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M L Chye

Publications and source records attributed to M L Chye.

12 recordsLinked to original sources
PubMed1997

Characterization of TSCL, a nonviral retroposon from Arabidopsis thaliana.

We isolated by differential screening a 1.2 kb cDNA from an Arabidopsis thaliana ecotype Columbia cDNA library that is highly expressed in stem and root. In situ hybridization studies on stem sections and root sections showed that the mRNA is expressed in stem sclerenchyma and root cortex, respectively. The isolation and sequence analysis of four other overlapping cDNA clones from two independent A. thaliana cDNA libraries confirmed that these cDNAs lack a significant open reading frame that has recognizable homology to any known proteins. We have obtained from A. thaliana ecotype Columbia three corresponding genomic clones and nucleotide sequence analysis of these clones revealed that we have isolated a retroposon, TSCL, that is flanked by two 13 bp direct repeats, is intronless, and has a poly(A)+ tract at the 3' end. The site of transcription initiation mapped by primer extension analysis lies 48 bp downstream from an external TATA box. Results from Southern blot analysis suggest that TSCL occurs as a single-copy insert in the genomes of A. thaliana ecotype Columbia (Col-0) and Col-2 but is absent in the genomes of Brassica napus. Brassica juncea and A. thaliana ecotypes Be-0, Oy-0 and Ler-0. This suggests that Col-0 and Col-2 are phylogenetically more closely related to each other than to Be-0, Oy-0 and Ler-0, and that the Laibach Landsberg seeds Redei received, from which ecotypes Col-0, Col-2 and Ler-0 originated, were heterogeneous for TSCL.

Amino Acid Sequence↗
PubMed1996

Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana.

Calcium-dependent protein kinases (CDPKs) belong to a unique family of enzymes containing a single polypeptide chain with a kinase domain at the amino terminus and a putative calcium-binding EF hands structure at the carboxyl terminus. From Arabidopsis thaliana, we have cloned three distinct cDNA sequences encoding CDPKs, which were designated as atcdpk6, atcdpk9 and atcdpk19. The full-length cDNA sequences for atcdpk6, atcdpk9 and atcdpk19 encode proteins with a molecular weight of 59343, 55376 and 59947, respectively. Recombinant atCDPK6 and atCDPK9 proteins were fully active as kinases whose activities were induced by Ca2+. Biochemical studies suggested the presence of an autoinhibitory domain in the junction between the kinase domain and the EF hands structure. Serial deletion of the four EF hands of atCDPK6 demonstrated that the integrity of the four EF hands was crucial to the Ca2+ response. All the three atcdpk genes were ubiquitiously expressed in the plant as demonstrated by RNA gel blot experiments. Comparison of the genomic sequences suggested that the three cdpk genes have evolved differently. Using antibodies against atCDPK6 and atCDPK9 for immunohistochemical experiments, CDPKs were found to be expressed in specific cell types in a temporally and developmentally regulated manner.

Amino Acid Sequence↗
PubMed1995

A cDNA clone encoding Brassica calmodulin.

A 834 bp cDNA encoding calmodulin (CaM) has been isolated from Brassica juncea. On Northern analysis this cDNA hybridises this cDNA to mRNAs of about 0.9 kb in leaf, silique and peduncle. Genomic Southern analysis indicates the presence of a CaM multigene family in Brassica juncea. Comparison of the predicted amino acid sequence of Brassica CaM with that of Arabidopsis CaM ACaM-2 and ACaM-3 showed 100% homology, which is not unusual, since both plants belong to the family Cruciferae. In situ hybridisation studies on Brassica seedlings using a digoxigenin-labelled RNA probe showed that high levels of CaM mRNA were detected in the leaf primordia and the shoot apical meristem, and to a lesser degree, in the zone of root elongation of the root tip. The occurrence of a higher rate of cell division and growth in these regions than its surrounding tissue may possibly be related to higher levels of CaM mRNA.

Amino Acid Sequence↗
PubMed1995

beta-1,3-Glucanase is highly-expressed in laticifers of Hevea brasiliensis.

Clones encoding beta-1,3-glucanase have been isolated from a Hevea cDNA library prepared from the latex of Hevea brasiliensis using a probe Nicotiana plumbaginifolia cDNA encoding beta-1,3-glucanase, gnl. Nucleotide sequence analysis showed that a 1.2 kb Hevea cDNA encoding a basic beta-1,3-glucanase showed 68% nucleotide homology to gnl cDNA. Northern blot analysis using the Hevea cDNA as probe detected a mRNA of 1.3 kb which was expressed at higher levels in latex than in leaf. In situ hybridization analysis using petiole sections from Hevea localized the beta-1,3-glucanase mRNA to the laticifer cells. Genomic Southern analysis suggested the presence of a low-copy gene family encoding beta-1,3-glucanases in H. brasiliensis.

Amino Acid Sequence↗
PubMed1992

Three genes encode 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hevea brasiliensis: hmg1 and hmg3 are differentially expressed.

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyses an important step in isoprenoid biosynthesis in plants. In Hevea brasiliensis, HMGR is encoded by a small gene family comprised of three members, hmg1, hmg2 and hmg3. We have previously described hmg1 and hmg2 (Plant Mol Biol 16: 567-577, 1991). Here we report the isolation and characterization of hmg3 genomic and cDNA clones. In comparison to hmg1 which is more highly expressed in laticifers than in leaves, the level of hmg3 mRNA level is equally abundant in laticifers and leaves. In situ hybridization experiments showed that the expression of hmg3 is not cell-type specific while hmg1 is expressed predominantly in the laticifers. Primer-extension experiments using laticifer RNA showed that hmg1 is induced by ethylene while hmg3 expression remains constitutive. The hmg3 promoter, like the promoters of most housekeeping genes, lacks a TATA box. Our results suggest that hmg1 is likely to encode the enzyme involved in rubber biosynthesis while hmg3 is possibly involved in isoprenoid biosynthesis of a housekeeping nature.

Amino Acid Sequence↗
PubMed1991

Characterization of cDNA and genomic clones encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Hevea brasiliensis.

Hevea brasiliensis is the major producer of natural rubber which is cis-1,4-polyisoprene. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is involved in the biosynthesis of rubber and other plant products. We have used a hamster HMGR cDNA clone as a heterologous hybridization probe to isolate and characterize cDNA and genomic clones of HMGR from H. brasiliensis. Sequence analysis revealed that these clones fall into two different classes, HMGR1 and HMGR2. Comparison of the two classes shows 86% nucleotide sequence homology and 95% amino acid homology. The carboxy-termini of Hevea HMGRs are highly homologous to those of hamster, yeast and Arabidopsis HMGR. The amino-terminus of Hevea HMGR contains two potential membrane-spanning domains as in Arabidopsis HMGR while seven such domains are found in the HMGRs of other organisms. The apparent molecular mass of Hevea HMGR was estimated in western blot analysis to be 59 kDa. Northern blot analysis indicated that the HMGR1 transcript of 2.4 kb is more highly-expressed in laticifer than in leaf. Genomic Southern analysis using 3'-end cDNA probes indicates the presence of at least two HMGR genes in Hevea.

Amino Acid Sequence↗
PubMed1989

The rice phytochrome gene: structure, autoregulated expression, and binding of GT-1 to a conserved site in the 5' upstream region.

We have isolated and characterized both cDNA and genomic clones encoding the apoprotein of rice phytochrome. The mRNA produced from this gene is expressed at a low level in etiolated leaves. Following a flash of red light, the steady-state mRNA level decreases within 15 minutes, and is barely detectable after 2 hours. This effect is partially reversed by far red light demonstrating autoregulation of phytochrome mRNA levels. Nuclear run-on experiments show that this effect is exerted on transcription of the phytochrome gene. In etiolated plants, phytochrome mRNA is twofold higher in leaves than in roots, whereas the reverse is true in fully green plants where phytochrome mRNA accumulates despite illumination of the leaves. DNA gel blots and screening of libraries indicate the presence of only a single gene, allowing convenient study of the autoregulatory phenomenon for a specific phytochrome gene. Gel retardation analysis using a fragment from the 5' upstream region reveals that GT-1 is present in nuclear extracts of etiolated rice leaves and binds to sites conserved between rice and oat phytochrome genes.

Base Sequence↗
PubMed1987

Transcription control of the aroP gene in Escherichia coli K-12: analysis of operator mutants.

The nucleotide sequence of the region containing the promoter-operator for the aroP gene was determined. The start site of aroP transcription was identified by using S1 nuclease mapping and primer extension techniques. Examination of the nucleotide sequence revealed the presence of two "TYR R" boxes which are similar to those identified in the regulatory regions of other genes in the tyrR regulon. Bisulfite-induced aroP operator-constitutive mutants were analyzed, and the base-pair changes responsible for alterations in aroP regulation were located within these boxes.

Acetyltransferases↗