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.

A consensus sequence for long-chain fatty-acid alcohol oxidases from Candida identifies a family of genes involved in lipid omega-oxidation in yeast with homologues in plants and bacteria.

The yeast Candida cloacae is capable of growing on alkanes and fatty acids as sole carbon sources. Transfer of cultures from a glucose medium to one containing oleic acid induced seven proteins of M(r) 102,000, 73,000, 61,000, 54,000, and 46,000 and two in the region of M(r) 45,000 and repressed a protein of M(r) 64,000. The induction of the M(r) 73,000 protein reached a 7-fold maximum 24 h after induction. The protein was confirmed by its enzyme activity to be a long-chain fatty-acid alcohol oxidase (LC-FAO) and purified to homogeneity from microsomes by a rapid procedure involving hydrophobic chromatography. An internal peptide of 30 amino acids was sequenced. A 1100-base pair cDNA fragment containing the LC-FAO peptide coding sequence was used to isolate a single exon genomic clone containing the full-length coding sequence of an LC-FAO (fao1). The fao1 gene product was expressed in Escherichia coli and was translated as a functional long-chain alcohol oxidase, which was present in the membrane fraction. In addition, full-length coding sequences for a Candida tropicalis LC-FAO (faoT) and a second C. cloacae LC-FAO (fao2) were isolated. The DNA sequences obtained had open reading frames of 2094 (fao1), 2091 (fao2), and 2112 (faoT) base pairs. The derived amino acid sequences of fao2 and faoT showed 89.4 and 76.2% similarities to fao1. The fao1 gene is much more highly induced on alkane than is fao2. Although this study describes the first known DNA sequences encoding LC-FAOs from any source, there are unassigned Arabidopsis sequences and an unassigned Mycobacterium sequence in the GenBank(TM) Data Bank that show strong homology to the described LC-FAO sequences. The conservation of sequence between yeast, plants, and bacteria suggests that an as yet undescribed family of long-chain fatty-acid oxidases exists in both eukaryotes and prokaryotes.

Re-evaluation of the primary structure of Ralstonia eutropha phasin and implications for polyhydroxyalkanoic acid granule binding.

Sequence analysis of several cDNAs encoding the phasin protein of Ralstonia eutropha indicated that the carboxyl terminus of the resulting derived protein sequence is different from that reported previously. This was confirmed by: (1) sequencing of the genomic DNA; (2) SDS-PAGE and peptide analysis of wild-type and recombinant phasin; and (3) mass spectrometry of wild-type phasin protein. The results have implications for the model proposed for the binding of this protein to polyhydroxyalkanoic acid granules in the bacterium.

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.

Studies on wheat acetyl CoA carboxylase and the cloning of a partial cDNA.

Wheat germ acetyl CoA carboxylase (ACCase) was purified by liquid chromatography and electroelution. During purification bovine serum albumin (BSA) was used to coat Amicon membranes used to concentrate partially pure ACCase. Despite further SDS-PAGE/electroelution and microbore HPLC steps BSA remained associated. This presented serious protein sequencing artefacts which may reflect the affinity of BSA for fatty acids bound to ACCase. To avoid these artefacts the enzyme was digested in gel with Endoproteinase LysC protease without the presence of BSA, and the resulting peptides blotted and sequenced. A partial cDNA (1.85 kb) encoding ACCase from a wheat embryo library was cloned, which hybridised to a 7.5 kb RNA species on northern blot of wheat leaf poly(A)+ RNA. The partial cDNA therefore represents about 0.25 of the full-length cDNA. The clone was authenticated by ACCase peptide sequencing and immuno cross-reactivity of the overexpressed clone. The derived amino acid sequence showed homology with both rat and yeast ACCase sequences (62%). Antibodies raised against wheat acetyl CoA carboxylase were specific for a 220 kDa protein from both wheat embryo and leaf. In addition, by using a novel quick assay for ACCase that utilised 125I-streptavidin, we showed the major biotin containing protein to be 220 kDa in both leaf and germ. This is in marked contrast to the previously published molecular mass of 75 kDa allocated to wheat leaf ACCase.

Resolution of synthetic Holliday junctions in DNA by an endonuclease activity from calf thymus.

Extracts of calf thymus have been fractionated to reveal a nuclease activity that specifically cleaves model Holliday junctions in vitro. The products of cleavage are unbranched linear duplex DNA molecules. Using synthetic four-way junctions, we show that the major sites of cutting are diametrically opposed, at sites one nucleotide from the base of the junction. Other types of four-way junctions, including pseudo-cruciform structures and cruciforms extruded from supercoiled plasmids, are also cleaved by the nuclease. The Mr of the partially purified activity, determined by gel filtration, is approximately 75,000. The calf thymus enzyme provides the first example of an endonuclease from a higher eukaryote that acts specifically on branch points in DNA, and indicates that junction-resolving proteins are normal constituents of somatic cells.

Specific binding of cruciform DNA structures by a protein from human extracts.

A gel electrophoresis binding assay has been used to probe extracts from cultured human lymphoblasts for proteins that bind cruciform structures in duplex DNA. Proteins have been detected that form complexes with synthetic X- and Y-junctions. Several lines of evidence suggest that binding is specific for DNA structure rather than sequence: (1) X- and Y-structures were bound whereas linear duplexes containing identical DNA sequences were not, (2) Binding occurred with equal efficiency to two X-junctions that were constructed from DNA strands of different sequence, (3) One X-junction successfully competed with another for binding whereas linear duplex DNA did not; and (4) protein-DNA complexes were observed at probe:non-specific competitor DNA ratios of 1:10,000.