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  • 1
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Crude protein extracts were made from kernels of 12 cultivars each of maize, sorghum and wheat. These preparations were fractionated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and subjected to Western blot analyses. Bands corresponding to chitinases and β-glucanases were identified immunologically (Western blots) and on activity gels. Ribosome Inactivating Protein(s) (RIP) and permatins were identified immunologically. In maize, two chitinase bands (25–29 kDa) were seen in all cultivars, and a third band of about 23 kDa was detected in 7 of the 12 cultivars. Two or three β-glucanase bands of sizes between 24 and 36 kDa (probably a mixture of 1,3–β- and 1,3–1,4-β-glucanases) were detected in blots of SDS gels, and one band was detected in activity gels (1,3-β-glucanase). In sorghum, one chitnase band of approximately 29 kDa, and two or three additional bands ranging in size from 21–24 kDa were observed. Only one β-glucanase band was identified, with an estimated molecular weight of 30 kDa. All bands that appeared on Western blots of SDS gels corresponded to bands detected on activity gels. In wheat, one chitmase band of around 20 kDa, one β-glucanase band of about 30 kDa and one RIP band of about 30 kDa were identified. Permatins (molecular weight about 22 kDa) were identified in maize, sorghum and wheat, with the different cultivars having varying amounts of permatins.
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  • 2
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Thaumatin-like proteins (TLPs) were shown to be induced in rice plants (cv. IR58) that were infected with the sheath blight fungus, Rhizoctonia solani. Western blot analysis revealed the presence of two TLPs with sizes of 25 and 24 kDa which are different from a previously reported TLP with a size of 15.6 kDa from rice plants infiltrated with the non-pathogenic bacterium, Pseudomonas syringae pv. syringae. By probing a cDNA expression library prepared from RNA isolated from R. solani-infected rice plants with a TLP antibody, several putative TLP cDNA clones were isolated and sequenced. The cDNA clones appeared to be derived from two different genes which shared only 77% sequence identity with each other and a lower percentage of sequence identity with the previously reported TLP cDNA clone. Southern blot analysis with the two TLP cDNAs revealed different rice genomic DNA fragments. Northern blot analysis also confirmed that a 1.1-kb RNA detectable by the TLP cDNA inserts was induced by fungal infection. Thus rice TLPs are encoded by a family of at least three genes which are differentially expressed in responses to bacterial or fungal pathogens.
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  • 3
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Extracts from several rice cultivars (Oryza sativa L. cvs IR58, 74586 and SC33) infected with the sheath blight pathogen Rhizoctonia solani, were analyzed to determine the isozyme distribution of chitinases (EC 3.2.1.14) and β-l,3-glucanases (EC 3.2.1.39). Upon infection by the fungal pathogen, two chitinases of 28 and 35 kDa and two β-l,3-glucanases of 30 and 32 kDa were shown to be induced in all cultivars. They resolved into multiple isozymes under nondenaturing electrophoretic conditions. Wounding, but not bacterial infection, resulted in the induction of these hydrolytic enzymes. Even though fungal infection resulted in induction of chitinases and β-glucanases in all cultivars, some cultivars that were moderately resistant to R. solani appeared to have higher levels of specific isozymes. A chitinase cDNA clone was identified by screening a library, prepared from RNA isolated from R. solani-infected rice plants, with an antibody to a bean chitinase. This cDNA encoded a 35-kDa chitinase which was significantly different in amino acid sequence from other rice chitinases described so far. Northern blot analysis of RNA from infected rice plants indicated that transcripts corresponding to this chitinase gene were induced upon fungal infection.
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  • 4
    ISSN: 1573-5028
    Keywords: aleurone protoplasts ; barley ; hormone-responsive α-amylase synthesis ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts were prepared from barley aleurone layers using ‘Onozuka’ cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of α-amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley α-amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormoneresponsive elements in hydrolase genes.
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  • 5
    ISSN: 1573-5028
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 12 (1989), S. 403-412 
    ISSN: 1573-5028
    Keywords: barley ; cDNA ; chromosomal location ; endochitinase ; sequence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A barley aleurone cDNA library was screened using 32P-labeled cDNA prepared by reverse transcription of mRNA from aleurone layers treated in the presence or absence of gibberellic acid (GA). Besides α-amylase cDNA clones, another set of clones representing an abundant mRNA in aleurone cells was identified. Messenger RNA hybrid-selected by a prototype clone of this group (clone 10) was translated in vitro to yield a 36 kDa protein. Analysis of the DNA sequence and the predicted amino acid sequence of the protein product of this clone indicates that this gene codes for a protein with homology to endochitinases from tobacco and bean. In addition, the predicted amino acid sequence includes a stretch that is closely related to a cyanogen bromide cleavage fragment from an endochitinase isolated from barley endosperm. The structural genes for endochitinase are present as multiple copies on barley chromosome 1. mRNA detectable by this clone increases in abundance in barley aleurone cells incubated in the absence and in the presence of GA. Western blot analysis of proteins from aleurone and endosperm tissues indicates the presence of multiple endochitinases differing in molecular size. Possible mechanisms for the occurrence of multiple forms of endochitinases and their biological significance are discussed.
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  • 7
    ISSN: 1573-5028
    Keywords: α-Globulin ; cDNA nucleotide sequence ; mRNA ; rice endosperm storage protein ; single-copy gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A λgt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosperm, was screened with affinity-purified antibodies against the rice storage protein called α-globulin (previously), or the 19 kDa globulin (our term). A positive clone was isolated and sequenced and shown to encode a 21 kDa precursor for the 19 kDa globulin, based on the identity of portions of the inferred amino acid sequence and the sequence of three cyanogen bromide peptides of the 19 kDa globulin. Analysis of genomic DNA by Southern blotting using the cDNA clone probe revealed one hybridizing band inEco RI,Hind III, andBam HI digests. This strongly suggests that the 19 kDa globulin is encoded by a single-copy gene. Because of its single-copy nature and its abundance of Arg and lack of Lys, the 19 kDa rice globulin appears to be a particularly attractive target for genetically engineering increased Lys content in rice seeds.
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  • 8
    ISSN: 1573-5028
    Keywords: maize ; protease inhibitor ; trypsin ; activated Hageman factor ; cDNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1573-5028
    Keywords: α-amylase genes ; barley ; DNA sequence ; genomic clones ; transcription starts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have isolated several α-amylase genomic clones from an Eco RI library of barley DNA in λ-Charon 32. Five of these clones exhibit unique restriction maps and differences in their abilities to hybridize with two previously characterized α-amylase cDNA probes representing two different loci, α-Amy 1 (high pI) and α-Amy 2 (low pI) on barley chromosomes 6 and 1, respectively. Stringent hybridizations indicate that four of the five genomic clones contain α-Amy 1 sequences and one contains α-Amy 2 sequences. The regions containing α-amylase genes from one representative genomic clone of each group have been sub-cloned, mapped and sequenced. S1-nuclease protection experiments indicate that the two α-amylase genes contained in these clones are functional in aleurone tissue. Transcription start sites in these genes were determined by primer extension using specific synthetic oligonucleotide primers. The DNA sequences of the two α-amylase genes, including promoter regions, are divergent, as are the predicted amino acid sequences of the mature proteins and the N-terminal “leader” peptides. The α-Amy 1 gene contains two introns while the α-Amy 2 gene has three introns. In the coding region, each gene shows 7–10% sequence divergence with respect to the previously characterized cDNA clones of the same gene type. Therefore, differences in nucleotide sequences can account for some of the isozyme variations seen between the sub-families of α-amylases and among members of the same subfamily. Although the nucleotide sequences of the promoter regions of α-Amy 1 and α-Amy 2 genes show little homology, both contain pairs of inverted repeat elements which could constitute regulatory sites.
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  • 10
    ISSN: 1573-9368
    Keywords: Bacillus thuringiensis toxin ; Heliothis virescens ; Manduca sexta ; Nicotiana tabacum ; tobacco budworm ; tobacco hornworm ; chitinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control
    Type of Medium: Electronic Resource
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