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D F Klessig

Publications and source records attributed to D F Klessig.

At least 73 records · Page 4Linked to original sources
PubMed1993

Purification and characterization of a soluble salicylic acid-binding protein from tobacco.

Previously, we identified a soluble salicylic acid (SA)-binding protein (SABP) in tobacco whose properties suggest that it may play a role in transmitting the SA signal during plant defense responses. This SA-binding activity has been purified 250-fold by conventional chromatography and was found to copurify with a 280-kDa protein. Monoclonal antibodies capable of immunoprecipitating the SA-binding activity also immunoprecipitated the 280-kDa protein, indicating that it was responsible for binding SA. These antibodies also recognized the 280-kDa protein in immunoblots of the partially purified SABP fraction or the crude extract. However, when the crude extract was prepared in the presence of antioxidants, only a 57-kDa protein was recognized. Since the SABP has a native molecular mass of 240 kDa, it appears that the SABP is a complex which contains a 57-kDa subunit and perhaps one or more additional proteins which are covalently crosslinked in the absence of antioxidants. The ability of a variety of phenolic compounds to compete with SA for binding to the SABP was both qualitatively and quantitatively correlated with their biological activity in inducing defense-related genes. Moreover, the inducibility of the pathogenesis-related (PR)-1 genes by SA was proportional to the abundance of the SABP in different organs. These correlations are consistent with a role for the SABP in perceiving and transducing the SA signal in plant defense.

Antibodies, Monoclonal↗
PubMed1993

Multiple functions of the adenovirus DNA-binding protein are required for efficient viral DNA synthesis.

Mutational analysis within the amino-terminal (N-t) domain of the adenovirus DNA-binding protein (DBP) defined a region (aa 2-38) important for DBP function. Several viruses carrying lesions in this region of DBP showed reduced accumulation of viral DNA and infectious virions. Characterization of one of these mutants, H5in800, indicated that the N-t domain affects viral DNA synthesis in vivo. The reduction in DNA synthesis was not due to a change in the amount or nuclear location of the H5in800 DBP. Expression of other early genes in H5in800-infected cells was similar to that seen in wild-type Ad5-infected cells, suggesting that the depression of DNA synthesis was not due to disruption of DBP's role in early gene expression. The H5in800 and wild-type DBP also had comparable affinities for single-stranded DNA and functioned with similar efficiencies in two DNA elongation assays. Prior studies have shown that the carboxyl-terminal (C-t) domain of DBP was responsible for these two activities. Together these results suggest that DBP has at least two separable functions in viral DNA replication in vivo and that both domains of the protein are necessary for full activity. The intragenic complementation between the N-t mutant H5in800 and the C-t mutant H5in804 supports this model.

Adenoviruses, Human↗
PubMed1993

Phosphorylation of the adenovirus DNA-binding protein and epitope mapping of monoclonal antibodies against it.

To facilitate structure/function studies of the adenovirus type 2 or 5 single-stranded DNA binding protein (DBP), the epitopes of five anti-DBP monoclonal antibodies were mapped. Antibodies 37-3, 38-2, and B6 mapped between DBP residues 131-174 which corresponds to the host-range or hinge region of the molecule. The epitopes for antibodies 16-5 and 18-9 mapped between residues 58-81, and included a phosphorylated serine at position 70. Using antibodies 16-5 or 18-9 as analytical reagents, approximately 1/2 of the DBP molecules present at late times in infected HeLa cells were found to be phosphorylated at serine 70. A similar pattern of serine 70 phosphorylation was observed in productively and abortively infected monkey cells as well as in an E2A-transformed, DBP expressing cell lines. This study also reports the preliminary identification of two new phosphorylation sites on Ad2 DBP at serines 160 and 161 (corresponding to serine 161 on Ad5 DBP). No differences were observed in the phosphorylation of serine 160/161 in DBP isolated from infected HeLa cells or from productively and abortively infected monkey cells. Therefore, phosphorylation of these sites cannot account for the block to wild-type adenovirus growth in monkey cells.

Adenovirus E2 Proteins↗
PubMed1993

Pathogen, salicylic acid and developmental dependent expression of a beta-1,3-glucanase/GUS gene fusion in transgenic tobacco plants.

The 5' flanking region of a gene encoding an acidic beta-1,3-glucanase from Nicotiana tabacum was isolated and characterized. A chimeric gene composed of 1759 bp of the promoter sequence from the PR-2 gene was fused to the beta-glucuronidase (GUS) coding region and used to transform tobacco. Transcriptional activation of the PR-2 promoter was investigated in response to inoculation with tobacco mosaic virus (TMV), after treatment of leaves with salicylic acid (SA), and in specific tissues during the normal development of healthy plants. In TMV-inoculated transgenic plants, GUS activity was induced locally around necrotic viral lesions and systemically in uninoculated leaves. GUS activity was also induced by treatment of leaves with SA. The chimeric gene was expressed in floral organs of healthy plants and in newly germinated seedlings. Analyses of a series of 5' deletions of the glucanase promoter indicated that the cis-acting elements necessary for induction by all these signals are localized in the region between -321 bp and -607 bp upstream of the transcription start site.

Amino Acid Sequence↗
PubMed1993

Interconversion of the salicylic acid signal and its glucoside in tobacco.

Salicylic acid (SA) has been proposed to play a role in the induction of pathogenesis-related (PR) proteins and systemic acquired resistance (SAR) in tobacco. Since SA is rapidly converted to salicylic acid beta-glucoside (SAG) in tobacco, we have attempted to assess the role of SAG in pathogenesis by application of chemically synthesized SAG to tobacco leaves. SAG was as active as SA in induction of PR-1 gene expression. This induction was preceded by a transient release of SA, which occurred in the extracellular spaces. The existence of a mechanism that releases SA from SAG suggests a possible role for SAG in SAR.

Culture Techniques↗
PubMed1992

Characterization of the nucleic acid binding region of adenovirus DNA binding protein by partial proteolysis and photochemical cross-linking.

The nucleic acid binding domain of the adenovirus type 2 (or type 5) DNA-binding protein (DBP) was characterized by using limited proteolysis and photochemical cross-linking. Three proteases were used to generate fragments of DBP which retained the ability to bind to single-stranded DNA. One fragment, a 35-kDa tryptic product, was partially sequenced and found to contain amino acid residues 153 to approximately 470. This fragment further defines the minimum region of the protein which is required for nucleic acid binding. The DNA binding pocket of DBP was defined by using ultraviolet irradiation to cross-link covalently the carboxyl-terminal portion of the protein to the oligonucleotide p(dT)14. Cross-linked complexes were digested with trypsin, and peptides which were associated with the oligonucleotide were isolated by anion-exchange and reverse-phase ion-pairing high performance liquid chromatography. Two DBP peptides comprised of residues 294-308 and 415-434 were isolated by this approach. Sequence analysis indicated that methionine 299 and phenylalanine 418 were probable sites of cross-linking between their respective peptides and the oligonucleotide; hence these residues may represent contact points between DBP and single-stranded DNA. Both residues are highly conserved and are near, but not identical to, regions of the protein implicated previously in DNA binding.

Adenoviruses, Human↗
PubMed1992

Adenovirus containing a deletion of the early region 2A gene allows growth of adeno-associated virus with decreased efficiency.

Efficient growth of adeno-associated virus (AAV) requires helper functions provided by a coinfecting adenovirus or herpesvirus. Earlier studies using adenoviruses having temperature-sensitive lesions in the early region 2A gene (E2A) produced contradictory evidence regarding the role of the E2A 72-kDa DNA-binding protein (DBP) in allowing efficient AAV growth. These disparate results may reflect varying levels of residual function in the temperature-sensitive DBP. We examined this issue using an adenovirus type 5 mutant (Add/802) that fails to produce any detectable DBP or any fragment of it. Our experiments show that AAV can carry out a full growth cycle in the complete absence of DBP. However, AAV DNA replication and rep and capsid protein synthesis were reduced several fold and the yield of infectious AAV was reduced by an order of magnitude. This appears to reflect mainly decreased post-transcriptional expression of AAV rep and capsid protein genes.

Adenoviridae↗
PubMed1992

A zinc-binding motif located between amino acids 273 and 286 in the adenovirus DNA-binding protein is necessary for ssDNA binding.

The human adenovirus single-stranded (ss) DNA-binding protein (DBP) possesses a highly conserved carboxyl domain which contains a putative zinc-binding motif between amino acids (aa) 273 and 286. Using a zinc blotting technique DBP was shown to bind 65Zn at levels similar to other documented zinc metalloproteins. In competition experiments, DBP bound specifically to the zinc ion even in the presence of other divalent ions such as Ca+2, Mg+2, Cd+2, Co+2, and Mn+2. The zinc-binding ability of DBP was also confirmed by zinc affinity chromatography. Site-directed mutagenesis was utilized to construct a mutant which deleted the entire zinc region (pKZNdl 273-286) and a mutant which contained a Cys to Ser substitution at aa residue 284 (pKZNpt 284). The deletion mutant was unable to bind zinc, and the point mutant showed limited binding suggesting that aa 273-286 are responsible for the interaction of DBP with zinc. The DBP zinc mutants were also examined for their ability to bind to ssDNA. The deletion mutant was unable to bind ssDNA cellulose while the point mutant exhibited decreased affinity. Thus, the region between aa 273 and 286 which mediates zinc binding also appears fundamental for the ssDNA-binding function of DBP.

Adenoviridae↗
PubMed1992

Construction, characterization, and utilization of cell lines which inducibly express the adenovirus DNA-binding protein.

To further our understanding of structure-function relationships within the multifunctional adenovirus DNA binding protein (DBP) a more diverse collection of mutants is necessary. DBP-expressing cell lines (gmDBP) were previously constructed that complemented DBP-negative mutants for viral growth. However, they did not allow severely defective viruses to form plaques. Since efficient mutant construction is reliant on plaque isolation of the desired mutant virus as a final step, additional gmDBP cell lines were constructed which allow all DBP-negative mutants to form plaques. Here we describe the construction and characterization of 12 new gmDBP cell lines. The utility of these lines was demonstrated by the efficient construction of a new defective mutant, H5in804, using a combination of DBP-expressing lines. The H5in804 mutation adds 22 amino acids at the carboxyl end of an otherwise wild type protein. Characterization of H5in804 revealed that it was altered in its ability to replicate viral DNA. The depression of DNA synthesis most probably results from a reduced ability of H5in804 DBP to bind ssDNA.

Adenovirus E2 Proteins↗
PubMed1991

Identification of a soluble salicylic acid-binding protein that may function in signal transduction in the plant disease-resistance response.

It has recently been demonstrated that salicylic acid (SA) may serve as an endogenous signal molecule in the induction of systemic acquired resistance in tobacco and cucumber. In addition, SA is an endogenous regulator of heat and odor production in the inflorescence of some thermogenic plants. No information, however, is currently available concerning the mode(s) of action of SA in plant signal transduction. In a search for possible cellular factors that directly interact with SA, we have detected and partially characterized a SA-binding protein in tobacco leaves. The SA-binding activity is both SDS and proteinase sensitive and behaves as a soluble protein with an apparent mass of 650 kDa. The protein has an apparent Kd of 14 microM for SA, which is consistent with the range of physiological concentrations of SA observed for the induction of plant resistance responses. Furthermore, the ability of SA analogues to compete with SA for binding to this soluble protein is strictly correlated with their biological activity to induce the expression of genes associated with disease resistance. Biologically active analogues effectively inhibit SA binding while biologically inactive analogues do not. These results collectively indicate that this SA-binding protein may play a role in perceiving and transducing the SA signal to appropriate response elements, which ultimately activate one or more of the plant disease-resistance responses.

Journal Article↗
PubMed1991

Differential targeting of the tobacco PR-1 pathogenesis-related proteins to the extracellular space and vacuoles of crystal idioblasts.

Several biochemical and localization studies have shown that the acidic isoforms of the tobacco pathogenesis-related (PR) proteins, PR-1a, -1b and -1c are secreted to the extracellular spaces of leaves in response to pathogen infection or chemical treatment. Here we report the differential accumulation of these proteins within the vacuoles of specialized cells known as crystal idioblasts. In situ hybridization analysis indicated that crystal idioblasts expressed the PR-1 genes at the mRNA level and suggested that PR-1 proteins were synthesized by these cells. Transgenic plants which constitutively express a chimeric gene encoding an acidic PR-1b isoform also accumulated PR-1 protein in the extracellular spaces and within crystal idioblast vacuoles. Analysis of mRNA derived from these transgenic plants indicated that expression of the introduced PR-1b gene was responsible for the accumulation of PR-1 protein in these two distinct locations. The synthesis and accumulation within crystal idioblasts of PR-1 proteins, which are secreted by other cell types, indicates that idioblasts sort these proteins in a unique manner. Moreover, this suggests that protein sorting in higher plants may be modulated in a cell specific manner.

Antibodies, Monoclonal↗
PubMed1990

Light-mediated control of translational initiation of ribulose-1, 5-bisphosphate carboxylase in amaranth cotyledons.

In cotyledons of 6-day-old amaranth seedlings, the large subunit (LSU) and the small subunit (SSU) polypeptides of ribulose-1,5-bisphosphate carboxylase are not synthesized in the absence of light. When dark-grown seedlings were transferred into light, synthesis of both polypeptides was induced within the first 3 to 5 hr of illumination without any significant changes in levels of their mRNAs. In cotyledons of light-grown seedlings and of dark-grown seedlings transferred into light for 5 hr (where ribulose-1,5-bisphosphate carboxylase synthesis was readily detected in vivo), the LSU and SSU mRNAs were associated with polysomes. In cotyledons of dark-grown seedlings, these two mRNAs were not found on polysomes. In contrast to the SSU message, mRNAs encoding the nonlight-regulated, nuclear-encoded proteins actin and ubiquitin were associated with polysomes regardless of the light conditions. Similarly, mRNA from at least one chloroplast-encoded gene (rpl2) was found on polysomes in the dark as well as in the light. These results indicate an absence of translational initiation in cotyledons of dark-grown seedlings which is specific to a subset of nuclear- and chloroplast-encoded genes including the SSU and LSU, respectively. Upon illumination, synthesis of both polypeptides, and possibly other proteins involved in light-mediated chloroplast development, was induced at the level of translational initiation.

Chloroplasts↗
PubMed1989

Are the PR1 proteins of tobacco involved in genetically engineered resistance to TMV?

Transgenic tobacco plants constitutively expressing the coat protein (CP) of tobacco mosaic virus (TMV) exhibit enhanced resistance ot TMV (P. Powell Abel, R. S. Nelson, B. De, N. Hoffman, S. G. Rogers, R. T. Fraley, and R. N. Beachy, Science, 232, 738-743, 1986; R. S. Nelson, P. Powell Abel, and R. N. Beachy, Virology 158, 128-132, 1987). To determine if this enhanced resistance might be mediated through the PR1 family of pathogenesis-related (PR) proteins, their synthesis was examined. In transgenic plants derived from NN genotypic tobacco, a high proportion (approximately 80%) of those producing CP also expressed the PR1 genes at low levels. However, this correlation between TMV CP and PR1 gene expression was not observed in similarly transformed nn genotypic tobacco plants. Therefore, it appears unlikely that PR1 proteins play a critical role in genetically engineered resistance in transgenic plants producing TMV CP.

Gene Expression Regulation↗
PubMed1989

Characterization of adenovirus type 5 insertion and deletion mutants encoding altered DNA binding proteins.

We have introduced insertion and deletion mutations in the cloned DNA binding protein (DBP) gene of adenovirus type 5. The mutated DBP genes were subsequently introduced in the viral genome by a combination of in vitro and in vivo methods. The resulting mutant viruses were tested for their viability in human 293 cells and an initial characterization of these viruses was performed. Viable mutants with insertions in the carboxyl-terminal portion of the gene could not be obtained. In contrast, a number of viable mutants were constructed that contained insertions or deletions in the amino-terminal half of DBP. Several of these, which covered the region between amino acid (aa) residues 39 and 81, were phenotypically wild type, implying that this segment is completely dispensable for DBP function. However, mutations altering the region encompassed by aa 2-38 were, at least, partially defective suggesting that this region is important for full activity of the protein.

Adenoviruses, Human↗
PubMed1989

Disease response to tobacco mosaic virus in transgenic tobacco plants that constitutively express the pathogenesis-related PR1b gene.

Correlation of the temporal and spacial pattern of induction of the pathogenesis-related (PR) genes PR1a, PR1b, and PR1c with viral infections in certain tobacco cultivars has implicated PR proteins in viral disease resistance. To test whether the PR1 proteins of tobacco are involved in viral resistance, transgenic Nicotiana tabacum plants were constructed which constitutively express the PR1b gene. This protein was secreted from cells of transgenic plants and accumulated in the extracellular space at levels equivalent to those found in nontransgenic plants in association with disease resistance. Transgenic plants derived from the cultivar (cv.) Xanthi (susceptible to tobacco mosaic virus [TMV] infection) exhibited no delayed onset or reduction in the severity of systemic symptoms after TMV infection. In transgenic plants derived from cv. Xanthi-nc (TMV resistant), the time of appearance, the size and general morphology, and the number of viral lesions produced were similar to the parental control plants after TMV infection. These data indicate that the PR1b protein of tobacco is not sufficient for TMV resistance, and imply that the PR1 proteins may not function as unique antiviral factors.

Cell Transformation, Viral↗
PubMed1989

Increased permissivity of monkey cells to human adenovirus multiplication is affected by culturing conditions and correlates with both synthesis of virion fiber protein and altered splicing of its mRNA.

Human adenoviruses (Ad2) grow poorly in CV-1 (monkey) cells, resulting in an abortive infection. During abortive infections synthesis of the fiber protein is approximately 100-fold depressed compared to its synthesis in CV-1 cells productively infected with a host range mutant of adenovirus (Ad2hr400), while the corresponding mRNA is reduced only 10-fold. We found that passage of CV-1 cells under modified culture conditions resulted in loss of restrictiveness for Ad2 growth. As cells became more permissive, fiber polypeptide synthesis was enhanced although fiber mRNA levels did not necessarily increase. Analysis of the 5' ends of fiber message isolated from cells in various stages of permissivity showed a direct correlation between efficient synthesis of the fiber protein and an altered splicing pattern of the fiber message.

Adenoviruses, Human↗