Genetic and physical mapping of the patatin genes in potato and tomato

Summary Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10–15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5 promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5 promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.     

Structural aspects and orientation mechanism of mitochondrial F1 adenosinetriphosphatase. Evidence for a negative electric birefringence due to a permanent moment perpendicular to the long axes of the particle

The electric birefringence technique was used to investigate the steady-state birefringence, the orientational relaxation time, and the orientation mechanism of pig heart mitochondrial F1 adenosine-5'-triphosphatase (F1-ATPase). The electrooptical properties of this enzyme in solution were studied as functions of pH, protein concentration, and applied electric field. The F1-ATPase exhibits a surprising negative electric birefringence with a specific Kerr constant of -1.5 X 10(-3) esu cgs. The field-independent relaxation time was found to be 0.65 +/- 0.05 microseconds, corresponding to a rotational diffusion constant of 2.55 X 10(5) s-1. The overall size and shape of F1-ATPase have been calculated from both translational and rotational diffusion constants. The enzyme may be assumed to be an oblate ellipsoid of revolution with dimensions of about 170 X 170 X 70 A. The orientation mechanism of F1-ATPase was analyzed by fitting experimental birefringence rising curves with theoretical rising functions. The ratio of the permanent to induced dipole moment is found to be very high; therefore, the birefringence of F1-ATPase is due to a strong permanent dipole moment in a direction perpendicular to the long axes of the particle. These particular electric properties can be explained by the oligomeric structure of the protein and seem likely to play a role in its mechanism of functioning.     

A variant form of the 1,25-dihydroxyvitamin D3 receptor with low apparent hormone affinity in cultured monkey kidney cells (LLC-MK2). A model for tissue resistance to vitamin D

Chandler et al. (Chandler, J.S., Chandler, S.K., Pike, J.W., and Haussler, M.R. (1984) J. Biol. Chem. 259, 2214-2222) previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) caused the induction of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) in a rhesus monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity 1,25-(OH)2D3 receptor. We have re-examined this phenomenon and report here that 24-hydroxylase induction is mediated by a receptor variant in LLC-MK2 cells with low hormone affinity. Dose response analysis showed that in contrast to LLC-PK1 (a typical receptor-positive cell line), the LLC-MK2 line was less sensitive to 1,25-(OH)2D3 by 2 orders of magnitude. Employing optimal concentrations of 1,25-(OH)2D3 for 24-hydroxylase induction in each cell type, the early time courses of this bioresponse were identical in LLC-MK2 and LLC-PK1 and were consistent with a nuclear action of hormone-receptor complexes. Moreover, the rank order of potency of vitamin D3 congeners as inducers of 24-hydroxylase activity in LLC-MK2 cells agreed well with their relative affinity for the 1,25-(OH)2D3 receptor. An examination of 1,25-(OH)2D3 receptor content via DNA-cellulose chromatography in LLC-MK2 cells incubated at ligand concentrations 10-25-fold higher than the normal 2 nM revealed a minimum of 1600 receptor-like molecules/LLC-MK2 cell. These results show that LLC-MK2 cells possess a variant receptor form with apparent low affinity for 1,25-(OH)2D3. This system should serve as a model for clinical syndromes characterized by the requirement for massive doses of vitamin D to prevent rickets.     

Characterization of a Marine Bacterium Associated with Crassostrea virginica (the Eastern Oyster)

A gram-negative bacterium found to be closely associated with oysters has been isolated and characterized. The organism, designated LST, has a generation time of 106 min in Marine broth under optimal growth conditions at 25°C. During the decline phase of growth, it exhibits a morphological transition from a motile rod (ca. 1 μm in length) to an elongated, 3- to 40-μm, nonmotile, tightly coiled helix. LST synthesizes and releases a pigment in the stationary and decline phases of growth. Identified as melanin on the basis of chemical properties and UV absorbance maxima, the pigment comprises polymers of heterogeneous molecular weights, ranging from 12,000 to 120,000. The guanosine-plus-cytosine content of the LST DNA is 46%, and results of phenetic analysis and DNA-DNA hybridization indicate that this bacterium represents a new species. LST adheres to a variety of surfaces, including glass, plastics, and oyster shell, and has been shown to promote the settlement of oyster larvae.     

The effect of histone H1 on the compaction of oligonucleosomes. A quasielastic light scattering study

The structural properties of H1-depleted oligonucleosomes are investigated by the use of quasielastic laser light scattering, thermal denaturation and circular dichroism and compared to those of H1-containing oligomers. To obtain information on the role of histone H1 in compaction of nucleosomes, translational diffusion coefficients (D) are determined for mono-to octanucleosomes over a range of ionic strength. The linear dependences of D on the number of nucleosomes show that the conformation of stripped oligomers is very extended and does not change drastically with increasing the ionic strength while the rigidness of the chain decreases due to the folding of linker DNA. The results prove that the salt-induced condensation is much smaller for H1-depleted than for H1-containing oligomers and that histone H1 is necessary for the formation of a supercoiled structure of oligonucleosomes, already present at low ionic strength.     

Absence of genotoxic effects of nonasbestos mineral fibers

The biological activity of natural and synthetic mineral fibers has been examined. Natural attapulgite [(Mg, Al)₂Si₄O₁₀(OH).4H₂0], synthetic xonotlite [Ca₃Si₃O₈(OH)₂] and natural sepiolite [Mg₂Si₃O₈.2H₂O] were selected. Genotoxic effects were investigated by means of a well established cellular model based upon the measurement of unscheduled DNA synthesis (UDS) in rat hepatocytes in primary culture. The intrinsic capacity of the fibers (1 and 10 µ/ml) to induce UDS was first tested. None of the fiber types showed detectable UDS-eliciting activity. Also, the possible modulation of the cellular response to genotoxic agents by the materials was examined by exposing the cells to mixtures of 2-acetylaminofluorene (AAF) (0.05 and 0.25 µg/ml) and fibers (1 and 10 µg/ml). In these experiments, the UDS response was significantly diminished in the presence of xonotlite. This phenomenon may reflect changes in the uptake and/or metabolism of AAF or may result from an inhibition of DNA repair processes, the latter suggesting a possible cocarcinogenic potential for this synthetic silicate. These results point to the immediate necessity of studying more extensively the biological effects of fibrous materials that can be used as substitutes for asbestos.Abbreviations AAF 2-acetylaminofluorene - DMSO dimethyl-sulfoxide - FBS fetal bovine serum - IRDA Institut de Recherche et de Développement sur l'Amiante - LDH lactate dehydrogenase - UDS unscheduled - DNA synthesis - WME Williams' Medium E This work was supported by the Institut de Recherche et de Développement sur l'Amiante (IRDA), Sherbrooke, Canada.     

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora

Summary The organization of the ribosomal DNA (rDNA) repcat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, N. tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11+0.21 kb; standard error=0.038; coefficient of variation (C.V.)=2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5 end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.