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Biomedical subjects

R B Turley

Publications and source records attributed to R B Turley.

17 recordsLinked to original sources
PubMed2001

Ultrastructural effects of cellulose biosynthesis inhibitor herbicides on developing cotton fibers.

Cotton fibers are often utilized as a model system to investigate cellulose biosynthesis and cell wall elongation. In this study, we grew cotton fibers in vitro, with ovules dissected at day zero post anthesis as the explant source, in the presence of three herbicides that inhibit cellulose biosynthesis. Cultures were sampled for electron microscopy and immunocytochemistry 1-2 days after beginning the treatments. After dichlobenil treatment, the fibers were much shorter than the controls and assumed a variety of abnormal shapes, from shortened versions of the control fiber to nearly spherical. The inner layers of the fiber wall often contained juxtaposed electron-translucent and -transparent areas; this layer reacted strongly with antibodies to callose. Cellulase-gold labeling in these newly developed fibers grown in the presence of dichlobenil was present at only about 3% of the control labeling. After treatment with either isoxaben or flupoxam, the fibers assumed spherical shapes and frequently (more than 60% of fibers) exhibited a new cell plate within the fiber, indicating that cell division had occurred, a process that rarely occurred in the controls. Unlike the dichlobenil-treated fibers, fibers grown in the presence of isoxaben or flupoxam contained an extensive accumulation of chiefly deesterified pectins, replacing the entire wall with an elaborated version of the pectin sheath found in control cotton fibers. These data indicate that all three herbicides are effective disrupters of cellulose biosynthesis and cause radical changes in cell wall structure and composition. Moreover, these data indicate that the composition of the walls may influence indirectly cell cycle kinetics, keeping these fiber cells in a more meristematic mode.

Benzamides↗
PubMed1999

Identification and expression of cotton (Gossypium hirsutum L.) plastidial carbonic anhydrase.

Four carbonic anhydrase (CA) cDNA clones were isolated from a 48 h dark-grown cotton (Gossypium hirsutum L.) seedling cDNA library. Nucleotide sequence analysis revealed two different CA isoforms designated GhCA1 and GhCA2. The encoded polypeptides possess N-terminal serine/threonine-rich regions indicative of plastid transit peptides, and approximately 80% sequence identity to other plant plastidial beta-CAs. The GhCA1 cDNA encodes a nearly complete preprotein of 323 amino acids with a molecular mass of 34.9 kDa and a predicted mature protein of 224 amino acids with a molecular mass of 24.3 kDa. Eleven nucleotide differences within ORFs of GhCA1 and GhCA2 result in 5 conservative amino acid substitutions. The 3' GhCA2 untranslated region contains five additional substitutions and one single nucleotide addition. GhCA1 clones, nearly full-length or with 70% of the transit peptide deleted, were expressed as LacZ alpha fusion proteins in E. coli. Lysates of these strains contained 9-fold higher levels of CA activity as compared to untransformed controls and this activity was inhibited by CA-specific inhibitors. Sulfanilamide, acetazolamide, ethoxyzolamide, each at 10 mM, inhibited recombinant CA activity approximately 50%, 65%, and 75%, respectively. In plant tissue homogenates these inhibitors reduced CA activity by 50%, 70%, and 95%, respectively. Although CA activity was bighest in extracts of mature cotton leaves, probing total RNA with GhCA1 revealed CA transcript levels to be highest in the cotyledons of dark-grown cotton seedlings. Collectively, our data indicate the presence of a plastid-localized CA in cotyledons of germinated seeds, suggesting a role for CA in postgerminative growth.

Amino Acid Sequence↗
PubMed1997

Identification of a delta-TIP cDNA clone and determination of related A and D genome subfamilies in Gossypium species.

Tonoplast intrinsic proteins (TIPs) have been implicated in the process of cell elongation, such as occurs in the developing cotton fiber. We have isolated a cDNA clone (997 bp in length) from a cotton (Gossypium hirsutum L.) library which putatively encodes a protein of 248 residues (Mr 25079) with 85% identity to Arabidopsis delta-TIP. The derived amino acid sequence included two conserved sequences associated with major intrinsic proteins (SGxHxNPA at residues 78 to 85, NPA residues at 197 to 199) and a cysteine residue at 116 which is reported to bind mercury in Arabidopsis delta-TIP. The polymerase chain reaction was used to generate partial genomic clones of the cotton delta-TIP. In comparison to other genomic TIP sequences, the number (two) and position of the introns were conserved in cotton. Comparing the TIP sequences from cotton revealed two subfamilies, which were consistently distinguished by a Tsp45I restriction site polymorphism. This polymorphism was used to demonstrate that TIP subfamilies were specific to either the A or D genomes of Gossypium. When delta-TIP DNA fragments were amplified from cDNA of fiber 14 days after anthesis, the A and D were found, indicating the presence of delta-TIP transcripts in these elongating cells.

Amino Acid Sequence↗
PubMed1996

Ribosomal protein RL44 is encoded by two subfamilies in upland cotton (Gossypium hirsutum L.).

We have isolated 4 cDNA clones encoding the full-length sequence of the eukaryotic ribosomal protein RL44 from upland cotton (Gossypium hirsutum L.). Sequencing of these clones resulted in the classification of 2 subfamilies of RL44; these subfamilies had coding regions (315 bp) which were 92% identical. RL44-1 (454 bp) and RL44-2 (485 bp) constitute subfamily 1, whereas RL44-3 (913 bp) and RL44-5 (541 bp) constitute subfamily 2. The differences in nucleotide sequences, however, occurred only at third codon positions and the resulting amino acid sequences of the two RL44 subfamilies were identical. The ORF encoded a protein of 105 residues with a Mr = 12029. A bipartite nuclear targeting sequence was identified from residues 29 to 43.

Amino Acid Sequence↗
PubMed1990

Characterization of a cDNA encoding cottonseed catalase.

A 1.7 kb cDNA clone was isolated from our lambda gt11 library constructed from poly(A) RNA of 24-h-old cotyledons. The cDNA encodes a full-length catalase peptide (492 amino acid residues). The calculated molecular mass is 56,800, similar to that determined for purified enzyme (57,000 SDS-PAGE). Among higher plant catalases, this cotton catalase shows the highest amino acid sequence identity (85%) to the subunit of homotetrameric maize CAT 1, a developmental counterpart to the homotetrameric CAT A isoform of cotton seeds. Comparison of sequences from cotton, sweet potato, maize CAT 1, and yeast with bovine catalase revealed that the amino acid residues and regions that are involved in catalytic activity and/or required to maintain basic catalase structure, are highly conserved. The C-terminus region, which has the lowest nucleotide sequence identity between plant and mammalian catalases, does not terminate with a tripeptide, S-K/R/H-L, a putative targeting signal for peroxisomal proteins.

Amino Acid Sequence↗
PubMed1990

Characterization of a cDNA clone encoding the complete amino acid sequence of cotton isocitrate lyase.

A cDNA clone encoding the glyoxysomal enzyme isocitrate lyase (ICL) (EC 4.1.3.1) was isolated from a library prepared from cotton (Gossypium hirsutum L.) cotyledon poly(A)+ RNA. The clone is 1893 basepairs (bp) in length and contains a 1728 bp open reading frame encoding a polypeptide of 576 residues (Mr = 64,741). The deduced amino acid sequence of cotton ICL is 85.2%, 90.3% and 41.1% identical to ICL from rapeseed, castor bean and E. coli, respectively. Cotton ICL has a C-terminal tripeptide of A-R-M which is a putative trafficking signal for peroxisome (glyoxysome) proteins.

Amino Acid Sequence↗
PubMed1990

Development and regulation of three glyoxysomal enzymes during cotton seed maturation and growth.

The temporal, nonconcerted development of activities of malate synthase (MS), isocitrate lyase (ICL), and catalase (Cat) was explored in more detail in maturing and germinated cotton (Gossypium hirsutum L.) seeds. RNA was extracted at six intervals beginning at 17 days post anthesis (DPA) through 72 hours post imbibition (HPI). In vitro translations revealed that mRNAs for each enzyme were translatable at all intervals. Enzyme activities and immunoselected proteins also were found at all intervals. Similar specific activities throughout maturation indicated that embryo cells were not accumulating inactive protein. The steady-state level of mRNAs encoding each enzyme exhibited different patterns of change during seed maturation, and each peaked at least 24 h before peak enzyme activities in germinated seeds. All three enzymes occur together as early as 17 DPA in a coordinate manner; however, the subsequent, nonconcerted increases in protein, activity, and mRNA for each enzyme indicate that developmental expression in cotton seed embryos is regulated in a noncoordinate fashion by as yet unidentified specific control mechanism(s).

Blotting, Western↗
PubMed1989

Relationship between Cottonseed Malate Synthase Aggregation Behavior and Suborganellar Location in Glyoxysomes and Endoplasmic Reticulum.

Malate synthase (EC 4.1.3.2) (MS), an enzyme unique to the glyoxylate cycle, was studied in cotyledons of dark-grown cotton (Gossypium hirsutum, L.) seedlings. MS has generally been regarded as a peripheral membrane protein in glyoxysomes and believed by some to be synthesized on rough ER. Immunocyto-chemical localization of MS in both in situ and isolated cottonseed glyoxysomes, however, showed that MS was located throughout the matrix of glyoxysomes, not specifically associated with their membranes. Biochemical data also supported matrix localization. Isolated glyoxysomes were diluted in variously-buffered salt solutions (200 millimolar KCl or 100 millimolar K-phosphate) or detergents (0.1% Triton X-100, 10 millimolar deoxycholate, or 1.0% Triton X-114) and centrifuged to pellet membranes. Greater than 70% of the MS was recovered in supernatants after treatment with salt solutions, whereas generally less than 30% was released following detergent treatments. MS in pellets derived from glyoxysomes burst in low ionic strength buffer solutions was aggregated (observed on rate-zonal gradients). MS released following salt treatments was the 20S nonaggregated form indicating that salt solutions either disaggregated (or prevented aggregation of) glyoxysomal MS rather than releasing it from membranes. We confirmed reports by others that MS comigrated with ER (NADH: cytochrome c reductase) in sucrose (20-40% w/w) gradients buffered with 100 millimolar Tricine (pH 7.5) after 3 hours centrifugation. However, cottonseed MS did not comigrate with ER in gradients buffered with 10 millimolar Hepes (pH 7.0) or 20 millimolar K-phosphate (pH 7.2) after 3 hours centrifugation, or after 22 hours centrifugation in Tricine or Hepes. Collectively, our data with cotton seeds indicate that MS is not a peripheral membrane protein, and that the aggregation behavior of MS (in various buffers) very likely has led to misinterpretations of its putative associations with ER and glyoxysomal membranes.

Journal Article↗
PubMed1988

Purification and biosynthesis of cottonseed (Gossypium hirsutum L.) catalase.

As part of our research on peroxisome biogenesis, catalase was purified from cotyledons of dark-grown cotton (Gossypium hirsutum L.) seedlings and monospecific antibodies were raised in rabbits. Purified catalase appeared as three distinct electrophoretic forms in non-denaturing gels and as a single protein band (with a subunit Mr of 57,000) on silver-stained SDS/polyacrylamide gels. Western blots of crude extracts and isolated peroxisomes from cotton revealed one immunoreactive polypeptide with the same Mr (57,000) as the purified enzyme, indicating that catalase did not undergo any detectable change in Mr during purification. Synthesis in vitro, directed by polyadenylated RNA isolated from either maturing seeds or cotyledons of dark-grown cotton seedlings, revealed a predominant immunoreactive translation product with a subunit Mr of 57,000 and an additional minor immunoreactive product with a subunit Mr of 64000. Labelling studies in vivo revealed newly synthesized monomers of both the 64000- and 57,000-Mr proteins present in the cytosol and incorporation of both proteins into the peroxisome without proteolytic processing. Within the peroxisome, the 57,000-Mr catalase was found as an 11S tetramer; whereas the 64,000-Mr protein was found as a relatively long-lived 20S aggregate (native Mr approx. 600,000-800,000). The results strongly indicate that the 64,000-Mr protein (catalase?) is not a precursor to the 57,000-Mr catalase and that cotton catalase is translated on cytosolic ribosomes without a cleavable transit or signal sequence.

Catalase↗
PubMed1987

Cottonseed malate synthase : purification and immunochemical characterization.

Malate synthase (EC 4.1.3.2), an enzyme unique to the glyoxylate cycle, was purified to homogeneity from cotyledons of 72-hours, darkgrown cotton (Gossypium hirsutum L.) seedlings. Homogeneity of the enzyme was assessed by silver staining SDS-PAGE gels. Purification was accomplished by using a single buffer medium through six steps involving one ammonium sulfate fractionation and chromatography on three columns (Sephacryl S-300, DEAE Sephacel, Phenyl Sepharose). Large-scale preparation of glyoxysomes, a main step in all other published procedures, was not involved. The purified enzyme and that extracted from glyoxysomes appears to be a dodecamer with a native molecular weight of 750,000 (sedimentation coefficient of >20 Svedberg units [S] on sucrose gradients) composed of identical subunits (molecular weight approximately 63,000). The monomer (5S) occurs in the cytosol. Polyclonal antibodies raised in rabbits were judged to be monospecific for malate synthase by immunotitration, double immunodiffusion, and western blotting. Double immunodiffusion experiments revealed only partial immunological identity between the 5S (cytosolic) and 20S (glyoxysomal forms, although complete identity was observed between the 5S form in immature and germinated seeds, and the 20S form in immature and germinated seeds. Cross-reactivity of the cotton antimalate synthase serum was observed with extracts from five other oilseeds. Western blot analyses showed that malate synthase protein was not present in immature seeds prior to appearance of enzyme activity, but when present, subunit molecular weight was indistinguishable in immature, desiccated, and germinated seeds.

Journal Article↗
PubMed1987

Cottonseed malate synthase : biogenesis in maturing and germinated seeds.

The activity of malate synthase (MS) (EC 4.1.3.2) appears and increases during cotton (Gossypium hirsutum L.) seed maturation, persists through desiccation and imbibition, then increases again following germination. The research reported herein is a comparative study of the synthesis and acquisition of MS into glyoxysomes as they occur in maturing and germinated seeds. Rate-zonal centrifugation of cotyledon extracts revealed that the 5 Svedberg unit (S) cytosolic form of MS was the only form present at 42 days postanthesis (DPA) when activity was first detectable. At later stages (48 DPA, 0 day, 26 hours, and 48 hours), both the 5S and glyoxysomal 20S forms were present, with the 20S form becoming much more prevalent. Western blot analyses revealed that no other form(s) of MS were present in the phosphate-buffered gradients, and that 5S and 20S forms had the same subunit molecular weight in maturing and germinated seeds. Comparisons of radiospecific activity of MS immunoprecipitates following in vivo labeling with [(35)S]methionine for varying time intervals provided strong evidence for a 5S-precursor to 20S-product relationship during both seed maturation and seedling growth. Comparisons of MS labeled in vivo and in vitro in wheat germ and rabbit reticulocyte lysates programmed with poly(A)(+)RNA (from maturing and germinated seeds) revealed no detectable differences in subunit molecular weights. These results reinforced our other data indicating that MS was synthesized in the cytosol and acquired by glyoxysomes in both maturing and germinated cotton seeds without involvement of an intervening aggregate pool in the endoplasmic reticulum, or via processing of a cleavable precursor molecule. MS was translated from poly(A)(+)RNA extracted from 28 DPA cotton seeds. This was nearly 2 weeks before MS activity or protein was detected in vivo. This finding invites further study on the regulation of RNA transcripts during maturation.

Journal Article↗
PubMed1986

Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions.

The bincinchoninic acid protein assay has been adapted for use with microtiter plates and a plate reader to reduce the time needed for analyses. The total volume of the assay was reduced to 0.21 ml with various ratios of sample to reagent volume being used. The assay is sensitive to a limit of less than 1 microgram of bovine serum albumin. The assay is insensitive to the presence of the detergents sodium dodecyl sulfate, Triton X-100, and deoxycholate. In addition, the method has been adapted for determining the protein concentrations of sucrose density gradient fractions, which eliminates the need for removing the sucrose prior to assay.

Animals↗
PubMedDate not supplied

Identification of a third fuzzless seed locus in upland cotton (Gossypium hirsutum L.).

Segregating populations were developed to evaluate the inheritance of the fuzzless seed phenotypes in upland cotton (Gossypium hirsutum L.). Accession 143 of the Mississippi Obsolete Variety Collection (MOVC) has a fuzzless seed phenotype. This line carries the n(2) locus which is recessive to the seed fuzz phenotype. Data from the F(2), BC(1)F(1), F(2:3), and BC(1)F(2) populations of DP 5690 x 143 fit a two-loci model for expression of the recessive fuzzless seed phenotype. Fuzzless seeds were obtained in n(2)n(2) plants when a second recessive locus (n(3)) was present. The dominant N(3) allele found in DP 5690 confers the fuzzy seed phenotype in homozygous n(2) plants. Accession 243 of the MOVC carries the N(1) locus, which is dominant to the presence of seed coat fuzz. No variation from expected ratios was observed in the F(2), BC(1)F(1), F(2:3), and BC(1)F(2) populations of the DP 5690 x 243 cross. The N(3) allele had no apparent effect on the expression of the N(1) locus. In a cross between accessions 243 x 143, a few plants were observed which were completely devoid of lint and fuzz fiber (fiberless). A fiberless line was developed from one of these fiberless plants. This line was designated MD 17 fiberless. In a cross between DP 5690 x MD 17 fiberless, we demonstrated that at least three loci were involved in the expression of the fiberless phenotype. The involvement of n(2) and n(3) in the expression of this fiberless phenotype was demonstrated in the F(2) progeny of the cross between 143 x MD 17 fiberless. This is the first demonstration that N(1), n(2), and n(3) interacted to produce fiberless seed.

Crosses, Genetic↗