RFLP tagging of a salt tolerance gene in rice

A salt tolerant rice mutant (M-20) was obtained through selection in vitro. Its tolerance was stably inherited over eight generations and most traints between M-20 and its sensitive original 77–170 (Oryza sativa) were very similar. By deriving an F₂ population of M-20 × 77–170 and splitting every F₂ individual into two parts, with one part planted in normal conditions and another part in saline conditions, the inheritance of salt tolerance in rice was studied. Under normal conditions, there was no apparent segregation among F₂ individuals. Under saline conditions, however, the segregation of traits was obvious. According to our standards, the ratio of salt sensitive:moderately-tolerant:tolerant plants was 25:42:18, in accordance with a 1:2:1 ratio. It suggested that the improvement of salt tolerance in our materials was induced by the mutation of a major tolerant gene which showed incomplete dominance. By use of 130 RFLP probes distributed throughout the rice genome, the gene was tagged by a single copy DNA probe, RG4, which was located on chromosome 7. The genetic distance between the salt tolerant gene and RG4 was 7.0 ± 2.9 cM. Based on the split method, a method which could be currently used to evaluate the damage of salt stress in rice was proposed.     

ATP,pH and Mg2+ modulate a cation current in Beta vulgaris vacuoles: A possible shunt conductance for the vacuolar H+-ATPase

Macroscopic instantaneous and time-dependent currents have been measured in the vacuolar membrane of Beta vulgaris using a patch clamp configuration analogous to whole cell mode. At low cytosolic Ca²⁺ and in the absence of Mg²⁺, only an instantaneous current was observed. This current is carried predominantly by cations (P_KP_Cl 71, p_nap_cl 41 and arginine is also conducted). The instantaneous current can be activated by ATP^4– (e.g., ATP-activated mean K⁺ current density was –20 mA.m^–2 at a membrane voltage of –20 mV) and by increasing cytosolic pH and Mg²⁺ (raising Mg²⁺ from 0 to 0.4 mm induced a mean current density increase of –7 mA.m^–2 at –20 mV). Such current can be activated by simultaneous addition of putative in vivo concentrations of ATP^4–/MgATP/Mg _free ²⁺ (in the presence of bafilomycin to inhibit the vacuolar ATPase) and further modulated by cytosolic pH. With vacuolar K⁺ concentration greater than that of the cytosol, activation of the instantaneous current would mediate vacuolar K⁺ release over the range of physiological membrane voltage. It is argued that the ATP^4–-activated current, in addition to acting as a K⁺ mobilization pathway, could provide a counter-ion (shunt) conductance, allowing the two electrogenic H⁺ pumps which reside in the vacuolar membrane to acidify the vacuolar lumen.A separate time-dependent current, which was not observed at low Ca²⁺ concentrations (less than 500 nm) could also be elicited by addition of Mg²⁺ at the cytoplasmic membrane face. This current was stimulated by increasing cytoplasmic pH.The authors are grateful to the BBSRC for financial support (Grant PG87/529) and to the Royal Society (University Research Fellowship to J.M.D.). We thank C. Abbott, K. Partridge and J. Robinson for plant cultivation; A. Amtmann, A. Bertl, D. Gradmann and G. Thiel for helpful discussion.     

A BC200-derived element and Z-DNA as structural markers in annexin I genes: Relevance to Alu evolution and annexin tetrad formation

We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.     

Molecular cloning and complete nucleotide sequence of the repeated unit and flanking gene of the scallop Pecten maximus mitochondrial DNA: Putative replication origin features

In the bivalve mollusc Pecten maximus, the size of the mitochondrial DNA molecules ranges from 20 to 25.8 kbp. This variability is mainly correlated with the occurrence of a variable domain composed with two to five 1.6-kbp repeated units tandemly arrayed in the genome. DNA fragments spanning the 1,586-base-pair-long repeated element and the nearest flanking gene have been cloned and sequenced. This sequence was analyzed regarding its base composition and potential secondary structures. The repeated unit domain was positioned and oriented with regard to the known flanking gene. It ends 2 base pairs upstream relative to the beginning of the tRNA^gly gene. The peculiar properties of the repeated unit were compared with those of the 1,442-bp repeated element found in the mitochondrial genome of the deep sea scallop Placopecten magellanicus. This comparison provided evidence for the absence of nucleotide conservation, except for a small sequence engaged in a secondary structure, but argued for a strong pressure maintaining domains with specific nucleotide content. A possible role for the conserved sequence is discussed.Correspondence to: A. Rigaa     

Nuclear counterparts of the cytoplasmic mitochondrial 12S rRNA gene: A problem of ancient DNA and molecular phylogenies

Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results.Correspondence to: A.C. van der Kuyl     

Identification of two Drosophila TGF-β family members in the grasshopper Schistocerca americana

Intercellular signaling molecules of the transforming growth factor- (TGF-) superfamily are required for pattern formation in many multicellular organisms. The decapentaplegic (dpp) gene of Drosophila melanogaster has several developmental roles. To improve our understanding of the evolutionary diversification of this large family we identified dpp in the grasshopper Schistocerca americana. S. americana diverged from D. melanogaster approximately 350 million years ago, utilizes a distinct developmental program, and has a 60-fold-larger genome than D. melanogaster. Our analyses indicate a single dpp locus in D. melanogaster and S. americana, suggesting that dpp copy number does not correlate with increasing genome size. Another TGF- superfamily member, the D. melanogaster gene 60A, is also present in only one copy in each species. Comparison of homologous sequences from D. melanogaster, S. americana, and H. sapiens, representing roughly 900 million years of evolutionary distance, reveals significant constraint on sequence divergence for both dpp and 60A. In the signaling portion of the dpp protein, the amino acid identity between these species exceeds 74%. Our results for the TGF- superfamily are consistent with current hypotheses describing gene duplication and diversification as a frequent response to high levels of selective pressure on individual family members.