PubMed Health⌕ Search

Biomedical subjects

J Ryals

Publications and source records attributed to J Ryals.

At least 37 records · Page 2Linked to original sources
PubMed1992

Acquired resistance in Arabidopsis.

Acquired resistance is an important component of the complex disease resistance mechanism in plants, which can result from either pathogen infection or treatment with synthetic, resistance-inducing compounds. In this study, Arabidopsis, a tractable genetic system, is shown to develop resistance to a bacterial and a fungal pathogen following 2,6-dichloroisonicotinic acid (INA) treatment. Three proteins that accumulated to high levels in the apoplast in response to INA treatment were purified and characterized. Expression of the genes corresponding to these proteins was induced by INA, pathogen infection, and salicylic acid, the latter being a putative endogenous signal for acquired resistance. Arabidopsis should serve as a genetic model for studies of this type of immune response in plants.

Amino Acid Sequence↗
PubMed1991

Structural and functional conservation of histidinol dehydrogenase between plants and microbes.

The partial amino acid sequence of histidinol dehydrogenase (L-histidinol:NAD+ oxidoreductase, EC 1.1.1.23) from cabbage was determined from peptide fragments of the purified protein. The relative positions of these peptides were deduced by aligning their sequences with the sequence of the HIS4C gene product of Saccharomyces cerevisiae. cDNA encoding histidinol dehydrogenase was then amplified from a library using a polymerase chain reaction primed with degenerate oligonucleotide pools of known position and orientation. By using this amplified fragment as a probe, an apparently full-length cDNA clone was isolated that is predicted to encode a proenzyme having a putative 31-amino acid chloroplast transit peptide and a mature molecular mass of 47.5 kDa. The predicted protein sequence was 51% identical to the yeast enzyme and 49% identical to the Escherichia coli enzyme. Expression of the cDNA clone in an E. coli his operon deletion strain rendered the mutant able to grow in the presence of histidinol.

Alcohol Oxidoreductases↗
PubMed1991

Comparison of cloned genes provides evidence for intergenomic exchange of DNA in the evolution of a tobacco glucan endo-1,3-beta-glucosidase gene family.

Two genes for prepro glucan endo-1,3-beta-glucosidase (1,3-beta-glucanase; 1,3-beta-D-glucan glucanohydrolase, EC 3.2.1.39) of tobacco were cloned and their sequences were compared with cDNA clones. Southern analysis indicates that the genomic clones represent genes derived from ancestral parents of tobacco similar to the present day species Nicotiana sylvestris and Nicotiana tomentosiformis, whereas the genes represented by two of the cDNA clones appear to be unique to tobacco. The coding sequences of the genomic clones and cDNA clones differed at less than 2.2% of the positions, indicating that the tobacco 1,3-beta-glucanase gene family is highly conserved. Alternating blocks of sequence in the cDNA clones were identical to the coding sequence of the two genomic clones. These results and an analysis of evolutionary distances for nucleotide substitution are consistent with the hypothesis that the evolution of the tobacco 1,3-beta-glucanase gene family has involved exchange of DNA between members of the tomentosiformis and sylvestris subgenomes by recombination or gene conversion.

Amino Acid Sequence↗
PubMed1991

Pathogenesis-related protein 4 is structurally homologous to the carboxy-terminal domains of hevein, Win-1 and Win-2.

The extracellular, acidic pathogenesis-related protein, PR-4, was purified to homogeneity from leaves of Nicotiana tabacum infected with tobacco mosaic virus (TMV) and characterized by partial amino acid sequencing. Complementary DNA clones encoding PR-4 were isolated using an oligonucleotide probe based on the sequence of one of the peptides. The deduced PR-4 protein sequence was found to be related to a family of proteins including hevein and Win-1, which have an amino-terminal lectin domain and a carboxy-terminal domain of unknown function. PR-4 is homologous to the carboxy-terminus of these proteins but does not contain the lectin domain. Thus, the organization of the PR-4 family of proteins is similar to that of the plant chitinase family, in that both contain structural subclasses characterized by the presence or absence of an amino-terminal lectin domain. This observation is consistent with the proposal that the DNA encoding the lectin domain may be capable of transposing to form new genes encoding proteins of more complex, multi-domain structure. The expression of PR-4 mRNA was found to increase dramatically in response to TMV infection and the time course of RNA accumulation was similar to that of other PR proteins.

Amino Acid Sequence↗
PubMed1990

Structure of a tobacco endochitinase gene: evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain.

The endochitinases (E.C. 3.2.1.14, chitinase) are a structurally diverse group of enzymes believed to be important in the biochemical defense of plants against potential pathogens. The gene for a chitinase of Nicotiana tabacum L. cv. Havana 425 has been cloned and sequenced. The major transcription start is 11 bp upstream of the ATG codon and 28 bp downstream of the TATA box. The gene contains two introns and encodes a basic chitinase of 329 amino acids with a 23 amino acid N-terminal signal peptide followed by a 43 amino acid, cysteine-rich domain, which is linked by a hinge region to the main structure of the enzyme. This gene appears to be expressed because the exons are identical to the coding sequence of a cDNA which was isolated. Comparison of chitinase amino acid sequences from different plants indicates there are at least three classes of these enzymes: class I, basic chitinases with an N-terminal cysteine-rich domain and a highly conserved main structure; class II, chitinases similar to the main structure of class I chitinases but lacking the cysteine-rich domain; and, class III, chitinases with conserved sequences different from those of the class I and II enzymes. The sequences encoding the cysteine-rich domain in class I chitinases are flanked by 9-10 bp imperfect direct repeats suggesting that these domains arose from a common ancestral gene and were introduced into genes for class I enzymes by transposition events.

Amino Acid Sequence↗
PubMed1990

Evidence for a third structural class of beta-1,3-glucanase in tobacco.

Glucan endo-1,3-beta-glucosidases (beta-1,3-glucanases) have been implicated in several developmental processes and they may also play a direct role in the plant's defense against fungal pathogens. In an effort to characterize the glucanase gene family, complementary DNA clones encoding an acidic form of beta-1,3-glucanase have been isolated from tobacco. The cDNA was expressed in E. coli and shown to encode a beta-1,3-glucanase activity. The protein sequence encoded by the cDNA was found to match the partial protein sequence of PR-35, a previously characterized beta-1,3-glucanase. The protein encoded by the cDNA was purified from the extracellular fluid of TMV-infected tobacco leaves and found by immunological methods to correspond to glucanase PR-Q'. From a detailed analysis of the cDNA it is clear that this glucanase represents a third structural class of enzyme which differs substantially from both the basic, vacuolar glucanase and the acidic, extracellular forms (PR-2, PR-N and PR-O). It has previously been demonstrated that the basic form of beta-1,3-glucanase is synthesized as a pre-pro-enzyme and upon maturation the 21 amino acid signal peptide and a 22 amino acid carboxy-terminal peptide are removed. This processing event has been proposed to be involved with the vacuolar localization of the enzyme. By comparing the deduced protein structure of PR-Q' to that of the basic form it is evident that this extracellular enzyme is missing the carboxy-terminal 22 amino acids. The role of a conserved phenylalanine-glycine dipeptide in the processing of glucanases and other pathogenesis-related proteins from tobacco is discussed.

Amino Acid Sequence↗
PubMed1990

Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco.

Complementary DNA clones encoding two isoforms of the acidic endochitinase (chitinase, EC 3.2.1.14) from tobacco were isolated. Comparison of amino acid sequences deduced from the cDNA clones and the sequence of peptides derived from purified proteins show that these clones encode the pathogenesis-related proteins PR-P and PR-Q. The cDNA inserts were not homologous to either the bacterial form of chitinase or the form from cucumber but shared significant homology to the basic form of chitinase from tobacco and bean. The acidic isoforms of tobacco chitinase did not contain the amino-terminal, cysteine-rich "hevein" domain found in the basic isoforms, indicating that this domain, which binds chitin, is not essential for chitinolytic activity. The accumulation of mRNA for the pathogenesis-related proteins PR-1, PR-R, PR-P, and PR-Q in Xanthi.nc tobacco leaves following infection with tobacco mosaic virus was measured by primer extension. The results indicate that the induction of these proteins during the local necrotic lesion response to the virus is coordinated at the mRNA level.

Amino Acid Sequence↗
PubMed1989

Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase.

An extracellular, acidic chitinase was purified to homogeneity from tobacco necrosis virus-infected leaves of Cucumis sativis. The amino acid sequences of the intact protein and of peptides isolated following endoproteinase Lys-C digestion, cyanogen bromide cleavage, and trypsin digestion were determined. Oligonucleotide probes derived from this sequence were used to isolate a cDNA clone encoding this protein. No significant homology was found between this chitinase and either the basic chitinase isolated from bean or tobacco or the chitinase isolated from Serratia marcescens; however, strong homology was found between the cucumber chitinase and a lysozyme/chitinase from Parthenocissus quinquifolia. The induction of the protein by tobacco necrosis virus infection or salicylate was found to be at the level of RNA accumulation. Genomic Southern analysis indicates that a single gene in the cucumber genome encodes this protein.

Amino Acid Sequence↗
PubMed1987

Reversible silencing of enhancers by sequences derived from the human IFN-alpha promoter.

The virus-responsive element of the IFN-alpha 1 promoter, VRE(IFN alpha), comprises two imperfect 19 bp repeats, repA and repB. VRE(IFN alpha), tetrameric repA, and tetrameric GAAAGT (a subsequence of repB) or tetrameric AAGTGA conferred inducibility on a reporter gene when placed upstream of a complete or truncated promoter. Induced transcription was weak with a minimal promoter (TATA box only), but was strongly stimulated by the SV40 enhancer placed immediately upstream of the inducible element. Surprisingly, under noninduced conditions, tetrameric repA, GAAAGT, and AAGTGA (but not VRE(IFN alpha)) completely silenced enhancement of constitutive transcription by the SV40 72 bp repeat when interposed between the latter and the TATA box; silencing was fully abrogated by induction.

Animals↗
PubMed1985

A 46-nucleotide promoter segment from an IFN-alpha gene renders an unrelated promoter inducible by virus.

A hybrid gene consisting of the human IFN-alpha 1 promoter and a beta-globin transcription unit is expressed correctly only after viral induction. To determine the region required for inducibility, 25 hybrid promoters consisting of varying upstream IFN-alpha 1 and downstream beta-globin promoter moieties were analyzed, and 5'-deletion analysis was performed on an inducible hybrid promoter. An IFN promoter region from position -109 to -64 conferred maximal inducibility on downstream beta-globin promoter segments and even on the intact beta-globin promoter. This region is strikingly conserved among human IFN-alpha and -beta genes. As constitutive expression of the beta-globin gene was not diminished by placing IFN promoter fragments in various positions, induction is attributed largely to positive, rather than to negative control.

Animals↗
PubMed1984

The expression of human interferon alpha genes.

We have determined the levels of mRNAs for IFN-beta, IFN-gamma and various alpha-IFNs (IFN-alpha 1, -alpha 2, -alpha 4, -alpha 5, -alpha 6, -alpha 7, -alpha 8 and -alpha 14) in normal and leukaemic human blood leucocytes and several cell lines induced in different fashions. The ratio of alpha to beta IFN transcripts varied greatly, depending on the cell type. The levels of the individual IFN-alpha RNAs were very different: IFN-alpha 1, -alpha 2 and -alpha 4 RNAs constituted the major fraction of the IFN-alpha transcripts measured, while IFN-alpha 6, -alpha 7, -alpha 8 and -alpha 14 were minor components in normal, induced leucocytes. Moreover, there was a striking difference in the proportion of individual IFN-alpha mRNA species in different cell types, in particular between normal and leukaemic cells; for example all cases of myeloblastic leukaemia examined showed a high expression of IFN-alpha 14. Use of different induction protocols did not significantly affect the proportion of IFN mRNAs. Analysis of the human IFN-alpha 1 gene by reversed genetics led to the identification of a segment of 5' flanking sequence between positions 117 and 68 upstream of the cap site which is required for inducibility by virus.

Cell Line↗
PubMed1983

rpoB mutation in Escherichia coli alters control of ribosome synthesis by guanosine tetraphosphate.

An isogenic pair of relA+ and relA strains of Escherichia coli B/r with a mutation in the RNA polymerase subunit gene rpoB (Rifr) was isolated in which the relationship between guanosine tetraphosphate (ppGpp) concentration and stable RNA (rRNA, tRNA) gene activity was altered. The RNA polymerase in the rpoB strains was found to be about 20-fold more sensitive to ppGpp with respect to its stable RNA promoter activity than was the wild-type enzyme. The existence of such mutants is consistent with the idea that ppGpp interacts with the RNA polymerase enzyme and thereby alters its promoter selectivity, i.e., reduces its affinity for the stable RNA promoters. Under most conditions, the rpoB mutants had a reduced rate of growth and about a 10-fold-reduced intracellular concentration of ppGpp compared with the rpoB wild-type strains. The reduction of the level of ppGpp in the rpoB mutants during exponential growth was presumably a reflection of an indirect effect of the rpoB mutation on the control of relA-independent ppGpp metabolism.

DNA-Directed RNA Polymerases↗
PubMed1983

Physiological characterization of Escherichia coli rpoB mutants with abnormal control of ribosome synthesis.

We have previously reported the isolation of Escherichia coli rpoB mutants in which the control of ribosome synthesis by the nucleotide effector guanosine tetraphosphate (ppGpp) is altered, owing to a 20-fold increased sensitivity of the mutant RNA polymerases to ppGpp. In these mutants, the level of ppGpp during exponential growth is decreased about 10-fold, relative to that of rpoB+ wild-type strains, such that a near normal partitioning of RNA polymerase occurs with respect to stable RNA (rRNA and tRNA) gene activity. Here, the physiological effects of two different rpoB alleles in a relA+ and relA background were analyzed in greater detail by comparison with their isogenic rpoB+ wild-type parents. For a given growth medium, the rpoB mutations were found to affect four parameters which resulted in a reduction of growth rate. The results reinforce a previous conclusion that a key element in control of the bacterial growth rate is a mutual relationship between control of ribosome synthesis by ppGpp and control of relA-independent ppGpp metabolism by the concentration and function of ribosomes.

DNA-Directed RNA Polymerases↗
PubMed1982

relA-dependent RNA polymerase activity in Escherichia coli.

Parameters relating to RNA synthesis were measured after a temperature shift from 30 to 42 degrees C, in a relA+ and relA- isogenic pair of Escherichia coli strains containing a temperature-sensitive valyl tRNA synthetase. The following results were obtained: (i) the rRNA chain growth rate increased 2-fold in both strains; (ii) newly synthesized rRNA became unstable in both strains; (iii) the stable RNA gene activity (rRNA and tRNA, measured as stable RNA synthesis rate relative to the total instantaneous rate of RNA synthesis) decreased 1.7-fold in the relA+ strain and increased 1.9-fold in the relA mutant; and (iv) the RNA polymerase activity (measured by the percentage of total RNA polymerase enzyme active in transcription an any instant) decreased from 20 to 3.6% in the relA+ strain and remained unchanged (or increased at most to 22%) in the relA mutant. It is suggested that both rRNA gene activity and the RNA polymerase activity depend on the intracellular concentration of guanosine tetraphosphate, whereas the altered chain elongation rate and stability of rRNA are temperature or amino acid starvation effects, respectively, without involvement of relA function.

Amino Acids↗
PubMed1982

Temperature dependence of RNA synthesis parameters in Escherichia coli.

For Escherichia coli B/r growing in glucose minimal medium, the following parameters of RNA synthesis remained invariant between 20 and 40 degrees C: RNA polymerase concentration (RNA polymerase/mass), rRNA and tRNA concentration (RNA/mass), RNA polymerase activity (fraction of total RNA polymerase actively engaged in RNA chain elongation), and stable RNA synthesis relative to total RNA synthesis. The following parameters increased 3.4-fold over the same temperature range: rRNA chain elongation rate, guanosine tetraphosphate (ppGpp) concentration, and culture growth rate. Above 40 degrees C, the changes became more complex, and the growth rate began to decrease. The observation that most RNA synthesis parameters are temperature invariant despite the increase of ppGpp suggests that the mechanism of RNA synthesis control by ppGpp, assumed to involve an interaction of RNA polymerase wtih ppGpp, is itself temperature dependent such that, with increasing temperature, higher concentrations of ppGpp are required to affect the RNA polymerase.

Bacterial Proteins↗
PubMed1982

Isolation of single-site Escherichia coli mutants deficient in thiamine and 4-thiouridine syntheses: identification of a nuvC mutant.

A method is described to rapidly select and classify many independent near-UV irradiation-resistant Escherichia coli mutants, which include tRNA modification and RNA synthesis control mutants. One class of these mutants was found to be simultaneously deficient in thiamine biosynthesis and in the ability to modify uridine in tRNA to 4-thiouridine, known to be the target for near-UV irradiation. These mutants were found to be unable to make thiazole, a thiamine precursor. The addition of thiazole restores the thiamine deficiency but does not render the cells near-UV irradiation sensitive. In vitro studies on one of these mutants indicated a deficiency in protein factor C (nuvC), required for the 4-thiouridine modification of tRNA. In P1 transduction, the thiazole marker cotransduced with the histidine marker, which places the thiazole marker between 42 and 46 min on the E. coli chromosome map. Both thiamine production and 4-thiouridine production were resumed by 87% of the spontaneous reversions, suggesting a single-point mutation. Our results indicate that we have isolated nuvC mutants and that the nuvC polypeptide is involved in two functions, tRNA modification and thiazole biosynthesis.

Chromosome Mapping↗
PubMed1982

Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate.

The expression of stable RNA (rRNA and tRNA) genes and the concentration of guanosine tetraphosphate (ppGpp) were measured in an isogenic pair of relA+ and relA derivatives of Escherichia coli B/r. The cells were either growing exponentially at different rates or subject to amino acid starvation when they were measured. The specific stable RNA gene activity (rs/rt, the rate of rRNA and tRNA synthesis relative to the total instantaneous rate of RNA synthesis) was found to decrease from 1.0 at a ppGpp concentration of 0 (extrapolated value) to 0.24 at saturating concentrations of ppGpp (above 100 pmoles per optical density at 460 nm unit of cell mass). The same relationship between the rs/rt ratio and ppGpp concentration was obtained independent of the physiological state of the bacteria (i.e., independent of the growth rate or of amino acid starvation) and independent of the relA allele. It can be concluded that ppGpp is an effector for stable RNA gene control and that stable RNA genes are not controlled by factors other than the ppGpp-mediated system. The results were shown to be qualitatively and quantitatively consistent with data on in vitro rRNA gene control by ppGpp, and they were interpreted in the light of reported ideas derived from those in vitro experiments.

Escherichia coli↗