A framework linkage map of perennial ryegrass based on SSR markers.

A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third (124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci were generated from either SSR-enriched genomic libraries (247) or ESTs (5). Forty-five percent of the GeneThresher SSRs were associated with an expressed gene. Unlike EST-derived SSR markers, GeneThresher SSRs were often associated with genes expressed at a low level, such as transcription factors. The map constructed here fulfills 2 definitions of a "framework map". Firstly, it is composed of codominant markers to ensure map transferability either within or among species. Secondly, it was constructed to achieve a level of statistical confidence in the support-for-order of marker loci. The map consists of 81 framework SSR markers spread over 7 linkage groups, the same as the haploid chromosome number. Most of the remaining 295 SSR markers have been placed into their most likely interval on the framework map. Nine RFLP markers and 1 SSR marker from another map constructed using the same pedigree were also incorporated to extend genome coverage at the terminal ends of 5 linkage groups. The final map provides a robust framework with which to conduct investigations into the genetic architecture of trait variation in this commercially important grass species.

Endoplasmic reticulum Ca2+-ATPase pump is up-regulated in calcium-transporting dental enamel cells: a non-housekeeping role for SERCA2b.

Dental enamel-forming cells face a major challenge to avoid the cytotoxic effects of excess calcium. We have characterized sarcoplasmic/endoplasmic reticulum calcium-ATPase pumps (SERCA) in rat enamel cells to address the proposal that non-mitochondrial calcium stores play a dominant role in transcellular calcium transport. A single major isoform, SERCA2b, was detected during the protein-secretory and calcium-transport stages of enamel formation using reverse-transcriptase PCR, cDNA cloning, Northern analysis and immunoblotting. Most importantly, SERCA2b exhibited a specific 3-fold up-regulation to high expression levels during calcium transport, as determined by quantitative immunoblotting and ATPase assays. Sensitivity of the calcium-dependent ATPase to thapsigargin and three other SERCA inhibitors was characterized. These findings indicate that enamel cells are well-equipped to sequester calcium in endoplasmic reticulum stores and so protect against calcium toxicity, associate SERCA with transcellular calcium transport for the first time, and establish SERCA2b as a molecular and pharmacological target for future investigations of calcium transcytosis. The observed physiological regulation in enamel cells contradicts the widespread perception that SERCA2b is restricted to general housekeeping duties.

Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. IV. Insect-Induced ethylene reduces jasmonate-induced nicotine accumulation by regulating putrescine N-methyltransferase transcripts.

Attack by the specialist herbivore, Manduca sexta, on its native host Nicotiana attenuata Torr. ex Wats. produces a dramatic ethylene release, a jasmonate burst, and a suppression of the nicotine accumulation that results from careful simulations of the herbivore's damage. Methyl-jasmonate (MeJA) treatment induces nicotine biosynthesis. However, this induction can be suppressed by ethylene as pretreatment of plants with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene receptors, restores the full MeJA-induced nicotine response in herbivore attacked plants (J. Kahl, D.H. Siemens, R.J. Aerts, R. Gäbler, F. Kühnemann, C.A. Preston, I.T. Baldwin [2000] Planta 210: 336-342). To understand whether this herbivore-induced signal cross-talk occurs at the level of transcript accumulation, we cloned the putrescine methyltransferase genes (NaPMT1 and NaPMT2) of N. attenuata, which are thought to represent the rate limiting step in nicotine biosynthesis, and measured transcript accumulations by northern analysis after various jasmonate, 1-MCP, ethephon, and herbivory treatments. Transcripts of both root putrescine N-methyltransferase (PMT) genes and nicotine accumulation increased dramatically within 10 h of shoot MeJA treatment and immediately after root treatments. Root ethephon treatments suppressed this response, which could be reversed by 1-MCP pretreatment. Moreover, 1-MCP pretreatment dramatically amplified the transcript accumulation resulting from both wounding and M. sexta herbivory. We conclude that attack from this nicotine-tolerant specialist insect causes N. attenuata to produce ethylene, which directly suppresses the nitrogen-intensive biosynthesis of nicotine.

Biotin carboxyl carrier protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from Brassica napus: cloning and analysis of expression during oilseed rape embryogenesis.

In the oilseed rape Brassica napus there are two forms of acetyl-CoA carboxylase (ACCase). As in other dicotyledonous plants there is a type I ACCase, the single polypeptide 220 kDa form, and a type II multi-subunit complex analogous to that of Escherichia coli and Anabaena. This paper describes the cloning and characterization of a plant biotin carboxyl carrier protein (BCCP) from the type II ACCase complex that shows 61% identity/79% similarity with Anabaena BCCP at the amino acid level. Six classes of nuclear encoded oilseed rape BCCP cDNA were clones, two of which contained the entire coding region. The BCCP sequences allowed the assignment of function to two previously unassigned Arabidopsis expressed sequence tag (EST) sequences. We also report the cloning of a second type II ACCase component from oilseed rape, the beta-carboxyltransferase subunit (betaCT), which is chloroplast-encoded. Northern analysis showed that although the relative levels of BCCP and betaCT mRNA differed between different oilseed rape tissues, their temporal patterns of expression were identical during embryo development. At the protein level, expression of BCCP during embryo development was studied by Western blotting, using affinity-purified anti-biotin polyclonal sera. With this technique a 35 kDa protein thought to be BCCP was shown to reside within the chloroplast. This analysis also permitted us to view the differential expression of several unidentified biotinylated proteins during embryogenesis.

Isolation of cDNAs from Brassica napus encoding the biotin-binding and transcarboxylase domains of acetyl-CoA carboxylase: assignment of the domain structure in a full-length Arabidopsis thaliana genomic clone.

One independent and two overlapping rape cDNA clones have been isolated from a rape embryo library. We have shown that they encode a 2.3 kb and a 2.5 kb stretch of the full-length acetyl-CoA carboxylase (ACCase) cDNA, corresponding to the biotin-binding and transcarboxylase domains respectively. Using the cDNA in Northern-blot analysis we have shown that the mRNA for ACCase has a higher level of expression in rape seed than in rape leaf and has a full length of 7.5 kb. The level of expression during rape embryogenesis was compared with both oil deposition and expression of two fatty acid synthetase components enoyl-(acyl-carrier-protein) reductase and 3-oxoacyl-(acyl-carrier-protein) reductase. Levels of ACCase mRNA were shown to peak at 29 days after anthesis during embryonic development, similarly to enoyl-(acyl-carrier-protein) reductase and 3-oxoacyl-(acyl-carrier-protein) reductase mRNA. In addition, a full-length genomic clone (19 kb) of Arabidopsis ACCase has been isolated and partially sequenced. Analysis of the clone has allowed the first plant ACCase activity domains (biotin carboxylase-biotin binding-transcarboxylase) to be ordered and assigned. Southern-blot analysis using the Arabidopsis clone indicates that ACCase is a single-copy gene in Arabidopsis but is encoded by a small gene family in rape.

Unique structural features and differential phosphorylation of the 280-kDa component (isozyme) of rat liver acetyl-CoA carboxylase.

Rat liver acetyl-CoA carboxylase (ACC, EC 6.4.1.2) exhibits major and minor subunits (M(r) of 265,000 and 280,000 respectively), the structure and function of which are compared in this study. The two subunits copurified and each contained biotin as demonstrated by avidin reactivity and direct determination of biocytin. In agreement with previous studies, the ACC subunits could be distinguished with specific monoclonal antibodies and differential tissue expression. We now report extensive differences in primary structure revealed by peptide mapping, mass spectrometric analysis of peptides following reverse phase high performance liquid chromatography, and microsequencing of selected peptides. Four peptides derived from the 265-kDa subunit were sequenced and matched sequences within the predicted structure of rat 265-kDa ACC. Although one identical peptide sequence was detected within both subunits (residues 2009-2024 of the 265-kDa subunit), 12 peptides derived from the 280-kDa subunit exhibited entirely novel sequences or matched partially (average 70% identity) with sequences within the 265-kDa subunit. The 280-kDa subunit may also exhibit distinct functional properties, since the initial rate of phosphorylation was at least 10-fold greater than that of the 265-kDa subunit in the presence of cAMP-dependent protein kinase. Two-dimensional mapping demonstrated that the tryptic phosphopeptides released from the two ACC subunits are distinct. These structural studies suggest that the 265- and 280-kDa components (isozymes) of ACC are so distinct they may be encoded by separate genes, while the differential phosphorylation observed in vitro suggests a key role for the 280-kDa subunit in regulating enzyme activity within intact cells.

Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis.

We report the use of microbore reverse-phase high performance liquid chromatography connected on-line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow-splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer. The method was used to analyze the 265-kDa and the 280-kDa isoforms of the enzyme acetyl-CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error-free data interpretation in chemical high-sensitivity peptide sequence analysis.