Systematics within Gyps vultures: a clade at risk

Background  

Populations of the Oriental White-backed Vulture (Gyps bengalensis) have declined by over 95% within the past decade. This decline is largely due to incidental consumption of the non-steroidal anti-inflammatory veterinary pharmaceutical diclofenac, commonly used to treat domestic livestock. The conservation status of other Gyps vultures in southern Asia is also of immediate concern, given the lack of knowledge regarding status of their populations and the continuing existence of taxonomic uncertainties. In this study, we assess phylogenetic relationships for all recognized species and the majority of subspecies within the genus Gyps. The continuing veterinary use of diclofenac is an unknown but potential risk to related species with similar feeding habits to Gyps bengalensis. Therefore, an accurate assessment of the phylogenetic relationships among Gyps vultures should aid in their conservation by clarifying taxonomic uncertainties, and enabling inference of their respective relatedness to susceptible G. bengalensis.     

Sequence specificity of DNA cleavage by bis(1,10-phenanthroline)copper(I)

The bis(1,10-phenanthroline)copper(I) complex is a relatively simple molecule previously shown to cause DNA cleavage with a strong preference for gene control regions such as the Pribnow box. Sequence level mapping of sites of [(Phen)2CuI]+ cleavage in greater than 2000 bases in histone genes and the plasmid pUC9 showed that the specificity for control regions is related to a predominant preference for minor groove binding at TAT triplets, which were cleaved most strongly at the adenosine sugar ring. The related sequences TGT, TAAT, TAGPy, and CAGT (Py = pyrimidine) were moderately preferred, while CAT and TAC triplets, PyPuPuPu quartets, PuPuPuPy quartets, and CG-rich PyPuPuPy quartets were cleaved with low to average frequency. Polypurine and polypyrimidine sequences were cleaved with low frequency. The sequence preferences of [(Phen)2CuI]+ can be ascribed predominantly to (i) a requirement for binding in the minor groove at a pyrimidine 3'----5' step and (ii) stereoelectronic effects of the 2-amino group of guanine in the minor groove, which inhibit binding. Although the reagent appears primarily to recognize sequence features at the triplet or quartet level, lower than expected cleavage was observed for two TAT sequences adjacent to several other preferred sequences and higher than expected cleavage was observed at CAAGC sequences, suggesting that longer range sequence-dependent DNA conformational effects influence specificity in certain cases.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Mitochondrial DNA polymorphisms in subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel, and its peripheral isolates

Patterns of mitochondrial DNA (mtDNA) variation were examined in 133 mole-rats constituting all four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of the Spalax ehrenbergi superspecies in Israel, as well as the peripheral isolates of 2n = 60. In the main range of the complex, a total of 28 mtDNA haplotypes were found in 64 mole-rats, with most haplotypes being unique to either a single chromosomal species or population. mtDNA divergence increased from low to high diploid number in a north-to-south direction in Israel. Overall levels of mtDNA diversity were unexpectedly the highest in the 2n = 60, the youngest species of the complex. The mtDNA haplotypes can be separated into two major groups, 2n = 52-54 and 2n = 58-60, and a phylogenetic analysis for each group revealed evidence of a few haplotypes not sorted by diploid number. The overall patterns of mtDNA divergence seen within and among the four chromosomal species are consistent with the parapatric mode of speciation as suggested from previous studies of allozyme and DNA hybridization. In a separate data set the patterns of mtDNA variation were examined across the main geographic range and across peripheral semi-isolates and isolates of the 2n = 60 chromosomal species. Fifteen haplotypes were found in 69 mole-rats. High levels of mtDNA diversity characterized the main range, semi-isolated, and even some desert isolated populations. The peripheral isolates contain much mtDNA diversity, including novel haplotypes.      

Histones H2A and H2B are neighbors along DNA in chromatin: characterization of subnucleosomal particles containing H2A+H2B.

Two specific slow sedimenting nucleoprotein particles containing equimolar amounts of histones H2A and H2B and 38 or 49 base pair (bp) lengths of DNA have been isolated by centrifugation on sucrose gradients. The 3.4S particles containing 38 bp DNA and H2A+H2B thermally denature at 61 degrees, considerably higher than Proteinase K treated particles (44 degrees), but lower than 11S nucleosomes (76 degrees). Treatment with Proteinase K increases the circular dichroism of 3.4S particles at 280 nm by 63% and decreases the sedimentation coefficient to 2.1S. These results indicate that H2A and H2B are proximate along DNA in nucleosomes and alone can alter the optical activity and perhaps conformation of local regions of DNA.     

Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed.     

Sequence preferences of covalent DNA binding by anti-(+)- and anti-(-)-benzo[a]pyrene diol epoxides

The sequence preferences of formation of piperidine-labile adducts of guanine by individual (+)- and (-)-isomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [anti-(+)- and anti-(-)-BPDE] were examined by techniques analogous to chemical DNA sequencing. Data were obtained on over 1200 bases with anti-(-)-BPDE and 1000 bases with anti-(+)-BPDE. Guanines on average yielded more labile adducts than other bases, and the reactivities of guanines with both anti-(+)- and anti-(-)-BPDE isomers were found to be distinctly nonrandom with respect to DNA sequence. The most and least reactive guanines, defined in terms of the upper and lower 10 percentiles of reactivity, differed on average by a factor of 17. This range of guanine reactivities was correlated with distinct sequence preferences, which differed in part for the two isomers. The strongest determinant for preferred reaction of anti-(-)-BPDE to form a labile adduct at a guanine was the presence of a 3'-flanking guanine, but a thymine 5'-flanking a guanine also generally enhanced reactivity. The triplets containing central guanines most preferred by anti-(-)-BPDE were AGG, CGG, and TG(G greater than T greater than C,A). anti-(+)-BPDE also formed labile adducts preferentially at AGG and CGG triplets, but not at TGN triplets. Significant effects of next-nearest-neighbor bases on guanine reactivities were also noted.     

A 23Na-NMR study of sodium-DNA interactions in concentrated DNA solutions at low-supporting electrolyte concentration

Aqueous solutions of DNA fragments with a contour length (500 A) near the persistence length at DNA concentrations ranging from 10 to 290 mg/mL solvent and a constant supporting electrolyte concentration of 0.01 M (predominantly NaCl) were examined by 23Na-nmr spectroscopy at temperatures of 20, 40, and 60 degrees C. Over the higher portion of this concentration range (greater than 100 mg/ml) the DNA solutions undergo a complex series of transitions between different anisotropic, liquid crystalline phases (T. E. Strzelecka and R. L. Rill, Biopolymers, in press). Counterions in solutions of strong polyelectrolytes are usually described in terms of a two-state model as free or "bound" (influenced by the electrostatic field of the polyanion). The longitudinal relaxation rate (R1 = 1/T1) at all DNA concentrations decreased with increasing temperature, demonstrating fast exchange between free and bound counterions. R1 increased nearly linearly with increasing DNA phosphate/sodium ratio in the isotropic domain until the onset of anisotropic phase formation, in agreement with similar nmr studies conducted at low DNA concentrations. The value of R1,b = 194 +/- 7 Hz obtained for the isotropic phase from 10 to 100 mg DNA/mL at 20 degrees C was in agreement with values reported previously. A nonlinear increase in R1 with DNA concentration was observed upon onset of anisotropic phase formation, indicating an increase in the product of the fraction of bond ions times their relaxation rate (r.R1,b). The spectral lineshape of all isotropic samples was Lorentzian. Spectra of anisotropic samples exhibited low magnitude quadrupole splitting of less than or equal to 400 Hz correlated with appearance of a cholesteric phase with pitch approximately 2 microns. The magnitude of the quadrupole splitting decreased with increasing DNA concentration at low temperatures and increased with concentration at high temperatures. At all concentrations the quadrupole splitting decreased then increased with temperature. These temperature- and concentration-dependent changes in quadrupole splitting are consistent with an angle between the DNA helix axis and the principal component (VZZ) of the local electric field gradient tensor near the "magic angle" of 54.7 degrees.