Genome-wide mapping with biallelic markers in Arabidopsis thaliana.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

Reversible inhibition of tomato fruit senescence by antisense RNA.

Ethylene controls fruit ripening. Expression of antisense RNA to the rate-limiting enzyme in the biosynthetic pathway of ethylene, 1-aminocyclopropane-1-carboxylate synthase, inhibits fruit ripening in tomato plants. Administration of exogenous ethylene or propylene reverses the inhibitory effect. This result demonstrates that ethylene is the trigger and not the by-product of ripening and raises the prospect that the life-span of plant tissues can be extended, thereby preventing spoilage.

Ethylene signalling: redundant receptors all have their say.

In Arabidopsis, the hormone ethylene is sensed by five related receptors, all of the 'two-component' variety. The receptors constitutively suppress a downstream signalling pathway, and are inactivated by ethylene, leading to the activation of genes necessary for the various ethylene-regulated biological responses.

Anaerobiosis and plant growth hormones induce two genes encoding 1-aminocyclopropane-1-carboxylate synthase in rice (Oryza sativa L.).

The plant hormone ethylene is believed to be responsible for the ability of rice to grow in the deepwater regions of Southeast Asia. Ethylene production is induced by hypoxia, which is caused by flooding, because of enhanced activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, the key enzyme in the ethylene biosynthetic pathway. We have cloned three divergent members, (OS-ACS1, OS-ACS2, and OS-ACS3), of a multigene family encoding ACC synthase in rice. OS-ACS1 resides on chromosome 3 and OS-ACS3 on chromosome 5 in the rice genome. The OS-ACS1 and OS-ACS3 genes are induced by anaerobiosis and indoleacetic acid (IAA) + benzyladenine (BA) + LiCl treatment. The anaerobic induction is differential and tissue specific; OS-ACS1 is induced in the shoots, whereas OS-ACS3 is induced in the roots. These inductions are insensitive to protein synthesis inhibitors, suggesting that they are primary responses to the inducers. All three genes are actually induced when protein synthesis is inhibited, indicating that they may be under negative control or that their mRNAs are unstable. The OS-ACS1 gene was structurally characterized, and the function of its encoded protein (M(r) = 53 112 Da, pI 8.2) was confirmed by expression experiments in Escherichia coli. The protein contains all eleven invariant amino acid residues that are conserved between aminotransferases and ACC synthases cloned from various dicotyledonous plants. The amino acid sequence shares significant identity to other ACC synthases (69-34%) and is more similar to sequences in other plant species (69% with the tomato LE-ACS3) than to other rice ACC synthases (50-44%). The data suggest that the extraordinary degree of divergence among ACC synthase isoenzymes within each species arose early in plant evolution and before the divergence of monocotyledonous and dicotyledonous plants.

Placement of iliosacral screws using 3D image-guided (O-Arm) technology and Stealth Navigation: comparison with traditional fluoroscopy.

AIMS: We compared the accuracy, operating time and radiation exposure of the introduction of iliosacral screws using O-arm/Stealth Navigation and standard fluoroscopy. MATERIALS AND METHODS: Iliosacral screws were introduced percutaneously into the first sacral body (S1) of ten human cadavers, four men and six women. The mean age was 77 years (58 to 85). Screws were introduced using a standard technique into the left side of S1 using C-Arm fluoroscopy and then into the right side using O-Arm/Stealth Navigation. The radiation was measured on the surgeon by dosimeters placed under a lead thyroid shield and apron, on a finger, a hat and on the cadavers. RESULTS: There were no neuroforaminal breaches in either group. The set-up time for the O-Arm was significantly longer than for the C-Arm, while total time for placement of the screws was significantly shorter for the O-Arm than for the C-Arm (p = 0.001). The mean absorbed radiation dose during fluoroscopy was 1063 mRad (432.5 mRad to 4150 mRad). No radiation was detected on the surgeon during fluoroscopy, or when he left the room during the use of the O-Arm. The mean radiation detected on the cadavers was significantly higher in the O-Arm group (2710 mRem standard deviation (sd) 1922) than during fluoroscopy (11.9 mRem sd 14.8) (p < 0.01). CONCLUSION: O-Arm/Stealth Navigation allows for faster percutaneous placement of iliosacral screws in a radiation-free environment for surgeons, albeit with the same accuracy and significantly more radiation exposure to cadavers, when compared with standard fluoroscopy. TAKE HOME MESSAGE: Placement of iliosacral screws with O-Arm/Stealth Navigation can be performed safely and effectively. Cite this article: Bone Joint J 2016;98-B:696-702.

Rapid induction of specific mRNAs by auxin in pea epicotyl tissue.

DNA sequences complementary to three indoleacetic acid (IAA)-inducible mRNAs in pea epicotyl tissue were isolated by differential plaque filter hybridization of cDNA libraries constructed in the vector lambda gt10. Clone pIAA6 hybridized to an mRNA encoding the previously identified translational product polypeptide 6 (Mr 22,000), and clone pIAA4/5 hybridized to one or two mRNAs, encoding polypeptides 4 and 5 (Mr 23,000 and 25,000, respectively). The cDNA clones were subsequently used to characterize the hormonally mediated mRNA accumulation. The induction of the mRNAs was rapid, within 15 minutes of exposure to the IAA, and specific to auxins. Anaerobiosis, heat and cold stress did not induce the mRNAs. Other plant hormones, such as gibberellic acid, kinetin, abscisic acid and ethylene were also unable to cause or interfere with the IAA-induced mRNA accumulation. The hormonally regulated mRNAs were induced at least 50 to 100-fold above control levels after two hours of treatment with IAA and the accumulation was (1) independent of protein synthesis, (2) completely abolished by alpha-amanitin, (3) not due to polyadenylylation of pre-existing RNAs, and (4) independent of IAA and fusicoccin-induced H+ secretion. The IAA-induced mRNAs returned to control levels within three hours after removal of IAA, and the hormonally regulated genes were primarily expressed in the third and second internode of the seven-day-old etiolated pea seedling. The data indicate that IAA increases the amount of specific mRNAs rather than alters the translatability of pre-existing mRNAs. Auxin-induced H+ secretion appears not to have a potential role in mediating the induction and perhaps is a consequence of the enhanced biosynthetic activity induced by the hormone. The IAA-mediated mRNA induction is the fastest known for any plant growth regulator and may represent a primary hormonal response to auxin.

The PS-IAA4/5-like family of early auxin-inducible mRNAs in Arabidopsis thaliana.

The plant hormone auxin transcriptionally activates early genes. We have isolated a 14-member family of DNA sequences complementary to indoleacetic acid (IAA)-inducible transcripts in Arabidopsis thaliana. The corresponding genes, IAA1 to IAA14, are homologs of PS-IAA4/5 and PS-IAA6 from pea, Aux22 and Aux28 from soybean, ARG3 and ARG4 from mungbean, and AtAux2-11 and AtAux2-27 from Arabidopsis. The members of the family are differentially expressed in mature Arabidopsis plants. Characterization of IAA gene expression in etiolated seedlings demonstrates specificity for auxin inducibility. The response of most family members to IAA is rapid (within 4 to 30 minutes) and insensitive to cycloheximide. Cycloheximide alone induces all the early genes. Auxin-induction of two late genes, IAA7 and IAA8, is inhibited by cycloheximide, indicating requirement of protein synthesis for their activation. All IAA genes display a biphasic dose response that is optimal at 10 microM IAA. However, individual genes respond differentially between 10 nM and 5 microM IAA. Expression of all genes is defective in the Arabidopsis auxin-resistant mutant lines axr1, axr2 and aux1. The encoded polypeptides share four conserved domains, and seven invariant residues in the intervening regions. The spacers vary considerably in length, rendering the calculated molecular mass of IAA proteins to range from 19 kDa to 36 kDa. Overall sequence identity between members of the family is highly variable (36 to 87%). Their most significant structural features are functional nuclear transport signals, and a putative beta alpha alpha-fold whose modeled three dimensional structure appears to be compatible with the prokaryotic beta-ribbon DNA recognition motif. The data suggest that auxin induces in a differential and hierarchical fashion a large family of early genes that encode a structurally diverse class of nuclear proteins. These proteins are proposed to mediate tissue-specific and cell-type restricted responses to the hormone during plant growth and development.

Identification of the auxin-responsive element, AuxRE, in the primary indoleacetic acid-inducible gene, PS-IAA4/5, of pea (Pisum sativum).

The plant hormone auxin transcriptionally activates early genes in pea. We have developed a transient assay system using protoplasts of auxin-responsive pea seedling cells to define the auxin-responsive element, AuxRE, of the early auxin-induced PS-IAA4/5 gene. The auxin responsive protoplasts show an authentic hormonal response identical to that observed in intact pea tissue, with respect to rapidity, specificity and cycloheximide (CHX) inducibility of the PS-IAA4/5 transcript. The hormone also mediates rapid and specific induction of chloramphenicol acetyltransferase (CAT) activity in protoplasts transfected with a chimeric IAA4/5-CAT gene. The IAA-induced CAT activity is developmentally regulated and is observed only in protoplasts derived from auxin-responsive regions of the pea seedling. Extensive deletion analysis of the PS-IAA4/5 promoter defined a promoter region between -318 and -154 that confers auxin inducibility. This AuxRE mediates auxin-inducible CAT activity in pea cells driven by the non auxin-responsive CaMV 35S minimal promoter. The functionality of this promoter region as an AuxRE was further verified in tobacco plants using IAA4/5-GUS gene fusions. The AuxRE contains two domains: Domain A acts as an auxin switch; domain B has an enhancer-like activity. The A and B domains contain the highly conserved sequences found in various auxin-regulated genes (T/GGT-CCCAT (domain A) and C/AACATGGNC/AA/GTGTT/CT/CC/A (domain B)). DNase I footprinting reveals binding of nuclear proteins to the highly conserved sequence found in A and B domains. The sequence of the A domain does not correspond to any known regulatory elements found in other eukaryotic genes, and the data suggest that this conserved motif functions as an AuxRE. A model for the early transcriptional activation of the PS-IAA4/5 gene by IAA is discussed.

Transcriptional regulation of PS-IAA4/5 and PS-IAA6 early gene expression by indoleacetic acid and protein synthesis inhibitors in pea (Pisum sativum).

The transcription of two genes, PS-IAA4/5 and PS-IAA6, in pea is induced by indoleacetic acid (IAA) and protein synthesis inhibitors such as cycloheximide (CHX) and anisomycin (ANI). Induction by IAA is rapid, taking 5 and 7.5 minutes for PS-IAA4/5 and PS-IAA6, respectively, and is independent of IAA concentration and whether IAA has a free or esterified carboxyl group (ethyl-IAA). The rate of mRNA accumulation, however, is dependent on hormone concentration, and is greater with IAA than with ethyl-IAA. The turnover rates (t1/2) of the PS-IAA4/5 and PS-IAA6 mRNAs are 60 and 75 minutes, respectively, and are not affected by IAA. CHX or ANI induce the transcription of PS-IAA4/5 and PS-IAA6 more slowly than IAA (5 to 10 minutes for PS-IAA4/5 and 20 minutes for PS-IAA6). While protein synthesis inhibitors stabilize both mRNAs, the rapidity of induction by CHX and ANI cannot be accounted for solely by mRNA stabilization. The relationship between mRNA induction and protein synthesis inhibition does not obey Michaelis-Menten kinetics, but rather is best described by a hyperbolic curve, suggesting the release of transcriptional repression by the inhibition of protein synthesis. RNA expression experiments with transgenic tobacco seedlings or with transfected pea protoplasts using PS-IAA4/5 promoter GUS or CAT fusions reveal that CHX transcriptionally activates PS-IAA4/5 gene expression. Thus, protein synthesis inhibitors have a dual effect on PS-IAA4/5 and PS-IAA6. (1) They stabilize both mRNAs (possibly by a translational arrest-linked process or by preventing the synthesis of a labile nuclease(s)). (2) They activate transcription (possibly by preventing the synthesis or function of a repressor).

Structural characterization of the early indoleacetic acid-inducible genes, PS-IAA4/5 and PS-IAA6, of pea (Pisum sativum L.).

Two early auxin-inducible genes (PS-IAA4/5 and PSIAA6) from pea were cloned using previously isolated complementary DNA sequences. They are present in single copy per haploid genome, and are members of a large divergent multigene family that encodes similar proteins. The genes were structurally characterized and sequence analysis of their 5'-flanking regions revealed the presence of several highly conserved sequences found in various auxin-regulated genes from other plant species. Their coding regions are interrupted by three and two introns, respectively. Introns two and three of PS-IAA4/5 and introns one and two of PS-IAA6 are located in identical positions. These genes encode proteins of 189 (21,036 Da) and 179 (20,330 Da) residues that are 46% identical. They also share a significant degree of identity (42 to 80%) with other proteins encoded by auxin regulated genes in soybean, mungbean and Arabidopsis thaliana. All proteins contain four conserved domains ranging in size from 9 to 43 amino acids. Their most prominent feature is the presence of a highly charged N terminus consisting of two clusters of acidic residues separated by a cluster of basic amino acids.

1-aminocyclopropane-1-carboxylate synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence.

The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.1.1.14). It catalyzes the conversion of S-adenosylmethionine to ACC, the precursor of ethylene. We isolated complementary DNA sequences, ptACC2 and ptACC4, for two distinct and differentially regulated ACC synthase mRNAs expressed in ripe tomato fruit. The authenticity of the clones has been confirmed by expression experiments in E. coli. The predicted size of the encoded polypeptides (54,690 and 53,519 Da) is similar to that of the primary in vitro translation products and to the proteins found in vivo. The sequence of the gene encoding one mRNA, LE-ACC2, has been determined and its transcription initiation site defined. Four additional genes, LE-ACC1A, LE-ACC1B, LE-ACC3 and LE-ACC4, have also been identified and the sequence of their coding regions determined. The LE-ACC1A and LE-ACC1B genes are adjacent to each other and are convergently transcribed. Their encoded polypeptides are 96% identical; the identity of the other polypeptides to each other varies between 50 and 70%. The proteins predicted to be encoded by the ACC synthase genes so far cloned from tomato and zucchini contain 11 of the 12 conserved amino acid residues found in various aminotransferases involved in the binding of the substrate and the cofactor pyridoxal-5'-phosphate. The data indicate that ACC synthase is encoded by a divergent multigene family in tomato that encodes proteins related to aminotransferases.

When do anterior external or internal fixators provide additional stability in an unstable (Tile C) pelvic fracture? A biomechanical study.

PURPOSE: This study aimed at evaluating the additional stability that is provided by anterior external and internal fixators in an unstable pelvic fracture model (OTA 61-C). METHODS: An unstable pelvic fracture (OTA 61-C) was created in 27 synthetic pelves by making a 5-mm gap through the sacral foramina (posterior injury) and an ipsilateral pubic rami fracture (anterior injury). The posterior injury was fixed with either a single iliosacral (IS) screw, a single trans-iliac, trans-sacral (TS) screw, or two iliosacral screws (S1S2). Two anterior fixation techniques were utilized: external fixation (Ex-Fix) and supra-acetabular external fixation and internal fixation (In-Fix); supra-acetabular pedicle screws connected with a single subcutaneous spinal rod. The specimens were tested using a nondestructive single-leg stance model. Peak-to-peak (P2P) displacement and rotation and conditioning displacement (CD) were calculated. RESULTS: The Ex-Fix group failed in 83.3 % of specimens with concomitant single-level posterior fixation (Total: 15/18-7 of 9 IS fixation, 8 of 9 TS fixation), and 0 % (0/9) of specimens with concomitant two-level (S1S2) posterior fixation. All specimens with the In-Fix survived testing except for two specimens treated with In-Fix combined with IS fixation. Trans-sacral fixation had higher pubic rotation and greater sacral and pubic displacement than S1S2 (p < 0.05). Rotation of the pubis and sacrum was not different between In-Fix constructs combined with single-level IS and TS fixation. CONCLUSION: In this model of an unstable pelvic fracture (OTA 61-C), anterior fixation with an In-Fix was biomechanically superior to an anterior Ex-Fix in the setting of single-level posterior fixation. There was no biomechanical difference between the In-Fix and Ex-Fix when each was combined with two levels of posterior sacral fixation.

Characterization of two members (ACS1 and ACS3) of the 1-aminocyclopropane-1-carboxylate synthase gene family of Arabidopsis thaliana.

The nucleotide sequences of two highly homologous 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14)-encoding genes, ACS1 and ACS3, of Arabidopsis thaliana (At) have been determined. The sequence analysis shows that ACS3 is a pseudogene representing a truncated version of ACS1. The missing region of ACS3 corresponding to the fourth exon of ACS1 has been shown by Southern analysis to be absent in the At genome. The chromosomal locations of the five members of the At ACS multigene family have been determined. The results show that each family member resides on a different chromosome. This observation suggests that the ACS3 pseudogene originated by a partial inter-chromosomal gene duplication. The ACS1 polypeptide contains all the conserved and characteristic domains found in the ACC synthase isoenzymes from various plant species, but is unable to express ACS activity in Escherichia coli and yeast. The predicted amino-acid sequence of ACS1 is missing the highly conserved tripeptide, Thr-Asn-Pro (TNP), between Ile204 and Ser205. Introduction of TNP into ACS1 restores the ACS activity, whereas its removal from the enzymatically active ACS2 results in a loss of activity. The results suggest that TNP is crucial for expression of ACS activity in E. coli.

Biochemical characterization of recombinant polypeptides corresponding to the predicted betaalphaalpha fold in Aux/IAA proteins.

The plant hormone indoleacetic acid (IAA or auxin) transcriptionally activates a select set of early genes. The Aux/IAA class of early auxin-responsive genes encodes a large family of short-lived, nuclear proteins. Aux/IAA polypeptides homo- and heterodimerize, and interact with auxin-response transcription factors (ARFs) via C-terminal regions conserved in both protein families. This shared region contains a predicted betaalphaalpha motif similar to the prokaryotic beta-ribbon DNA binding domain, which mediates both protein dimerization and DNA recognition. Here, we show by circular dichroism spectroscopy and by chemical cross-linking experiments that recombinant peptides corresponding to the predicted betaalphaalpha region of three Aux/IAA proteins from Arabidopsis thaliana contain substantial alpha-helical secondary structure and undergo homo- and heterotypic interactions in vitro. Our results indicate a similar biochemical function of the plant betaalphaalpha domain and suggest that the betaalphaalpha fold plays an important role in mediating combinatorial interactions of Aux/IAA and ARF proteins to specifically regulate secondary gene expression in response to auxin.

The nucleotide sequence of the 5' flanking region of the Arabidopsis ACS2 gene.

The ACS2 gene of Arabidopsis thaliana encodes 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.1.1.14) - the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene. The gene is unique among ACC synthase genes isolated thus far in that it is highly expressed in both reproductive (e.g. flower) and vegetative tissues (e.g. leaves and roots). In addition, the accumulation of ACS2 transcripts is greatly induced by protein synthesis inhibition (Liang et al 1992). We report here the DNA sequence of the 5' flanking region of the ACS2 gene and an analysis of the sequence with respect to the potential cis-acting regulatory elements that can be recognized by DNA-binding proteins from plants and other organisms.

Goodbye to 'one by one' genetics.

The completion of the Arabidopsis thaliana (mustard weed) genome sequence constitutes a major breakthrough in plant biology. It will revolutionize how we answer questions about the biology and evolution of plants as well as how we confront and resolve world-wide agricultural problems.