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Biomedical subjects

B H Taylor

Publications and source records attributed to B H Taylor.

13 recordsLinked to original sources

Analysis of tomato root initiation using a normal mixture distribution.

We attempt to identify the number of underlying physical phenomena behind tomato lateral root initiation by using a normal mixture distribution coupled with the Box-Cox power transformation. An initial analysis of the data suggested the possibility of two (possibly more) subpopulations, but upon taking reciprocals, the data appear to be very nearly Gaussian. A simulation study explores the possibility of erroneously detecting a second subpopulation by fitting data which are improperly scaled. A power calculation suggests that only unrealistically large sample sizes can detect the unbalanced mixtures one might expect with data of this type.

Biometry↗

A molecular marker for lateral root initiation: the RSI-1 gene of tomato (Lycopersicon esculentum Mill) is activated in early lateral root primordia.

The frequency of lateral root initiation in tomato (Lycopersicon esculentum Mill cv. VFN8) seedling roots is increased over eightfold in response to 1.6 microM alpha-naphthalene-acetic acid (NAA). To identify genes that are activated during lateral root initiation, a cDNA library was made with RNA from roots treated with auxin and differentially screened with radioactive probes made from RNA isolated from treated and untreated roots. A cDNA clone, TR132, was identified that hybridized to a transcript that was induced within 4 h of auxin treatment and increased tenfold by 72 h. A gene (RSI-1) corresponding to the TR132 cDNA was cloned and characterized with regard to its nucleotide sequence, transcription start site and chromosomal map position. Approximately 1 kb of the 5' flanking DNA was linked to the beta-glucuronidase (GUS) protein coding region and tested for expression in transgenic tomato seedlings. GUS activity was observed in both lateral and adventitious root initials, including very early initials, and persisted until shortly after the lateral emerged from the parent tissue. In roots from seedlings with high activity, GUS expression was also observed in the root cap and vascular tissue. The predicted RSI-1 protein is rich in cysteine, lysine and proline, and includes an N-terminal region with characteristics of a signal peptide. The putative mature protein exhibits 79% amino acid identity to a protein encoded by a gene (GAST1) that is induced by gibberellic acid in tomato shoots.

Amino Acid Sequence↗

A dispersed family of repetitive DNA sequences exhibits characteristics of a transposable element in the genus Lycopersicon.

A segment of DNA 5' to the transcribed region of an auxin-regulated gene, ARPI, from Lycopersicon esculentum Mill. cv. VFN8 contains a sequence with the structural characteristics of a transposable element. The putative element (Lyt1) is 1340 bp long, has terminal inverted repeats of approximately 235 bp and is flanked by 9-bp direct repeats. Lyt1 has a structure similar to the Robertson's Mutator (Mu) family from maize. The terminal inverted repeats are 80% AT-rich, are 96.6% identical, and define a larger family of repetitive elements. Southern analysis and genomic dot-blot reconstructions detected at least 41 copies of Lyt1-hybridizing sequences in red-fruited Lycopersicon spp. (L. esculentum, L. pimpinellifolium and L. cheesmanii), and 2-8 copies in the green-fruited species (L. hirsutum, L. pennellii, L. peruvianum, L. chilense and L. chmielewskii). There were two to four copies in the Solanum spp. closely allied with the genus Lycopersicon (S. lycopersicoides, S. ochranthum and S. juglandifolium), while the more distantly related Solanum spp. showed little (one to two copies in S. tuberosum) to no (S. quitoense) detectable hybridization under stringent conditions. Linkage analysis in the F2 progeny of a cross between L. esculentum and L. cheesmanii indicated that at least six loci that hybridize to the Lyt1 sequence are dispersed in the genome. Polymerase chain reaction and Southern analyses revealed that some red-fruited accessions and L. chmielewskii lacked Lyt1 5' to the transcribed region of ARPI. Subsequent sequence analysis indicated that only one copy of the 9-bp direct repeat (target site) was present, suggesting that transposition of the element into the ARPI gene occurred after the divergence of the red-fruited and green-fruited Lycopersicon species.

Base Sequence↗

Induction of a proteinase inhibitor II-class gene by auxin in tomato roots.

A cDNA clone corresponding to a transcript that was induced by auxin in tomato seedling roots encodes a 223 amino acid protein belonging to the proteinase inhibitor II family of tomato and potato. The TR8 open reading frame has an N-terminal region with characteristics of a signal peptide and three copies of a 64 amino acid segment that is also present in multiple copies in other PI-II family members. Comparison to other serine proteinase inhibitors indicates that all three domains are trypsin-specific. Transcripts homologous to TR8 increased in abundance within 24 h of auxin treatment and continued to increase through 72 h, at which point the induction was approximately ten-fold. Auxin induction was observed in roots and hypocotyls, but not in cotyledons or epicotyls. Induction may therefore be correlated with lateral and adventitious root initiation, which occurs over a similar time frame in the same tissues. RFLPs detected with the TR8 probe were mapped to the long arm of chromosome 11.

Amino Acid Sequence↗

The maize transposable element Ac excises in progeny of transformed tobacco.

To assess the potential of the maize transposable element Ac for gene tagging in heterologous plant species we monitored transcription, excision and transposition of the element in transgenic tobacco plants and their selfed progeny. Ac excised in the majority of primary regenerants and continued to excise in the first-generation progeny plants. In one primary regenerant Ac was transcribed but did not excise. Fourteen of eighteen kanamycin-resistant progeny from this plant showed Ac excision, suggesting that excision of Ac may have been activated during meiosis or in embryo development. This finding, together with the more general observation of continued Ac mobility in the progeny of transformed plants in which Ac had excised, suggests that Ac will be useful for gene tagging.

Blotting, Southern↗

Transposable elements can be used to study cell lineages in transgenic plants.

The beta-glucuronidase reporter gene has been used to develop a sensitive assay for the excision of transposable elements introduced into transgenic plants. The reporter gene, inactivated by the insertion of the maize transposable element Activator (Ac) into the 5'-untranslated leader, was introduced into the genome of tobacco by Agrobacterium-mediated transformation. Reactivation of the beta-glucuronidase gene was detected in transgenic plants using a fluorometric or histochemical assay. Reactivation of the reporter gene was dependent on the presence of the transposase of Ac, and resulted from the excision of the Ac element. This assay, together with the improved methods for visualization, will provide a valuable and rapid method for studying the basic mechanism of transposition in plants and for developing modified transposable element systems suitable for gene tagging in transgenic plants.

Cloning, Molecular↗

Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes.

The T-DNA regions of the root-inducing (Ri) plasmid pRiA4b of Agrobacterium rhizogenes were characterized. Two regions, designated TL-DNA and TR-DNA, were found to be integrated and stably maintained in the plant genome. The TL-DNA spanned a 15- to 20-kilobase region of pRiA4b and was separated from the TR-DNA region by at least 15 kilobases of nonintegrated plasmid DNA. The TR-DNA region also spanned a 15- to 20-kilobase region of pRiA4b and included a region of homology to the tms morphogenic loci of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. Eighteen deletions and 95 transposon insertions were generated in the T-DNA regions and tested for alterations in virulence. Insertions into four loci in the TL-DNA affected the morphology of root formation of Kalanchoë diagremontiana leaves and stems, but had no visible effects on other host plants. Insertions into two loci (tms-1 and tms-2) in the TR-DNA eliminated virulence symptoms on all plants tested, with the exception of K. diagremontiana stems, where sparse root formation occurred. Complementation experiments with Ri and Ti plasmid T-DNA mutations indicate that the tms genes of the two plasmids serve similar functions and suggest a functional relationship between one or more genes of the TL-DNA and the cytokinin synthesis locus tmr of the Ti plasmid.

Chromosome Deletion↗

Covalent crosslinking of tRNA1Val to 16S RNA at the ribosomal P site: identification of crosslinked residues.

N-Acetylvalyl-tRNA1Val (AcVal-tRNA1Val) was bound to the P site of uniformly 32P-labeled 70S ribosomes from Escherichia coli and crosslinked to 16S RNA in the 30S ribosomal subunit by irradiation with light of 300-400 nm. To identify the crosslinked nucleotide in 16S RNA. AcVal-tRNA1Val-16S [32P]RNA was digested completely with RNase T1 and the band containing the covalently attached oligonucleotides from tRNA and rRNA was isolated by polyacrylamide gel electrophoresis. The crosslinked oligonucleotide, and the 32P-labeled rRNA moiety released from it by photoreversal of the crosslink at 254 nm, were then analyzed by secondary hydrolysis with pancreatic RNase A and RNase U2. The oligonucleotide derived from 16S RNA was found to be the evolutionarily conserved sequence, U-A-C-A-C-A-C-C-G1401, and the nucleotide crosslinked to tRNA1Val, C1400. The identity of the covalently attached residue in the tRNA was established by using AcVal-tRNA1Val-16S RNA prepared from unlabeled ribosomes. This complex was digested to completion with RNase T1 and the resulting RNA fragments were labeled at the 3' end with [5'-32P]pCp. The crosslinked T1 oligonucleotide isolated from the mixture yielded one major end-labeled component upon photoreversal. Chemical sequence analysis demonstrated that this product was derived from the anticodon-containing pentadecanucleotide of tRNA1Val, C-A-C-C-U-C-C-C-U-cmo5U-A-C-m6A-A-G39(cmo5U, 5-carboxymethoxyuridine). A similar study of the crosslinked oligonucleotide revealed that the residue covalently bound to 16S was cmo5U34, the 5' or wobble base of the anticodon. The adduct is believed to result from formation of a cyclobutane dimer between cmo5U34 of tRNA1Val and C1400 of the 16S RNA.

Base Sequence↗

Effects of cooling and intraluminally administered antiseptics on surgically induced ischemia of the intestine in dogs.

The efficacy of surface cooling and intraluminal antiseptic solutions for short term preservation of intestinal segments in preparation for free tissue transfer were investigated. Five segments of the small intestine and five segments of the colon were allocated to each group as follows: group 1, ischemia at 37 degrees C.; group 2, ischemia at 6 to 12 degrees C., and group 3, ischemia at 6 to 12 degrees C. plus povidone-iodine intraluminally. Histologic grading of segments allowed assessment of the efficacy of short term protection against ischemic changes. Surface cooling effectively prevented histologic evidence of ischemia. No additional benefit was observed using povidone-iodine intraluminally. Significant changes in tissue water content and evidence of bacterial invasion were not observed.

Animals↗