Chloroplast DNA Sequence Homologies among Vascular Plants

The extent of sequence conservation in the chloroplast genome of higher plants has been investigated. Supercoiled chloroplast DNA, prepared from pea seedlings, was labeled in vitro and used as a probe in reassociation experiments with a high concentration of total DNAs extracted from several angiosperms, gymnosperms, and lower vascular plants. In each case the probe reassociation was accelerated, demonstrating that some chloroplast sequences have been highly conserved throughout the evolution of vascular plants. Only among the flowering plants were distinct levels of cross-reaction with the pea chloroplast probe evident; broad bean and barley exhibited the highest and lowest levels, respectively. With the hydroxylapatite assay these levels decreased with a decrease in probe fragment length (from 1,860 to 735 bases), indicating that many conserved sequences in the chloroplast genome are separated by divergent sequences on a rather fine scale. Despite differences observed in levels of homology with the hydroxylapatite assay, S1 nuclease analysis of heteroduplexes showed that outside of the pea family the extent of sequence relatedness between the probe and various heterologous DNAs is approximately the same: 30%. In our interpretation, the fundamental changes in the chloroplast genome during angiosperm evolution involved the rearrangement of this 30% with respect to the more rapidly changing sequences of the genome. These rearrangements may have been more extensive in dicotyledons than in monocotyledons. We have estimated the amount of conserved and divergent DNA interspersed between one another.     

Purification and properties of 16 alpha-hydroxyprogesterone dehydroxylase from Eubacterium sp. strain 144

Eubacterium sp. strain 144 converts 16 alpha-hydroxyprogesterone to 17-isoprogesterone. The first step of this reaction is catalyzed by 16 alpha-hydroxyprogesterone dehydroxylase (16 alpha-dehydroxylase). This enzyme was purified 40-70-fold and characterized. 16 alpha-Dehydroxylase was found to be active in two molecular weight forms of Mr 181 000 and 326 000. A subunit relative molecular weight of 42 400 was determined by sodium dodecyl sulfate gel electrophoresis of the purified enzyme. Although active with both 16 alpha-hydroxyprogesterone and 16 alpha-hydroxypregnenolone, the affinity of 16 alpha-dehydroxylase for the latter steroid was twice that of the former based on the apparent Km values. Evidence of possible substrate inhibition at high concentrations was seen with 16 alpha-hydroxypregnenolone. 16-Ketoprogesterone was found to be a competitive inhibitor of 16 alpha-dehydroxylase with respect to both steroid substrates. Although generally unaffected by low concentrations of non-ionic detergents, 16 alpha-dehydroxylase activity was stimulated 3-7-fold by sodium dodecyl sulfate and inhibited strongly by cetyltrimethylammonium bromide.     

Immobilization of Burkholderia cepacia in polyurethane-based foams: embedding efficiency and effect on bacterial activity

Immobilization of the trichloroethylene-degrading bacterium Burkholderia cepacia was evaluated using hydrophilic polyurethane foam. The influence of several foam formulation parameters upon cell retention was examined. Surfactant type was a major determinant of retention; a lecithin-based compound retained more cells than pluronic- or silicone-based surfactants. Excessive amounts of surfactant led to increased washout of bacteria. Increasing the biomass concentration in the foam from 4.8 to 10.5% dry weight per wet weight of foam resulted in fewer cells being washed out. Embedding at reduced temperature did not significantly affect retention, while the use of a silane binding agent gave inconsistent results. The optimal formulation retained all but 0.2% of total embedded cells during passage of 2 L of water through columns containing 2 g of foam. All foam formulations tested reduced the culturability of embedded cells by several orders of magnitude, but O₂ consumption and CO₂ evolution rates of embedded cells were never less than 50% of those of free cells. Nutrient amendments stimulated an increase in cell volume and ribosomal activity in immobilized cells as indicated by hybridization studies using fluorescently labeled ribosomal probes. These results indicate that, although immobilized cells were mostly nonculturable, they were metabolically active and thus could be used for biodegradation of toxic compounds. Received 23 December 1996/ Accepted in revised form 13 March 1997     

Restriction-map variation associated with the G6PD polymorphism in natural populations of Drosophila melanogaster

Restriction-map variation was studied in 126 copies of the G6pd region in X chromosome lines of Drosophila melanogaster from North America, Europe, and Africa. Special attention was focused on the distribution of variation relative to the geographically variable polymorphism for two electrophoretic variants. Nucleotide heterozygosity as determined by eight six-cutter restriction enzymes for the 13-kb region is estimated, on the basis of the worldwide sample, to be 0.065%, which is the lowest value reported for any comparable region in the D. melanogaster genome. Significant linkage disequilibrium between electrophoretic alleles and restriction-site variation is observed for several sites. In contrast to published studies of other genetic regions, there are large insertions that reach significant frequencies and are found across considerable geographic distances. There is a clustering of this variation inside the first large intervening sequence of the G6PD gene.      

Fine scale interspersion of conserved sequences in the pea and corn chloroplast genomes

Summary A comparison is made of the chloroplast genomes of two divergent higher plants, pea and corn. Reassociation kinetics analysis shows that only 33–34% of the chloroplast DNA (ct DNA) sequences are conserved in these two plants, which is equal to about 43 kilobases (kb). The restriction enzyme patterns produced by Eco RI, Bam HI, and Sal I are different for each ct DNA, as expected from the low level of homology. The total length of cross-reacting Eco RI fragments, assessed by blot hybridization methods, exceeds the reassociation kinetics estimate by at least 20 kb. An electron microscopic analysis of ct DNA heteroduplexes shows that the conserved regions are surprisingly short, and consequently, they are interspersed with divergent DNA. Fifty percent of the conserved regions are less than 550 bases; 10 sites are less than 150 bases. The median length of a heterologous region is 250 bases. The heteroduplexes fall into 4 classes, established by the position and size of the conserved and divergent regions, totaling 61 kb. One class has been identified as the ribosomal gene region: the corn Eco RI fragment, Eco RI A, which codes for the 16S and 23S cistrons (Bedbrook and Bogorad 1976), was reassociated with total pea ct DNA, and the products analyzed by electron microscopy. Only one pattern of heteroduplexes was observed. A stretch of almost completely conserved DNA, equivalent to 6.9 kb, extends from the 16S gene through the 23S gene, and therefore, includes the transcribed spacer separating these two cistrons. Heterologous regions occur immediately outside of the 5 and 3 ends of the 16S and 23S genes, respectively. A set of the 16S and 23S genes contribute about 4%, and the spacer 1.6%, to the level of sequence homology in each genome.G.K.L. was supported by a National Research Service Award from the National Institutes of Health (GM 07270). This work was also supported by National Institutes of Health Grant GM 22870, and a Grant-in-Aid of Research to G.K.L. from Sigma Xi, The Scientific Research Society     

Mutations at the transit peptide-mature protein junction separate two cleavage events during chloroplast import of the chlorophyll a/b-binding protein

We have shown previously that during in vitro import into chloroplasts, the precursor of the major light-harvesting chlorphyll a/b-binding protein (LHCP) generated from a wheat gene gives rise to two mature forms (25 and approximately 26 kDa) which are inserted into the thylakoids. However, during incubation of the LHCP precursor with a chloroplast-soluble extract in an organelle-free processing reaction, the NH2 terminus is cleaved, yielding only a 25-kDa peptide. In the present study, mutations at the transit peptide-mature protein junction were introduced in the LHCP precursor to investigate the relationship between the two peptides and the determinants of proteolytic processing. Mutant p delta 3 lacks 3 amino acids including Met34 at the primary cleavage site thought to give rise to the 26-kDa peptide. It is still processed during import and in the organelle-free reaction yielding in both assays only a 25-kDa peptide. Mutant p + 4 has 4 amino acids inserted immediately after Met34 and a proline that disrupts the alpha-helix predicted by the Garnier-Osguthorpe-Robson method (Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97-120) to extend through this region. Although p + 4 is imported, it is inefficiently processed; both a 25- and 26-kDa peptide are found, but at least 60% of the imported precursor remains uncleaved. Less than 5% is processed in the organelle-free assay. Replacement of the predicted alpha-helix in the mutant p + 4 alpha restores processing upon import into the chloroplast, but this mutant, which also has a 4-amino acid insert, yields only a 26-kDa peptide. p + 4 alpha is not processed in the organelle-free reaction. These results provide evidence that the two forms of LHCP obtained during import are the result of independent processing at two cleavage sites: the first site at Met34, and a second approximately 10 amino acids downstream within what has been designated the NH2 terminus of the mature protein. Whereas p delta 3 has the first site removed but retains a functional second site, in p + 4 alpha only the first site, or one very near it, is accessible to the processing enzyme during import. The conditions of the organelle-free reaction are specific for processing at only the secondary site. We discuss the implications of these findings in terms of the heterogeneity of LHCP in vivo.     

Molecular evolution of the MAGUK family in metazoan genomes

Background  

Development, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla.     

A forward genetic screen to explore chloroplast protein import in vivo identifies Moco sulfurase,pivotal for ABA and IAA biosynthesis and purine turnover

A genetic screen in Arabidopsis was developed to explore the regulation of chloroplast protein import in vivo using two independent reporters representing housekeeping and photosynthetic pre‐proteins. We first used 5‐enolpyruvylshikimate 3‐phosphate synthase (EPSP synthase*), a key enzyme in the shikimic acid pathway, with a mutation that confers tolerance to the herbicide glyphosate. Because the EPSP synthase* pre‐protein must be imported for its function, the loss of glyphosate tolerance provided an initial indication of an import deficiency. Second, the fate of GFP fused to a ferredoxin transit peptide (FD5–GFP) was determined. A class of altered chloroplast import (aci) mutants showed both glyphosate sensitivity and FD5–GFP mislocalized to nuclei. aci2‐1 was selected for further study. Yellow fluorescent protein (YFP) fused to the transit peptide of EPSP synthase* or the small subunit of Rubisco was not imported into chloroplasts, but also localized to nuclei during protoplast transient expression. Isolated aci2‐1 chloroplasts showed a 50% reduction in pre‐protein import efficiency in an in vitro assay. Mutants did not grow photoautotrophically on media without sucrose and were small and dark green in soil. aci2‐1 and two alleles code for Moco‐sulfurase, which activates the aldehyde oxidases required for the biosynthesis of the plant hormones abscisic acid (ABA) and indole‐acetic acid (IAA) and controls purine nucleotide (ATP and GTP) turnover and nitrogen recycling via xanthine dehydrogenase. These enzyme activities were not detected in aci2‐1. ABA, IAA and/or purine turnover may play previously unrecognized roles in the regulation of chloroplast protein import in response to developmental, metabolic and environmental cues.