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Biomedical subjects

E Lam

Publications and source records attributed to E Lam.

At least 55 records · Page 3Linked to original sources
PubMed1995

Construction of a trans-dominant inhibitor for members of the TGA family of transcription factors conserved in higher plants.

Activating sequence factor 1 (ASF-1) is a conserved DNA-binding activity that interacts with sequence elements containing TGACG motifs, some of which have been demonstrated to respond to exogenous application of auxin and salicylic acid. Genes encoding transcription factors with similar DNA-binding specificity to ASF-1 have been cloned from diverse plant species and these factors all contain a distinct basic-leucine-zipper (bZIP) motif. Members of this family of DNA-binding proteins, designated as TGA factors, have been shown to interact with similar DNA sequences and at least seven distinct TGA genes are present in the Arabidopsis genome. To study the roles that this family of factors may play in plant development, a trans-dominant inhibitor of ASF-1 was constructed by deleting the basic portion of the bZIP domain in the tobacco factor TGA1a. In vitro co-expression studies demonstrated that this deletion mutant, named TGA1a-D, suppresses the DNA-binding activity of wild-type tobacco TGA1a, and three different members of the Arabidopsis TGA family. In contrast, co-expression of TGA1a-D with another class of bZIP proteins, the G-Box Binding Factor family, showed no suppression of DNA-binding activity. Over-expression of TGA1a-D in transgenic tobacco significantly decreased nuclear ASF-1 relative to several other known factors, indicating that the proteins comprising ASF-1 activity in vivo are likely TGA family members. Thus, TGA1a-D may be a family-specific inhibitor for the TGA family and should facilitate the study of ASF-1 function in vivo.

Amino Acid Sequence↗
PubMed1995

Targeted disruption of the TGA3 locus in Arabidopsis thaliana.

A major drawback to study gene functions in plant systems is the lack of an effective gene knockout strategy. With a large number of plant genes isolated and the accelerating pace by which this collection is growing, the need for their functional analyses at the whole plant level has become increasingly urgent. Here evidence is reported for the first successful disruption of a non-selectable gene in Arabidopsis thaliana by creating a mutant of the TGA3 locus via targeted insertion of the bacterial neo gene conferring kanamycin (Km) resistance. A beta-glucuronidase (GUS) expression unit outside the region of homology was used as a screenable marker to distinguish homologous recombination events from those of ectopic insertions. PCR amplification coupled with Southern blot screening identified two putative homologous recombination events among 2580 Kmr calli. One callus line was subsequently isolated and the structure of the targeted TGA3 allele confirmed by Southern blot analyses. This study demonstrates the feasibility of targeting a non-selectable locus in Arabidopsis. Combined with future improvements in negative selection strategies and efficient transformation methodologies, gene replacement studies in plants could become a routine technique.

Arabidopsis↗
PubMed1995

Identification, characterization, and purification of a tobacco endonuclease activity induced upon hypersensitive response cell death.

Programmed cell death (pcd) is activated during the hypersensitive response (HR) of plants to avirulent pathogens. We have recently shown that, similar to pcd in animal cells, nuclei of cells undergoing HR cell death contain fragmented nuclear DNA (nDNA). Here, we report that cell death occurring during the HR is accompanied by an increase in the activity of several deoxyribonucleases. Induction of nuclease activities was coordinated with cell death and may account for the degradation of nDNA during the HR. HR-associated nuclease activities were not induced during senescence, following necrotic cell death resulting from abiotic stress, or in response to induction of plant defense mechanisms by salicylic acid. HR-associated nuclease activities were stimulated by Ca2+ and inhibited by EGTA, EDTA, and Zn2+. At least one of the HR-associated nuclease activities was detected in nuclei purified from leaves undergoing pcd. A nuclease with an electrophoretic mobility similar to that of the nuclease activity found in nuclei isolated from leaves undergoing HR cell death was purified. Our findings are in accordance with some of the biochemical events that occur during pcd in animal cells. However, further analysis of the pattern of nDNA fragmentation and the corresponding structural changes that occur in the nuclei of tobacco cells undergoing HR cell death revealed that these features may have differences from those that take place during apoptosis in animal cells.

Animals↗
PubMed1995

Domain analysis of the plant DNA-binding protein GT1a: requirement of four putative alpha-helices for DNA binding and identification of a novel oligomerization region.

Light is an important environmental signal that can influence diverse developmental processes in plants. Many plant nuclear genes respond to light at the level of transcription initiation. GT-1 and GT2 are nuclear factors which interact with DNA sequences in many light-responsive gene promoters. cDNA clones which encode proteins with sequence binding specificities similar to those of these two factors have been isolated. They show significant amino acid sequence similarities within three closely spaced, putative alpha-helices that were predicted by secondary structure analysis but do not show significant homologies with any other reported DNA-binding protein. In this work, N- and C-terminal deletions of tobacco GT1a were generated by in vitro transcription and translation, and their DNA-binding activities and subunit structures were studied. The results suggest that the C-terminal domain of GT1a is critical for protein oligomerization, while a region predicted to contain four closely spaced alpha-helices is required for DNA binding. Direct chemical cross-linking and gel filtration analyses of full-length and truncated derivatives of GT1a suggest that this factor can exist in solution as a homotetramer and that oligomerization is independent of DNA binding. This study thus establishes two independent functional domains in this class of eukaryotic trans-acting factors. Possible implications of the multimeric nature of GT1a in relation to the known characteristics of light-responsive promoter architecture are discussed.

Amino Acid Sequence↗
PubMed1994

Abundance of nuclear DNA topoisomerase II is correlated with proliferation in Arabidopsis thaliana.

Topoisomerase II (TOPII) is an important enzyme involved in DNA replication and chromosome condensation. The level of TOPII expression has been correlated with the proliferative state of eukaryotic cells. Here we report the cloning and characterization of a cDNA clone AtTopII encoding the first reported TOPII from higher plants. AtTopII is 4603 base pairs (bp) in length and encodes an open reading frame of 1473 amino acid residues. One interesting feature of AtTopII is the presence of a 110 bp direct repeat in the last one-third of the cDNA. Analysis of the genomic sequence within this region by PCR revealed that this duplication includes a small intron of 89 bp. Conservation of sequences within this repeated intron suggests that this in-frame duplication may be a relatively recent event. The deduced amino acid sequence of AtTopII shows strong homologies to TOPII sequences reported from other eukaryotes, particularly in the regions that are highly conserved among different species. Southern blot analysis with Arabidopsis DNA indicates that AtTopII is a single-copy gene while Northern blots detected a 5.0 kb transcript, the level of which is substantially higher in young seedlings than in mature plants. Using a polyclonal antiserum raised against the C-terminal one-third of AtTOPII, we found that the protein is localized in the nucleus and its level is correlated with the proliferative state of the particular tissue.

Amino Acid Sequence↗
PubMed1994

Inhibition of calcineurin by a novel FK-506-binding protein.

FK-506, a potent immunosuppressive drug, acts during the commitment phase of T-lymphocyte activation to block a subset of calcium-associated events necessary for transcription of certain early lymphokine genes. The drug binds to an abundant, cytosolic 11.8-kDa protein termed the FK-506-binding protein (FKBP12). The FKBP12.FK-506 complex inhibits calcineurin, a calcium-dependent phosphatase that is a component of the signal transduction pathway leading to early lymphokine gene transcription. FKBP12 is one member of a growing gene family. Prior to this report, all other FKBP family members had been irrelevant to the mechanism of action of FK-506 because no other FKBP.FK-506 complexes were able to bind and inhibit calcineurin. Here, we report the purification and characterization of a novel FK-506-binding protein, FKBP12.6. Having 85% amino acid sequence identity to FKBP12, FKBP12.6 is, among the FKBPs, most closely related to FKBP12. When complexed with FK-506, FKBP12.6 binds to and inhibits calcineurin, making it only the second FKBP discovered thus far to do so. The ability to inhibit calcineurin establishes the potential relevance of FKBP12.6 to the immunosuppressive or toxic side effects of FK-506.

Amino Acid Isomerases↗
PubMed1994

Two binding sites for the plant transcription factor ASF-1 can respond to auxin treatments in transgenic tobacco.

The hormone, auxin, plays an important role in the differentiation and growth of plant cells. A number of auxin-responsive genes have been characterized but until now minimal auxin-responsive cis-elements within these promoter regions have not been identified. Here we show that two related DNA sequences of 21 base pairs can respond to auxin treatment in transgenic tobacco. In contrast, treatments with cytokinin or abscisic acid do not cause any apparent increase in promoter activity of these cis-acting elements. These sequences are present in the promoter regions of the nopaline synthase gene from the T-DNA of Agrobacterium tumefaciens and the 35 S promoter from cauliflower mosaic virus. Both sequences have been shown to be binding sites for the tobacco transcription factor ASF-1. Pretreatment of leaves with cycloheximide does not inhibit the response to auxin treatment, suggesting that hormone sensitivity of these promoter elements does not involve de novo synthesis of ASF-1. In addition, promoter elements from some auxin-responsive plant genes can bind ASF-1 in vitro. Based on these results, we propose that transcriptional activation by ASF-1 may be modulated by auxin through modification of pre-existing factors. Our results also suggest a role for ASF-1 in mediating some of the effects of auxin in vivo.

Agrobacterium tumefaciens↗
PubMed1994

TGA3 is a distinct member of the TGA family of bZIP transcription factors in Arabidopsis thaliana.

TGA1a is a well-characterized transcription factor that may mediate the root-specific and auxin-responsive expression of some plant genes. In tobacco, Southern blot and genomic cloning analyses have shown that TGA1a consists of at least four closely related genes. Since TGA1a belongs to the bZIP class of transcriptional factors, the protein products of the tobacco TGA1a family are likely to form hetero-dimers with each other in addition to the homo-dimers. In order to find a model plant system that may have less genomic complexity, we have now characterized a TGA1a-related gene (TGA3) from Arabidopsis thaliana. Southern blot analyses at high stringency suggest that Arabidopsis contains only one copy of TGA3 per haploid genome. However, low stringency Southern blot analyses with homologous and heterologous probes suggest that there is a multigene family of TGA1a-related genes present in Arabidopsis, of which TGA1, TGA2 and TGA3 are members. Although these gene members share a highly conserved bZIP region, they are not genes with high homologies at the nucleotide level. Similar to TGA1a of tobacco, TGA3 is most highly expressed in root tissues and recombinant TGA3 protein shows similar DNA-binding site specificity to that of TGA1a in vitro. Comparison of the genomic organization between TGA3 and the tobacco homologue PG13 reveals striking conservation in the sizes and positions of exons and introns in the region surrounding the bZIP domain.

Amino Acid Sequence↗
PubMed1994

Hypertriglyceridemic serum, very low density lipoprotein, and iron enhance Mycobacterium avium replication in human macrophages.

The growth of Mycobacterium avium 7497, serovar 4, in cultured human macrophages is enhanced by Fe3+ and serum lipids over 7 days. Iron (1-80 micrograms/mL) added to macrophages cultured in normal serum resulted in 10-fold increases in growth. If iron-supplemented macrophages were cultured in serum from hypertriglyceridemic donors after infection, M. avium growth increased 10(3)- to 10(4)-fold. Without macrophages, differences in bacterial growth between sera were not seen. Removal of very low density lipoprotein (VLDL) eliminated the differences between sera. Isolated VLDL from hyperlipidemic serum resulted in 10(5)-fold increases in growth over that seen with VLDL from normal sera. Accelerated M. avium growth in macrophages cultured with hyperlipidemic serum was partly inhibited by the addition of superoxide dismutase (1000 IU/mL). Results suggest that iron stimulates O2-induced oxidation of VLDL and its subsequent accumulation in macrophages. The resultant iron- and lipid-laden cells become excellent hosts for mycobacterial growth.

Acquired Immunodeficiency Syndrome↗
PubMed1993

The mechanism of action of FK-506 and cyclosporin A.

FK-506 and cyclosporin A (CsA) are potent immunosuppressive agents used clinically to prevent tissue rejection. Interest in the development of more effective immunosuppressive drugs has led to an intense effort toward understanding their biochemical mechanism of action with the result that these compounds have now become powerful tools used in deciphering the signal transduction events in T lymphocyte activation. Although chemically unrelated, FK-506 and CsA exert nearly identical biological effects in cells by inhibiting the same subset of early calcium-associated events involved in lymphokine expression, apoptosis, and degranulation. FK-506 binds to a family of intracellular receptors termed the FK-506 binding proteins (FKBPs). CsA binds to another family of intracellular receptors, the cyclophilins (Cyps), distinct from the FKBPs. The similarities between the mechanisms of action of CsA and FK-506 converge upon the calcium- and calmodulin-dependent serine-threonine protein phosphatase calcineurin (CaN). Both the FKBP/FK-506 complex and the Cyp/CsA complex can bind to calcineurin, thereby inhibiting its phosphatase activity. Calcineurin, a component of the signal transduction pathway resulting in IL-2 expression, catalyzes critical dephosphorylation events required for early lymphokine gene transcription.

Amino Acid Isomerases↗
PubMed1993

Guanine nucleotide binding protein involvement in early steps of phytochrome-regulated gene expression.

The transmission process of light signals from plant photoreceptors to target cellular events is largely unknown. In the present work, we show that treatment of dark-adapted soybean cells (SB-P) with cholera toxin or pertussis toxin uncouples phytochrome-dependent gene expression. Addition of as little as 10 ng of toxin per ml is sufficient to activate expression of genes encoding the major chlorophyll a/b-binding protein (cab) in the dark. Significant levels of cab transcript accumulation are detected within 0.5 h after addition of the toxins and expression of these genes is desensitized to further light treatments. Treatment of SB-P cells with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphtha-lenesulfonamide hydrochloride (W-7) prevents induction of the photoregulated gene by phytochrome or bacterial toxins. These results indicate the involvement of guanine nucleotide binding protein(s) in phytochrome-mediated cab gene activation. A likely site of action for this step is between the photoreceptor and a downstream W-7-sensitive effector.

Journal Article↗
PubMed1993

Analysis of three tissue-specific elements from the wheat Cab-1 enhancer.

The genes encoding the major light-harvesting antenna chlorophyll a/b-binding protein (Cab) of higher plants are regulated by light at the transcriptional level. In addition, their expression is largely restricted to photosynthetically competent organs such as leaves. A 268 bp fragment of the Cab-1 promoter from wheat functions as a light-responsive and organ-specific enhancer in transgenic tobacco. Using DNase I footprinting, four different regions have been mapped (Cab1-A, Cab1-B, Cab1-C and Cab1-D) in this fragment that bind to protein factors in tobacco nuclear extracts. Three of these regions (A, B and C) coincide with sequences that have been found to be functionally important from previous cis-element analyses. Synthetic tetramers of these three sites interact with different proteins in gel retardation assays. In addition, cross-competition analyses demonstrate that Cab1-C is likely to interact with ASF-2, a tobacco DNA-binding activity that binds to a conserved GATA element found in many dicot Cab promoters. In transgenic tobacco, a 95 bp fragment of the Cab-1 enhancer containing the A, B and C regions can confer leaf expression when fused upstream of a truncated derivative of the cauliflower mosaic virus (CaMV) 35S promoter. However, expression observed with this enhancer fragment in the promoter context of these studies does not appear to be significantly dependent on light. Similar results were obtained with synthetic tetramers of Cab1-A, -B or -C. These data thus suggest that the wheat Cab-1 enhancer contains at least three distinct elements that contribute to leaf-specific expression in transgenic tobacco. Interaction between factors binding to these positive elements and those that bind elsewhere in the Cab-1 enhancer may be necessary for light-responsive transcriptional activation.

Base Sequence↗
PubMed1992

A tobacco DNA binding protein that interacts with a light-responsive box II element.

Ribulose-1,5-bisphosphate carboxylase/oxygenase plays a key role in photosynthetic carbon fixation in higher plants. The small subunit of this chloroplast enzyme (rbcS), encoded by a family of nuclear genes, is regulated at the transcriptional level by light. Promoter analyses have previously identified the box II sequence as a cis element critical for the light-regulated expression of rbcS genes. Nuclear factor GT-1 binds specifically to this element and is one of the plant nuclear factors that has been detected and studied in great detail. Here we describe the cloning and characterization of a tobacco cDNA encoding a protein, designated B2F (Box II Factor), with similar binding specificity and mobility in gel retardation assays as nuclear GT-1. Steady state levels of mRNA encoding B2F do not appear to be regulated by light; this is consistent with the previous observation that nuclear GT-1 activity is present in extracts from both light-grown and dark-adapted plants. Sequence comparison with another plant trans-acting factor, GT-2, which binds to a GT-like element in the rice phytochrome promoter, shows striking homology in three putative alpha-helices that may be involved in DNA binding.

Amino Acid Sequence↗
PubMed1991

Tetramer of a 21-base pair synthetic element confers seed expression and transcriptional enhancement in response to water stress and abscisic acid.

A conserved 21-base pair element, designated as hex-1, located between -180 and -160 of the wheat histone H3 promoter, is known to interact with two tobacco nuclear factors, activating sequence factor 1 and hex-1-specific binding factor. We have shown previously that a mutant sequence (hex-3), which differs from hex-1 by three base pairs, can no longer bind these two factors significantly. In the present work, we examined the functional characteristics of these two sequences in transgenic tobacco. Surprisingly, we found that a tetramer of hex-3, but not of hex-1, confers high level expression in mature seeds. Expression of this synthetic promoter rapidly diminishes upon germination but can be reactivated in young seedlings and mature leaves by desiccation, NaCl, or the phytohormone abscisic acid (ABA). Treatment with auxin or cytokinin has no apparent effect on the expression. Since the endogenous ABA level of plant cells is known to increase upon water stress, our data suggest that hex-3, the mutated hex-1 sequence, is an abscisic acid-responsive element (abre). We propose that a tobacco nuclear factor, distinct from activating sequence factor 1 and hex-1-specific binding factor, interacts with this sequence and is involved in mediating the effects of ABA and water stress on gene expression.

Abscisic Acid↗
PubMed1991

Antibodies to hepatitis C virus in autologous blood donors.

Hepatitis C virus (HCV) is the major cause of posttransfusion hepatitis. Two anti-HCV enzyme immunoassay (EIA) kits and one recombinant immunoblot assay (RIBA) were used to test serum samples of 1476 donations from 692 autologous blood donors to assess the prevalence of anti-HCV and its relationship to transfusion history. Of all autologous blood donations, 23 (1.6%) reacted when tested with one EIA kit and 29 (2.0%) reacted when tested by the other EIA kit. Of the autologous donors, 12 (1.78%) reacted by the first EIA kit and 14 (2.02%) by the second. Discrepancies in the EIA results from different donations by the same donor were seen in seven donors. The RIBA was positive or indeterminate in 33 percent of the EIA-reactive donations and in 41 percent of EIA-reactive donors. All RIBA-positive and -indeterminate samples reacted with both EIA kits. There was no significant difference in the EIA-reactive rates of autologous and first-time homologous blood donors. Previously transfused autologous blood donors had a higher anti-HCV EIA-reactive rate than nontransfused autologous donors, but the difference was not significant. In regard to hepatitis C, the use of autologous blood for homologous transfusion appears to be as safe as the use of blood from first-time homologous donors. Universal testing of previously transfused patients for hepatitis C appears premature at this time. Discrepant anti-HCV EIA results from different donations from the same individual have implications regarding donor deferral.

Adolescent↗