, induced a low level of CAB mRNA accumulation in the dark which could be further enhanced by light treatment. We propose that calmodulin activation by light is necessary but not sufficient to induce maximal CAB expression.

Calcium↗
PubMed1989

Streptozotocin-induced deficits in sex behavior and neuroendocrine function in male rats.

The effects of streptozotocin-induced (STZ) diabetes on neuroendocrine and sexual function were evaluated in adult male rats. Adult male rats were injected with STZ (50 mg/kg) or vehicle and tested for copulatory behavior 7, 14, and 21 days later. The rats were killed 1 month after STZ or vehicle treatment for measurement of plasma hormone levels, hypothalamic catecholamine turnover, LHRH content, and in vitro pituitary function. The STZ rats showed significant deficits in mount, intromission, and ejaculatory behaviors. Plasma levels of testosterone, LH, FSH, and PRL were all significantly reduced in the STZ compared to the control rats, but in vitro LH secretion was enhanced after STZ treatment. In vitro PRL secretion and the inhibitory response to dopamine did not differ between the two groups. The levels of LHRH were reduced in the medial basal hypothalamus (MBH), but LHRH levels in the median eminence (ME) and anterior hypothalamus (AH) were unchanged after STZ treatment. Norepinephrine turnover was reduced in the ME, MBH, and AH of the STZ rats, while dopamine turnover was unchanged in the ME, increased in the MBH, and reduced in the AH of the STZ rats compared to those in the vehicle-treated controls. These results suggest that changes in pituitary and testicular function in rats made diabetic by STZ treatment are secondary to changes in hypothalamic catecholamine metabolism. Changes in copulatory behavior could be due to both reductions in plasma testosterone levels as well as changes in central neurotransmitter metabolism.

Animals↗
PubMed1988

Characterization of a chloroplast sequence-specific DNA binding factor.

The large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) and the beta subunit of chloroplast ATP synthase (atpB) are encoded by divergently transcribed genes on the plastid genome. We have identified DNA binding factors specific for sequences located in the intergenic region between these two genes. Soluble plastid extracts from pea or whole cell extracts from maize protected a maize chloroplast DNA probe containing the 160-base pair region between the 5' ends of rbcL and atpB genes from exonuclease III digestion between positions -16 and -101 relative to the rbcL gene transcription start site. Competition assay with partial sequences from this intergenic region demonstrated that specific sequence(s) are required for the protection. The borders of the binding domain are conserved among the homologous regions of maize, tobacco, spinach, and pea chloroplast genomes. Gel filtration chromatography revealed a molecular weight of about 115,000 for the active complex involved in DNA binding. Using the exonuclease III protection assay, we have also shown that purified Escherichia coli RNA polymerase protects from +25 to -20 of the rbcL gene and from +21 to -23 of the atpB gene relative to their respective transcription start sites. These regions are analogous to open complexes found when E. coli RNA polymerase interacts with the prokaryotic promoters and are consistent with the ability of E. coli RNA polymerase to initiate transcription correctly on linear templates containing these chloroplast promoters. Possible role(s) for the chloroplast DNA binding factor in chloroplast gene expression and its regulation are discussed.

Base Sequence↗
PubMed1986

Nearest-neighbor relationships of the constituent polypeptides in plastoquinol-plastocyanin oxidoreductase.

The nearest-neighbor relationship among the constituent polypeptides of the isolated plastoquinol-plastocyanin oxidoreductase from spinach chloroplasts has been investigated. (1) The isolated plastoquinol-plastocyanin oxidoreductase (the b6/f complex) is treated with various concentrations of the cross-linker glutaraldehyde. The treated b6/f complexes are then analyzed by SDS-polyacrylamide gel electrophoresis coupled with the immunodecoration of cross-link products by specific antibodies for each of the four prominent constituent polypeptides. Cytochrome b6 is found to be most resistant to forming any intermolecular cross-link products. At low concentrations of glutaraldehyde, the 'Rieske' iron-sulfur (Fe-S) protein and subunit IV of the b6/f complex, however, appear to form cross-link products with a relative molecular weight of 35 000. Dimers of cytochrome f and cytochrome f/Rieske protein cross-link products can also be detected. (2) When a Rieske Fe-S protein-depleted b6/f complex is used in place of the control b6/f complex, cytochrome b6 is less resistant to intermolecular cross-linking, while subunit IV does not form any 35 kDa cross-link product, unlike the case in control b6/f complex. Subunit IV is concluded to be closely associated with the Rieske Fe-S protein. This provides evidence that subunit IV is a bona fide component of the cytochrome b6/f complex, although no function can yet be assigned to it. The results are discussed in relationship to the spatial and functional relationships among the components of the b6/f complex.

Chloroplasts↗
PubMed1985

Characterization of a photosynthetic mutant of Lemna lacking the cytochrome b6-f complex.

A photosynthetic mutant of Lemna perpusilla (no. 1073) has been examined by spectrophotometric and immunoblotting techniques in order to localize the site of defect. In contrast to previous conclusions (Shahak, Y., Posner, H.B. and Avron, M. (1976) Plant Physiol. 57, 577-679), neither cytochrome f nor cytochrome b6 could be detected spectrophotometrically in the mutant. Furthermore, immunoblotting using antibodies specific for each of the four constituent subunits of the cytochrome b6-f complex demonstrate that the entire complex is absent in the mutant. The light-harvesting chlorophyll-protein complex of Photosystem II is present in similar amounts in wild-type and mutant Lemna. However, the total amount of plastoquinone-9 is reduced by approx. 65% in the mutant strain, while the photoreducible plastoquinone-9 pool is comparable in wild-type and mutant Lemna.

Antibodies↗
PubMed1985

Interactions between thylakoid electron transfer complexes. I. In vitro kinetic studies with isolated photosystem II and cytochrome b6-f complexes.

The cytochrome b6-f complex from spinach thylakoids has been reconstituted with an oxygen-evolving Photosystem II (PSII) preparation isolated from the same source to give oxygenic plastocyanin reductase activity. We observe that (i) mixing of the two complexes in concentrated form prior to dilution with the assay medium is necessary for optimal reconstitution of activity; (ii) incubation for longer times after dilution can also give substantial reconstitution if the two complexes are added separately to the assay mixture; (iii) either monovalent or divalent cations are required for optimum activity in the reconstituted system; (iv) titration of the cytochrome complex with varying amounts of the PSII complex gave a saturation of the plastocyanin reduction activity at a cytochrome complex/PSII ratio of 3-4; (v) kinetic analysis of plastocyanin photoreduction by Photosystem II shows nonlinearity, while first-order reduction kinetics are observed with duroquinol as electron donor; and (vi) as the concentration of plastocyanin is increased, the half-time of the reduction increases. These observations are considered in terms of a functional association between PSII and the cytochrome b6-f complex in this reconstituted system, and the relevance of these observations to the situation in vivo is discussed.

Chlorophyll↗
PubMed1985

Interactions between thylakoid electron transfer complexes. II. Modification studies with glutaraldehyde.

Photosystem I (PSI) and photosystem II (PSII) complexes have been isolated from stacked spinach thylakoid membranes that had been treated with varying amounts of glutaraldehyde. The concentrations of cytochrome f, Q, and P700 have been determined by spectrophotometric methods. It was found that at low concentrations of glutaraldehyde, the amount of cytochrome f associated with either PSII or PSI increased significantly while the amounts of Q and P700 stayed relatively constant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analyses indicated the presence of cytochrome f and other components of the cytochrome b6-f complex in the PSII and PSI preparations after glutaraldehyde treatment, but no intermolecular cross-linked polypeptides could be detected. Solubilization of the cytochrome b6-f complex was also inhibited after thylakoid membranes were treated with low concentrations of glutaraldehyde. These results are discussed in relation to current models for the organization of the membrane complexes, and relate to the location of the cytochrome b6-f complex in appressed and nonappressed membrane regions of thylakoids.

Aldehydes↗
PubMed1985

Topography of the protein complexes of the chloroplast thylakoid membrane : studies of photosystem I using a chemical probe and proteolytic digestion.

The transverse heterogeneity of the polypeptides associated with the Photosystem I (PSI) complex in spinach thylakoid membranes and in a highly resolved PSI preparation has been studied using the impermeant chemical modifier, 2,4,6-trinitrobenzenesulfonate (TNBS) and the proteolytic enzyme, Pronase E. The present study has shown that the PSI reaction center polypeptide of approximately 62 kilodaltons and the 22 and 20 kilodalton polypeptides of the PSI light-harvesting chlorophyll protein (LHCPI) complex are not labeled by [(14)C]TNBS in unfractionated thylakoids. On the other hand, the 23 kilodalton polypeptide of the PSI LHCP and the 19 and 14 kilodalton polypeptides associated with the PSI primary electron acceptor complex are readily labeled by [(14)C]TNBS and are exposed to the stromal side of the thylakoid. Differences and similarities in the labeling of polypeptides associated with the PSI complex in thylakoids and in the isolated PSI complex are also noted. Treatment of thylakoids with pronase had no effect on the organization of the polypeptides in the LHCPI or the reaction center core complex, as manifested by the separation of these two subcomplexes from pronase-treated membranes. The 62, 19, and 14 kilodalton polypeptides associated with the reaction center core complex and the 23 and 22 kilodalton polypeptides associated with LHCPI are sensitive to pronase treatment while the 20 kilodalton polypeptide of LHCPI was inaccessible to the protease. The proteolysis of the 62 kilodalton polypeptide generated first a single immunodetectable fragment at about 48 kilodaltons, and further proteolytic digestion generated two other fragments at 30 and 17 kilodaltons respectively. These results are discussed in relation to the organization of the PSI complex in spinach thylakoids. A model for the transmembrane topography of the polypeptide constituents of PSI has been developed.

Journal Article↗
PubMed1985

Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane : Studies of Photosystem II using Pronase Digestion and Chemical Labeling.

The accessibility of various Photosystem II (PSII)-associated polypeptides to the protease pronase and the chemical modifier trinitrobenzene-sulfonic acid (TNBS) has been investigated. Three polypeptides with apparent molecular weight of 32, 21, and 16 kilodaltons, known to be associated with O(2) evolution, are all resistant to pronase digestion and TNBS labeling in intact thylakoids. All the polypeptides in the isolated PSII preparation were labeled with TNBS while a different pattern of labeling was observed when the PSII complex was isolated from TNBS-modified thylakoids. Attempts to prepare PSII particles from pronase-treated thylakoids using the Triton X-100 solubilization method were unsuccessful. Pronase-treated thylakoids were probed with antisera against the chlorophyll proteins of PSII using immunoblotting techniques. This allowed for a positive identification of proteolytic fragments from the respective proteins. The results are discussed in relation to the transmembrane organization of PSII in spinach thylakoids.

Journal Article↗