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.     

Oxalyl-CPG: a labile support for synthesis of sensitive oligonucleotide derivatives.

A procedure is described for linking nucleosides covalently to controlled pore glass or cross-linked polystyrene supports by means of an oxalyl anchor. Though stable to triethylamine and diisopropylamine, the nucleoside-oxalyl link can be cleaved within a few minutes at room temperature with ammonium hydroxide in methanol. This new anchor can be used in automated synthesis of conventional oligonucleotides. The primary value, however, is that it enables one to employ solid support methodology to synthesize a variety of base-sensitive oligonucleotide derivatives, as illustrated here by synthesis of oligomers with base protecting groups intact and with methyl phosphotriester groups at the internucleoside links.     

Use of phosphorus oxychloride in synthesizing nucleotides and oligonucleotides

Procedures are described for phosphorylating protected nucleotides, oligonucleotides and phosphoramidate oligonucleotide derivatives at the 3′-hydroxyl group. The conditions (phosphorylation with phosphorus oxychloride and pyridine in dioxane followed by hydrolysis with aqueous pyridine) are sufficiently mild that base labile (trifluoroacetylamino; β-cyanoethyl phosphotriester) and acid labile (O-monomethoxytrityl; phosphoramidate) functions are retained intact. Application of the technique is illustrated by the synthesis of dpT, dTp, d(CF₃CONH)Tp, dTp_NTp, and dTp_NTp_NTp. In addition, the utilization of phosphorus oxychloride in joining thymidine derivatives and dinucleoside phosphotriester blocks via phosphodiester links is described.     

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.      

Enhancement of selectivity in recognition of nucleic acids via chemical autoligation.

A new approach to increase the selectivity of interaction between oligonucleotide probes and target nucleic acids is described. In place of a single, relatively long oligonucleotide probe, two or three short oligomers terminated by thiophosphoryl and bromoacetamido groups are employed. Fast and efficient autoligation takes place when the oligomers hybridize in a contiguous mode to the same complementary strand such that a thiophosphoryl group on one strand and a bromoacetamido group on another are brought into proximity. A single nucleotide mismatch for the short probes leads to marked reduction in the rate of autoligation. The binding affinity of the product is close to that for a natural probe of the same length. This approach could have potential in oligonucleotide-based diagnostics, chemical amplification systems, and therapeutic applications.     

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.      

Formation of internucleotide 3''-5'' phosphoramidate links by direct coupling of phosphoryl and amino groups.

The stepwise synthesis of oligomers derived from 5'-amino-5'-deoxythymidine 3'-phosphate and 5'-amino-5'-deoxy-3'-O-mono-p-methoxytrityl-thymidine is described. The internucleotide phosphoramidate links were formed by condensation of nucleoside 3'-phosphates with aminonucleosides by means of triphenylphosphine and dipridyl disulfide.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.