Mitochondrial DNA evolution in lagomorphs: Origin of systematic heteroplasmy and organization of diversity in European rabbits

Summary A characterization was conducted on mitochondrial DNA (mtDNA) molecules extracted separately from 107 European rabbits (Oryctolagus cuniculus) both wild and domestic, 13 European hares (Lepus capensis), and 1 eastern cottontail (Sylvilagus floridanus). Experimentally this study took into account restriction site polymorphism, overall length variation of the noncoding region, and numbers of repeated sequences. Nucleotide divergences indicate that the mtDNAs from the three species derived from a common ancestor some 6–8 million years (Myr) ago. Every animal appeared heteroplasmic for a set of molecules with various lengths of the noncoding region and variable numbers of repeated sequences that contribute to them. This systematic heteroplasmy, most probably generated by a rate of localized mtDNA rearrangements high enough to counterbalance the cellular segregation of rearranged molecules, is a shared derived character of leporids.The geographic distribution of mtDNA polymorphism among wild rabbit populations over the western European basin shows that two molecular lineages are represented, one in southern Spain, the second over northern Spain, France, and Tunisia. These two lineages derived from a common ancestor some 2 Myr ago. Their present geographical distribution may be correlated to the separation of rabbits into two stocks at the time of Mindel glaciation.Finally the distribution of mtDNA diversity exhibits a mosaic pattern both at inter- and intrapopulation levels.     

Sequences homologous to Tc(s) transposable elements ofCaenorhabditis elegans are widely distributed in the phylum nematoda

Summary To have a better understanding of the evolutionary history of mobile elements within the nematodes, we examined the distribution and the conservation of homologues to transposable elements fromCaenorhabditis elegans (Tc1, Tc2, Tc3, Tc4, Tc5, and FB1) in 19 nematode species belonging to the class Secernentea. Our results show that Tc1 elements display a distribution restricted to the family Rhabditidae with poor conservation. The Tc2 and FB1 homologous elements have the same patchy distribution within the Rhabditidae. They were only found inCaenorhabditis and inTeratorhabditis. The Tc3 element is widely distributed among nematode species. Tc3 homologous elements are present in the majority of the Rhabditidae but also in two genera within the family Panagrolaimidae, and inBursaphelenchus, which belongs to the order Aphelenchida. Tc4 and Tc5 homologues show the most limited distribution of all tested elements, being strictly limited toC. elegans. These data indicate that in some cases, the distribution of transposable elements in the nematode cannot be explained by strict vertical transmission. The distribution of Tc3, Tc4, and Tc5 suggests that horizontal transmission may have occurred between reproductively isolated species during their evolutionary history.     

Proteolytic degradation of heterologous proteins expressed inAspergillus niger

Summary Proteolytic degradation of heterologous proteins expressed in the filamentous fungusAspergillus niger reduces the yield of authentic target protein. The activities ofA. niger proteases are differentiated by their effects on two proteins expressed and secreted fromA. niger: hen egg-white lysozyme and porcine pancreatic phospholipase A₂.     

Agrobacterium rhizogenes mediated induction of apparently untransformed roots and callus in chrysanthemum

The agropine type Agrobacterium rhizogenes strain LBA9402 induced callus and roots on stems of greenhouse grown plants and on leaf disks of in vitro grown plantlets of chrysanthemum (Dendranthema grandiflora Tzvel.). In this callus and roots no opines were detected, nor were any of the other features of the hairy root syndrome observed. Experiments aimed to identify the nature of the tumour-like growth revealed that induction was correlated with the presence of the TR-DNA on the Ri-plasmid. Root induction was probably the result of auxin synthesis following transient expression of iaaM and iaaH genes, present on the TR-DNA. The chrysanthemum cultivar used, cv. Parliament, showed a high auxin sensitivity compared to tobacco. Analysis of early transformation events using the GUSintron reporter gene revealed that low efficiency gene transfer and transient gene expression took place, but most probably without stable integration of the T-DNA in the plant genome. The results presented here stress the fact that callus formation or root induction as measures for transformation efficiency should be used with caution.     

Comparison of amide proton exchange in reduced and oxidizedRhodobacter capsulatus cytochrome c2: A1H−15N NMR study

Summary The hydrogen-deuterium exchange rates of the reduced and oxidized forms ofRhodobacter' capsulatus cytochrome c₂ were studied by¹H–¹⁵N homonuclear multiple quantum correlation spectroscopy. Minimal differences were observed for the N- and C-terminal helices on changing redox state suggesting that although these helices are structurally important they do not affect the relative stability of the two redox states and hence may not be important in determining the redox potential differences observed amongst the class I c-type cytochromes. However, significant differences were observed for other regions of the protein. For example, all slow exchanging protons of the helix spanning Phe⁸² to Asp⁸⁷ are similarly affected on reduction indicating that the unfolding equilibrium of this helix is altered between the two redox states. Other regions are not as simple to interpret; however, the difference in NH exchange rates between the redox states for a number of residues including His¹⁷, Leu³⁷, Arg⁴³, Ala⁴⁵, Gly⁴⁶, Ile⁵⁷, Val⁵⁸, Leu⁶⁰, Gly⁶¹ and Leu¹⁰⁰ suggest that interactions affecting the causes of these differences may be important factors in determining redox potential.Abbreviations NMR nuclear magnetic resonance - HMQC homonuclear multiple quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

Pro----Ala-35 Rhodobacter capsulatus cytochrome c2 shows dynamic not structural differences. A 1H and 15N NMR study

Comparative analysis of nuclear Overhauser effects show that the time average conformation of the wild-type and mutant Pro----Ala-35 Rhodobacter capsulatus cytochrome c2 are indistinguishable. The ring resonances of Phe-51 and Tyr-53 show that their ring flip rates increase in P35A. NH proton exchange studies show that the exchange rates of the NH of Gly-34 and the NpiH of His-17 increase by approximately 10(2) in P35A suggesting that their respective hydrogen bonds are destabilized in this protein. However, 3JchiNH 1H and 15N chemical shift data argue that these bonds are intact. These data are compatible if the replacement of a Pro with an Ala residue forms a cavity or increases local flexibility thus reducing steric hinderance and increasing solvent accessibility.     

The human ryanodine receptor gene: Its mapping to 19q13.1, placement in a chromosome 19 linkage group, and exclusion as the gene causing myotonic dystrophy

The recent cloning of cDNA encoding the Ca++ release channel (ryanodine receptor) of human sarcoplasmic reticulum has enabled us to use somatic cell hybrids to localize the ryanodine receptor gene (RYR) to the proximal long arm of human chromosome 19. Studies with additional hybrids containing deletions or translocations in chromosome 19 enabled us to localize RYR to 19q13.1 in a region distal to GPI/MAG and proximal to D19S18/DNF11. On the basis that the myotonic dystrophy (DM) locus maps near this region and that myotonia could result from a defect in the ryanodine receptor, we examined the linkage between the DM locus and RYR. Our results, showing several DM-RYR recombinants, rule out an RYR defect as the cause of DM. However, localization of RYR to a region of human chromosome 19 which is syntenic to an area of pig chromosome 6 containing the HAL gene responsible for porcine malignant hyperthermia supports the candidacy of RYR for this disorder.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85.

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.     

Increased sensitivity of the rapid hydrophobic grid membrane filter enzyme-labeled antibody procedure for Escherichia coli O157 detection in foods and bovine feces.

Several strains of Escherichia coli O157:H7 artificially inoculated into vegetables and dairy products were recovered on hydrophobic grid membrane filters and enumerated by an enzyme-labeled antibody assay. The mean of the recoveries from 12 fresh vegetables was 108.8%, whereas that from 10 dairy products was 93.2%. Modified tryptic soy broth at 43 degrees C with shaking at 100 rpm provided optimum recovery of the organism from meat, with a sensitivity of less than or equal to 1 CFU/g, which is 10 times more sensitive than direct plating. The method performed equally well with vegetable and dairy products. Tryptic soy broth, however, under the same conditions gave the best results for fecal samples. Of 22 asymptomatic dairy cattle, reported as having positive Brucella titers when assayed with polyclonal antibodies, eight were found to contain E. coli O157 in their feces as demonstrated by the enzyme-labeled antibody assay by using monoclonal antibodies. This finding may explain some of the false-positive Brucella tests.