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T A Wilkins

Publications and source records attributed to T A Wilkins.

At least 19 recordsLinked to original sources
PubMed2003

Evolution and expression of MYB genes in diploid and polyploid cotton.

R2R3-MYB transcription factors have been implicated in a diversity of plant-specific processes. Among the functions attributed to myb factors is the determination of cell shape, including regulation of trichome length and density. Because myb transcription factors are likely to play a role in cotton fiber development, the molecular evolutionary properties of six MYB genes previously shown to be expressed in cotton fiber initiation were examined. In accordance with their presumed central role, each of the genes display conservative substitution patterns and limited sequence divergence in diploid members of the genus Gossypium, and this pattern is conserved in allotetraploid cottons. In contrast to highly reiterated rDNA repeats, GhMYB homologues (duplicated gene pairs) exhibit no evidence of concerted evolution, but instead appear to evolve independently in the allopolyploid nucleus. Expression patterns for the MYB genes were examined in several organs to determine if there have been changes in expression patterns between the diploids (G. raimondii and G. arboreum) and the tetraploid (G. hirsutum) or between the duplicated copies in the tetraploid. Spatial and temporal expression patterns appear to have been evolutionarily conserved, both during divergence of the diploid parents of allopolyploid cotton and following polyploid formation. However, the duplicated copies of MYB1 in the tetraploid are not expressed at equal levels or equivalently in all organs, suggesting possible functional differentiation.

Blotting, Southern↗
PubMed1999

Differential regulation of six novel MYB-domain genes defines two distinct expression patterns in allotetraploid cotton (Gossypium hirsutum L.).

A PCR-based strategy was employed to identify myb-related genes potentially involved in the differentiation and development of cotton seed trichomes. cDNA clones representing six newly identified cotton myb-domain genes (GhMYB) of the R2R3-MYB family were characterized in the allotetraploid species Gossypium hirsutum L. (2n = 4x = 52; AADD). Several interesting motifs and domains in the transregulatory region (TRR) were identified as potential candidates for modulating GhMYB activity. One such structural feature is a basic 40-amino acid stretch (TRR1) located immediately downstream of the DNA-binding domain (DBD) in five of the GhMYBs. Furthermore, the conserved motif GIDxxH identified in a subset of plant MYBs is also present in the same position in the TRR1 domains of GhMYB1 and GhMYB6, exactly 12 amino acid residues downstream of the last tryptophan in the R3 repeat of the DBD. At least two of the GhMYBs (GhMYB4 and GhMYB5) contain unidentified ORFS in the 5' leader sequence (5'-uORFs) that may serve to regulate the synthesis of these particular GhMYB proteins at the translational level. Multiple alignment of DBD sequences indicated that GhMYBs show structural similarity to plant R2R3-MYB factors implicated in phenylpropanoid biosynthesis. GhMYB5 is the most distantly related cotton R2R3-MYB and is found in an isolated cluster that includes the drought-inducible AtMYB2. Sequence comparisons of DBD and TRR domains from GhMYBs, MIXTA (AmMYBMx) and G11 (AtMYBG11) did not reveal any striking similarity beyond conserved motifs. However, based on earlier phylogenetic analysis, GhMYB2, GhMYB3, and GHMYB4 are members of a cluster that contains GLABROUS1, while GhMYB1 and GhMYB6 belong to a closely related cluster. Semi-quantitative RT-PCR analysis revealed two discrete patterns of GhMYB gene expression. Type I cotton MYB (GhMYB-1, -2, and -3) transcripts were found in all tissue-types examined and were relatively more abundant than those derived from type II GhMYB genes (GhMYB-4, -5, and -6), which showed distinct, tissue-specific expression patterns. The developmental regulation of GhMYBs is consistent with a role for these DNA-binding factors in the differentiation and expansion of cotton seed trichomes.

Amino Acid Sequence↗
PubMed1999

Functional complementation of yeast vma1 delta cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H(+)-ATPase activity.

The ability of a vacuolar H(+)-ATPase (V-ATPase) subunit homolog (subunit A) from plants to rescue the vma mutant phenotype of yeast was investigated as a first step towards investigating the structure and function of plant subunits in molecular detail. Heterologous expression of cotton cDNAs encoding near-identical isoforms of subunit A in mutant vma1 delta yeast cells successfully rescued the mutant vma phenotype, indicating that subunit A of plants and yeast have retained elements essential to V-ATPases during the course of evolution. Although vacuoles become acidified, the plant-yeast hybrid holoenzyme only partially restored V-ATPase activity (approximately 60%) in mutant yeast cells. Domain substitution of divergent N- or C-termini only slightly enhanced V-ATPase activity, whereas swapping both domains acted synergistically, increasing coupled ATP hydrolysis and proton translocation by approximately 22% relative to the native plant subunit. Immunoblot analysis indicated that similar amounts of yeast, plant or plant-yeast chimeric subunits are membrane-bound. These results suggest that subunit A terminal domains contain structural information that impact V-ATPase structure and function.

Adenosine Triphosphate↗
PubMed1999

Hmg-coA reductase gene family in cotton (Gossypium hirsutum L.): unique structural features and differential expression of hmg2 potentially associated with synthesis of specific isoprenoids in developing embryos.

As a first step towards understanding the biosynthesis of isoprenoids that accumulate in specialized pigment glands of cotton at the molecular level, two full-length genes (hmg1 and hmg2) were characterized encoding hmg-coA reductase (HMGR; EC 1.1.1.34), the enzyme that catalyzes the formation of a key isoprenoid precursor. Cotton hmgr genes exhibited features typical of other plant genes, however, hmg2 encodes the largest of all plant HMGR enzymes described to date. HMG2 contains several novel features that may represent functional specialization of this particular HMGR isoform. Such features include a unique 42 amino acid sequence located in the region separating the N-terminal domain and C-terminal catalytic domain, as well as an N-terminal hydrophobic domain that is not found in HMG1 or other HMGR enzymes. DNA blot analysis revealed that hmg1 and hmg2 belong to small subfamilies that probably include homeologous loci in allotetraploid cotton (Gossypium hirsutum L.). Ribonuclease protection assays revealed that hmg1 and hmg2 are differentially expressed in a developmentally- and spatially-modulated manner during morphogenesis of specialized terpenoid-containing pigment glands in embryos. Induced expression of hmg2 coincided with a possible commitment to sesquiterpenoid biosynthesis in developing embryos, although other developmental processes also requiring HMGR cannot be excluded.

Base Sequence↗
PubMed1998

A gel diffusion assay for quantification of pectin methylesterase activity.

Increased binding of ruthenium red to pectin as the number of methyl esters attached to the pectin decreases was used as the basis for a gel diffusion assay for pectin methylesterase (PME, EC 3.1.1.11) activity. The stained zone diameters resulting from the hydrolysis of 0.1% (w/v) 90% esterified pectin in an agarose gel by diffused, commercial PME were log-linear over 4 orders of magnitude, with a minimum detection limit of 3.6 pkatals. Pectin deesterification as the cause for a stained zone after PME incubation was confirmed when only 1 N NaOH, which will chemically deesterify the pectin, and not methanol or acid, the two products formed when PME acts on a methyl ester, resulted in the characteristic stained zone. The stained zone diameters decreased with increasing percentage of substrate esterification, were independent of pH, and were insensitive to simultaneous incubation with two forms of pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), or all combinations. PME extracted from tomato seeds, cotton fibers, and melon fruit showed pH optima of 6, 6, and 8, respectively. Using individual tomato seed parts, the assay was adapted to quantify diffusate activity and to localize activity in tissue prints. The sensitivity, specificity, and simplicity of this PME assay are superior to all others.

Carboxylic Ester Hydrolases↗
PubMed1998

Structural analysis of a hmg-coA-reductase pseudogene: insights into evolutionary processes affecting the hmgr gene family in allotetraploid cotton (Gossypium hirsutum L.).

Structural analysis of hmg-coA reductase (hmgr) genes in the allotetraploid cotton species Gossypium hirsutum L. revealed the first-known existence of a pseudogene, psihmg5, for this important enzyme. Complete sequencing of the genomic clone hmg5 unveiled several deleterious lesions, resulting in an organization that departed significantly from the linear canonical hmgr gene structure. Although analysis of the 5' flanking region indicated a promoter-like composition based on comparison with other known plant hmgr genes, the precise loss of intron 3, and putative poly-(A) signals, small poly-(A) tracts, and terminal repeats (TRs) found in the 3'-flanking region are characteristic features of retro-pseudogenes. DNA-blot analysis indicated that a psihmg5-related subfamily exists within a larger hmgr gene family in cotton. Several mechanisms are proposed to account for the formation of this partially intronless pseudogene, including intragenic homologous-replacement recombination and gene conversion involving a cDNA. Alignments of psihmg5 with functional cotton homologs also raised interesting possibilities for the formation of 'chimeric' gene structures, or differential intragenic mutation rates, as potential evolutionary mechanisms involved in shaping the hmgr gene family in cotton.

Amino Acid Sequence↗
PubMed1998

Genes involved in osmoregulation during turgor-driven cell expansion of developing cotton fibers are differentially regulated.

Cotton (Gossypium hirsutum L.) fibers are single-celled trichomes that synchronously undergo a phase of rapid cell expansion, then a phase including secondary cell wall deposition, and finally maturation. To determine if there is coordinated regulation of gene expression during fiber expansion, we analyzed the expression of components involved in turgor regulation and a cytoskeletal protein by measuring levels of mRNA and protein accumulation and enzyme activity. Fragments of the genes for the plasma membrane proton-translocating ATPase, vacuole-ATPase, proton-translocating pyrophosphatase (PPase), phosphoenolpyruvate carboxylase, major intrinsic protein, and alpha-tubulin were amplified by polymerase chain reaction and used as probes in ribonuclease protection assays of RNA from a fiber developmental series, revealing two discrete patterns of mRNA accumulation. Transcripts of all but the PPase accumulated to highest levels during the period of peak expansion (+12-15 d postanthesis [dpa]), then declined with the onset of secondary cell wall synthesis. The PPase was constitutively expressed through fiber development. Activity of the two proton-translocating-ATPases peaked at +15 dpa, whereas PPase activity peaked at +20 dpa, suggesting that all are involved in the process of cell expansion but with varying roles. Patterns of protein accumulation and enzyme activity for some of the proteins examined suggest posttranslational regulation through fiber development.

Blotting, Western↗
PubMed1995

Expression of two related vacuolar H(+)-ATPase 16-kilodalton proteolipid genes is differentially regulated in a tissue-specific manner.

The 16-kD proteolipid subunit is the principal integral membrane protein of the vacuolar H(+)-ATPase (V-ATPase) complex that forms the proton channel responsible for translocating protons across lipid bilayers. Two degenerate synthetic oligonucleotides, COT11 and COT12, corresponding to highly conserved transmembrane domains in all 16-kD subunits sequenced so far, were used to amplify a partial cDNA of the V-ATPase proteolipid subunit from cotton (Gossypium hirsutum L.) by polymerase chain reaction (PCR). These PCR products were used to isolate two full-length cDNAs from a -3 d postanthesis cotton ovule library. Both clones, CVA16.2 and CVA16.4, consisting of 816 and 895 bp, respectively, encode the 16-kD proteolipid subunit of the V-ATPase. At the nucleotide level, the complete sequences of the two clones show 73.5% identity, but share about 95% identity within the coding region, although the two polypeptides differ by only one amino acid. Comparison of deduced amino acid sequences of the proteolipid subunits revealed that the four transmembrane domains and the two cytosolic extramembrane domains are highly conserved in all eukaryotes. Southern blot analysis of cotton genomic DNA showed that these clones belong to small gene families in related diploid and allotetraploid species. Northern blot analysis suggested that the three major V-ATPase subunits (69, 60, and 16 kD) are coordinately regulated, in part, at the transcriptional level. RNA analysis and reverse-transcription PCR established that 16-kD proteolipid transcripts differentially accumulate in different tissues and increase dramatically in tissues undergoing rapid expansion, particularly in anthers, ovules, and petals. The CVA16.4 proteolipid transcript is the most prevalent of the two proteolipid messages in expanding ovules harvested 10 d post-anthesis. In contrast, the two proteolipid mRNAs accumulate to similar levels in developing petals.

Amino Acid Sequence↗
PubMed1994

A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.).

The isolation of biologically active RNA from cotton (Gossypium hirsutum L.) is difficult due to interference by high levels of endogenous phenolics, polysaccharides, and secondary metabolites. A modified hot borate procedure was developed to combat these cellular constituents during tissue homogenization, resulting in the quantitative recovery of RNA suitable for hybridization analysis, in vitro translation, and cDNA synthesis. The efficacy of several hot borate buffer adjuvants for the qualitative and quantitative recovery of leaf RNA was monitored by absorbance spectra, gel electrophoresis, protein, and cDNA synthesis. Of the buffer adjuvants evaluated, polyvinylpyrrolidone-40 (PVP-40) exhibited the single, most significant impact on the yield and quality of RNA isolated from cotton leaves, although inclusion of deoxycholate and/or Nonident-40 (NP-40) further enhanced the quality of the RNA. The unsurpassed qualitative and quantitative recovery of total RNA from cotton by hot borate buffer at alkaline pH, supplemented with PVP-40, deoxycholate, and/or NP-40 had also proven satisfactory for other recalcitrant plant species as well as for especially difficult tissue types.

Boric Acids↗
PubMed1990

A carboxyl-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco.

Barley lectin is synthesized as a preproprotein with a glycosylated carboxyl-terminal propeptide (CTPP) that is removed before or concomitant with deposition of the mature protein in vacuoles. Expression of a cDNA clone encoding barley lectin in transformed tobacco plants results in the correct processing, maturation, and accumulation of active barley lectin in vacuoles [Wilkins, T.A., Bednarek, S.Y., and Raikhel, N.V. (1990). Plant Cell 2, 301-313]. The glycan of the propeptide is not essential for vacuolar sorting, but may influence the rate of post-translational processing [Wilkins, T.A., Bednarek, S.Y., and Raikhel, N.V. (1990). Plant Cell 2, 301-313]. To investigate the functional role of the CTPP in processing, assembly, and sorting of barley lectin to vacuoles, a mutant barley lectin cDNA clone lacking the 15-amino acid CTPP was prepared. The CTPP deletion mutant of barley lectin was expressed in tobacco protoplasts, suspension-cultured cells, and transgenic plants. In all three systems, the wild-type barley lectin was sorted to vacuoles, whereas the mutant barley lectin was secreted to the incubation media. Therefore, we conclude that the carboxyl-terminal domain of the barley lectin proprotein is necessary for the efficient sorting of this protein to plant cell vacuoles.

Amino Acid Sequence↗
PubMed1990

Role of propeptide glycan in post-translational processing and transport of barley lectin to vacuoles in transgenic tobacco.

Mature barley lectin is a dimeric protein composed of two identical 18-kilodalton polypeptides. The subunits of barley lectin are initially synthesized as glycosylated proproteins, which are post-translationally processed to the mature protein preceding or concomitant with deposition of barley lectin in vacuoles. To investigate the functional role of the glycan in processing and intracellular transport of barley lectin to vacuoles, the sole N-linked glycosylation site residing within the COOH-terminal propeptide of barley lectin was altered by site-directed mutagenesis. cDNA clones encoding wild-type (wt) or glycosylation-minus (gly-) barley lectin preproproteins were placed under the transcriptional control of the cauliflower mosaic virus 35S promoter and introduced into Nicotiana tabacum cv Wisconsin 38. Barley lectin synthesized from both the wt and gly- constructs was processed and correctly targeted to vacuoles of tobacco leaves. Localization of barley lectin in vacuoles processed from the nonglycosylated gly- proprotein indicated that the high-mannose glycan of the barley lectin proprotein was not essential for targeting barley lectin to vacuoles. However, pulse-chase labeling experiments demonstrated that the glycosylated wt proprotein and the nonglycosylated gly- proprotein were differentially processed to the mature protein and transported from the Golgi complex at different rates. These results implicate an indirect functional role for the glycan in post-translational processing and transport of barley lectin to vacuoles.

Amino Acid Sequence↗
PubMed1989

Expression of rice lectin is governed by two temporally and spatially regulated mRNAs in developing embryos.

Two cDNA clones encoding rice lectin have been isolated and characterized to investigate the expression of rice lectin at the molecular and cellular levels. The two cDNA clones code for an identical 23-kilodalton protein which is processed to the mature polypeptide of 18 kilodaltons by co-translational cleavage of a 2.6-kilodalton signal sequence and selective removal of a 2.7-kilodalton COOH-terminal peptide which contains a potential N-linked glycosylation site. In addition, the mature 18-kilodalton lectin is post-translationally cleaved between residues 94 and 95 to yield polypeptides of 10 kilodaltons and 8 kilodaltons, corresponding to the NH2- and COOH-terminal portions of the mature subunit, respectively. RNA gel blot analysis established that rice lectin is encoded by two mRNA transcripts (0.9 kilobase and 1.1 kilobase). On DNA gel blots, the rice lectin cDNAs hybridize specifically to a single restriction fragment. In situ hybridization showed localization of the 1.1-kilobase rice lectin mRNA in root caps and specific cell layers of the radicle, coleorhiza, scutellum, and coleoptile. RNA gel blot analysis demonstrated that both the 0.9-kilobase and 1.1-kilobase mRNAs are present in developing rice embryos. The two lectin mRNAs are differentially expressed temporally such that the 1.1-kilobase lectin mRNA accumulates to levels twofold higher than the 0.9-kilobase mRNA.

Amino Acid Sequence↗
PubMed1988

High sensitivity, homogeneous particle-based immunoassay for thyrotropin (Multipact).

We describe the first homogeneous, nonradioactive, high-sensitivity assay for human thyrotropin (TSH). The assay is based on particle immunoassay techniques, wherein 800-nm particles form the basis for the immunochemistry, delivery, and the detection technologies, respectively. Our assay also is the first to involve the use of fragmented monoclonal antibodies (to eliminate serum interferences) covalently coupled to particles without loss of their binding properties. Assays are performed in a semiautomated mode with use of a new modular system (Multipact). Equilibrium is reached in less than 2 h. Precision profile, sensitivity, and clinical studies indicate that the assay is accurate, has good precision at low concentrations, and that detection-limit characteristics compare well with those of a leading commercial high-sensitivity immunoradiometric assay (IRMA) for TSH. Dilution characteristics were satisfactory down to the assay's detection limit for a range of clinical samples. Correlation studies vs a reference IRMA method yielded the regression equation, present method = 0.976 (IRMA) + 0.002 milli-int. unit/L (r = 0.98), for 223 samples with TSH concentrations in the range 0 to 30 milli-int. units/L. For 40 samples with TSH less than or equal to 1.0 milli-int. unit/L it was: present method = 0.94 (IRMA) + 0.005 milli-int. unit/L (r = 0.96).

Antibodies, Monoclonal↗
PubMed1987

Relationship between effects of added albumin, initial free thyroxine value and endogenous serum-binding protein concentrations on Amerlex free thyroxine estimations.

We studied the effect of adding purified human albumin to sera on free thyroxine (FT4) values obtained with Amerlex radioimmunoassays. Apparent FT4 values increased with progressive addition of albumin in vitro. The effect was smallest with low and greatest with high initial FT4 concentrations, which were also linearly correlated with the incremental increase in FT4 values per g/l albumin added. Wide variations in either endogenous thyroxine binding globulin (TBG) or albumin concentrations in patient serum had little effect on the rate of increase in FT4 values when albumin was added in vitro. From Mass Action theory, calculations of the binding affinity of the endogenous albumin for the analog (2.1 X 10(5) l/mol) gave values nearly half that of the added albumin (3.94 X 10(5) l/mol). Distortions in Amerlex FT4 values caused by adding albumin in vitro may exaggerate its importance as a tracer binder and such results may be unrepresentative of patient samples.

Albumins↗
PubMed1987

Isolation and characterization of a cDNA clone encoding wheat germ agglutinin.

Two sets of synthetic oligonucleotides coding for amino acids in the amino- and carboxyl-terminal portions of wheat germ agglutinin were synthesized and used as hybridization probes to screen cDNA libraries derived from developing embryos of tetraploid wheat. The nucleotide sequence for a cDNA clone recovered from the cDNA library was determined by dideoxynucleotide chain-termination sequencing in vector M13. The amino acid sequence deduced from the DNA sequence indicated that this cDNA clone (pNVR1) encodes isolectin 3 of wheat germ agglutinin. Comparison of the deduced amino acid sequence of clone pNVR1 with published sequences indicates isolectin 3 differs from isolectins 1 and 2 by 10 and 8 amino acid changes, respectively. In addition, the protein encoded by pNVR1 extends 15 amino acids beyond the carboxyl terminus of the published amino acid sequence for isolectins 1 and 2 and includes a potential site for N-linked glycosylation. Utilizing the insert of pNVR1 as a hybridization probe, we have demonstrated that the expression of genes for wheat germ agglutinin is modulated by exogenous abscisic acid. Striking homology is observed between wheat germ agglutinin and chitinase, both of which are proteins that bind chitin.

Journal Article↗