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T A Wilkins

Publications and source records attributed to T A Wilkins.

At least 19 recordsLinked to original sources
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↗
PubMed1986

Assay performance and tracer properties for two analog-based assays of free triiodothyronine.

We determined binding characteristics of the triiodothyronine (T3) analog tracer used in the Amerlex and Amerlex-M FT3 radioimmunoassay for the three endogenous binding proteins in serum: thyroxin-binding globulin (TBG), thyroxin binding prealbumin (PA), and albumin. Both T3 and its analog bind to the same sites on TBG and PA. However, the analog has significantly lower association constants (1.0% and 3.8%, respectively, of T3 binding affinity) and it binds to different sites on albumin. Analog binding is characterized by two (weak) specific binding sites [K = 0.46 (SD 0.03) X 10(5) L/mol]; T3 is bound at about 28 very weak, nonspecific sites [K = 0.41 (SD 0.03) X 10(4) L/mol]. Sera from healthy subjects with a wide range of concentrations of binding proteins showed no interference from analog binding in the FT3 assay. In contrast, in vitro studies of albumin binding revealed a weak dependence of both assays on albumin concentration (0.05 pmol of FT3 per gram of albumin per liter), an interference probably unimportant for most laboratory samples. Nonesterified fatty acids (NEFA) and the T3 analog apparently bind to different sites on albumin; thus the Amerlex FT3 assay is insensitive to moderately increased concentrations of NEFA in serum.

Binding Sites↗