DNA methylation in sugarcane somaclonal variants assessed through methylation-sensitive amplified polymorphism.

Micropropagation is an important tool for large-scale multiplication of plant superior genotypes. However, somaclonal variation is one of the drawbacks of this process. Changes in DNA methylation have been widely reported as one of the main causes of somaclonal variations in plants. In order to investigate the occurrence of changes in the methylation pattern of sugarcane somaclonal variants, the MSAP (methylation-sensitive amplified polymorphism) technique was applied to micro-propagated plantlets sampled at the third subculture phase. The mother plant, in vitro normal plantlets, and in vitro abnormal plantlets (somaclonal variants) of four sugarcane clones were screened against 16 MSAP selective primers for EcoRI/MspI and EcoRI/HpaII restriction enzymes. A total of 1005 and 1200 MSAP-derived markers with polymorphism percentages of 28.36 and 40.67 were obtained for EcoRI/HpaII and EcoRI/MspI restriction enzyme combinations, respectively. The genetic similarity between the mother plant and the somaclonal variants ranged from 0.877 to 0.911 (EcoRI/MspI) and from 0.928 to 0.955 (EcoRI/HpaII). Most of the MASPs among mother plant and micro-propagated plantlets were derived from EcoRI/MspI restriction enzymes suggesting alteration due to gain or loss of internal cytosine methylation. A higher rate of loss of methylation (hypomethylation) than gain of methylation (hypermethylation) was observed in the abnormal in vitro sugarcane plantlets. Although changes in the methylation pattern were also observed in the in vitro normal plantlets, they were lower than those observed for the in vitro abnormal plantlets. The MASP technique proved to be a promising tool to early assessment of genetic fidelity of micro-propagated sugarcane plants.

Fastidian gum: the Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity.

The Gram-negative bacterium Xylella fastidiosa was the first plant pathogen to be completely sequenced. This species causes several economically important plant diseases, including citrus variegated chlorosis (CVC). Analysis of the genomic sequence of X. fastidiosa revealed a 12 kb DNA fragment containing an operon closely related to the gum operon of Xanthomonas campestris. The presence of all genes involved in the synthesis of sugar precursors, existence of exopolysaccharide (EPS) production regulators in the genome, and the absence of three of the X. campestris gum genes suggested that X. fastidiosa is able to synthesize an EPS different from that of xanthan gum. This novel EPS probably consists of polymerized tetrasaccharide repeating units assembled by the sequential addition of glucose-1-phosphate, glucose, mannose and glucuronic acid on a polyprenol phosphate carrier.

The mitochondrial genome of the primary screwworm fly Cochliomyia hominivorax (Diptera: Calliphoridae).

The complete sequence of the mitochondrial genome of the screwworm Cochliomyia hominivorax was determined. This genome is 16,022 bp in size and corresponds to a typical Brachycera mtDNA. A Serine start codon for COI and incomplete termination codons for COII, NADH 5 and NADH 4 genes were described. The nucleotide composition of C. hominivorax mtDNA is 77% AT-rich, reflected in the predominance of AT-rich codons in protein-coding genes. Non-optimal codon usage was commonly observed in C. hominivorax mitochondrial genes. Phylogenetic analysis distributed the Acalypterate species as a monophyletic group and assembled the C. hominivorax (Calyptratae) and the Acalyptratae in a typical Brachycera cluster. The identification of diagnostic restriction sites on the sequenced mitochondrial genome and the correlation with previous RFLP analysis are discussed.

Regulation of lysine catabolism in higher plants.

Lysine is an essential amino acid for mammals but its concentration in cereals, one of our main food sources, is low. Research over the past 40 years has unraveled many biochemical and molecular details of the aspartic acid pathway, which is the main route of lysine biosynthesis in plants. However, genetic manipulation of this pathway has not been successful at producing high-lysine seeds. This is because lysine, instead of being accumulated, is degraded via the saccharopine pathway. Recent work has increased our knowledge of this pathway, including both the enzymes involved and their regulation.

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Lysine degradation through the saccharopine pathway in mammals: involvement of both bifunctional and monofunctional lysine-degrading enzymes in mouse.

Lysine-oxoglutarate reductase and saccharopine dehydrogenase are enzymic activities that catalyse the first two steps of lysine degradation through the saccharopine pathway in upper eukaryotes. This paper describes the isolation and characterization of a cDNA clone encoding a bifunctional enzyme bearing domains corresponding to these two enzymic activities. We partly purified those activities from mouse liver and showed for the first time that both a bifunctional lysine-oxoglutarate reductase/saccharopine dehydrogenase and a monofunctional saccharopine dehydrogenase are likely to be present in this organ. Northern analyses indicate the existence of two mRNA species in liver and kidney. The longest molecule, 3.4 kb in size, corresponds to the isolated cDNA and encodes the bifunctional enzyme. The 2.4 kb short transcript probably codes for the monofunctional dehydrogenase. Sequence analyses show that the bifunctional enzyme is likely to be a mitochondrial protein. Furthermore, enzymic and expression analyses suggest that lysine-oxoglutarate reductase/saccharopine dehydrogenase levels increase in livers of mice under starvation. Lysine-injected mice also show an increase in lysine-oxoglutarate reductase and saccharopine dehydrogenase levels.

The role of opaque2 in the control of lysine-degrading activities in developing maize endosperm.

We have isolated a cDNA clone, designated ZLKRSDH, encoding the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) from maize. The predicted polypeptide has an N-terminal LKR domain and a C-terminal SDH domain that are similar to the yeast LYS1 and LYS9 monofunctional proteins, respectively. The maize LKR/SDH protein is located in the cytoplasm of subaleurone endosperm cell layers. Transcripts and polypeptides as well as enzyme activities showed an upregulation and downregulation during endosperm development. The developmental expression of ZLKRSDH was examined in normal and opaque2 seeds. In the mutant endosperm, mRNA levels were reduced by >90%, with concomitant reductions in polypeptide levels and LKR/SDH activity. These results suggest that lysine levels in the endosperm are likely to be controlled at the transcriptional level by the Opaque2 transcription factor.

Structure and regulation of the bifunctional enzyme lysine-oxoglutarate reductase-saccharopine dehydrogenase in maize.

The lysine-oxoglutarate reductase (LOR) domain of the bifunctional enzyme lysine-oxoglutarate reductase-saccharopine dehydrogenase (LOR/SDH) from maize endosperm was shown to be activated by Ca2+, high salt concentration, organic solvents and Mg2+. The Ca2+-dependent enhancement of LOR activity was inhibited by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and calmidazolium. Limited proteolysis was used to assess the structure/function relationship of the enzyme. Digestion with elastase separated the bifunctional 125-kDa polypeptide into two polypeptides of 65 kDa and 57 kDa, containing the functional domains of LOR and SDH, respectively. Proteolysis did not affect SDH activity, while LOR showed a time-dependent and protease-concentration-dependent inactivation followed by reactivation. Prolonged digestion or increasing amounts of elastase produced a complex pattern of limit polypeptides derived from additional cleavage sites within the 65-kDa (LOR) and 57-kDa (SDH) domains. The SDH-containing polypeptides inhibited the enzymatic activity of LOR-containing polypeptides. When separated from the SDH domain by limited proteolysis and ion-exchange chromatography, the LOR domain retained its Ca2+ activation property, but was no longer activated by high salt concentrations. These results suggest that the LOR activity of the native enzyme is normally inhibited such that after modulation, the enzyme undergoes a conformational alteration to expose the catalytic domain for substrate binding.