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.     

Detection of dense intra- and perinuclear 10 nm filament systems by whole mount and embedment-free electron microscopy in several species of the green algal order Dasycladales

Summary In contrast to the immense body of evidence supporting the occurrence of intermediate filament (IF) proteins in the animal kingdom, there is only limited information on their distribution in plants. Nevertheless, a number of immunocytochemical and electron microscopical observations indicate that particularly in higher plant cells IFs contribute to the construction of the cyto- and karyoskeleton. Here we show by whole mount electron microscopy of the giant nuclei extruded together with adhering cytoplasm from the rhizoids of some species of the algal order Dasycladales that cytoplasmic 10 nm filament networks also occur in unicellular, mononucleated green organisms of early evolutionary origin. The filament systems were associated with the residual nuclear envelope which consisted of a dense arrangement of pore complexes suspended by a meshwork of short 5 to 6 nm filaments; structurally it was very similar to the nuclear envelopes obtained from mammalian cells. When the Dasycladales nuclei were processed side by side with mouse skin fibroblasts, the algal filament systems were physically almost indistinguishable from the mammalian vimentin filament network. Embedment-free thin sections of rhizoids have not only confirmed the existence of the perinculear 10 nm filaments and their seamless association with the nuclear envelope, but have demonstrated the existence of an extensive intranuclear meshwork of 10 nm filaments. The latter were morphologically indistinguishable from the perinuclear 10 nm filaments and seem to be connected to these via the nuclear envelope to form a continuum. Among a variety of antibodies directed against mammalian IF proteins, only polyclonal anti-mouse lamin B antibodies decorated the cytoplasmic filaments of the Dasycladales cells. Surprisingly, none of the antibodies decorated the thinner filaments of the nuclear envelope, which possibly represent the nuclear lamina. In accord with this observation, one anti-lamin B antibody recognized in Western blot analysis of a urea extract ofAcetabularia acetabulum rhizoids three polypeptides with M_rs of approximately 47,000, 64,000, and 76,000. The proteins did not react with the -IFA antibody. Since the Dasycladales have a fossil record of nearly 600 million years — an extant genus, Acicularia, also investigated here, evolved about 170 million years ago -, the molecular characterization of the subunit proteins of their cytoplasmic filament systems might throw further light on the evolution and biological role of IFs.Dedicated to Professor Sir Henry Harris on the occasion of his 70th birthday     

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.      

Characterization of the nucleic acid binding region of the intermediate filament protein vimentin by fluorescence polarization

Employing deletion mutant proteins and fluorescein-labeled oligodeoxyribonucleotides in a fluorescence polarization assay, the nucleic acid binding site of the intermediate filament (IF) subunit protein vimentin was localized to the middle of the arginine-rich, non-alpha-helical, N-terminal head domain. While deletion of the first few N-terminal residues (up to amino acid 17) had almost no effect, deletions of residues 25-64 or 25-68 essentially abolished the binding of nucleic acids by the respective proteins. Proteins with smaller deletions, of residues 25-39 or 43-68, were still able to bind nucleic acids quite well at low ionic strength, but only the proteins containing the first DNA-binding wing (residues 27-39) retained the ability to stably bind nucleic acids at physiological ionic strength. These results were confirmed by data obtained with two synthetic peptides whose sequences correspond to the smaller deletions. Nitration experiments showed that one or more of the tyrosines in the head domain are responsible for the stable binding by intercalation. Interestingly, the residues responsible for binding nucleic acids can be deleted without major influence on the in vivo polymerization properties of the mutant proteins. Only the protein with the largest internal deletion, of residues 25-68, failed to form filaments in vivo. Since the N-terminal head domains of IF proteins are largely exposed on the filament surface, but nevertheless essential for filament assembly, these results support the model that the middle of the head domain of vimentin may loop out from the filament surface and thus be available for interactions with other cellular structures or molecules.     

Amino-terminal polypeptides of vimentin are responsible for the changes in nuclear architecture associated with human immunodeficiency virus type 1 protease activity in tissue culture cells

Electron microscopy of human skin fibroblasts syringe-loaded with human immunodeficiency virus type 1 protease (HIV-1 PR) revealed several effects on nuclear architecture. The most dramatic is a change from a spherical nuclear morphology to one with multiple lobes or deep invaginations. The nuclear matrix collapses or remains only as a peripheral rudiment, with individual elements thicker than in control cells. Chromatin organization and distribution is also perturbed. Attempts to identify a major nuclear protein whose cleavage by the protease might be responsible for these alterations were unsuccessful. Similar changes were observed in SW 13 T3 M [vimentin(+)] cells, whereas no changes were observed in SW 13 [vimentin(-)] cells after microinjection of protease. Treatment of SW 13 [vimentin(-)] cells, preinjected with vimentin to establish an intermediate filament network, with HIV-1 PR resulted in alterations in chromatin staining and distribution, but not in nuclear shape. These same changes were produced in SW 13 [vimentin(-)] cells after the injection of a mixture of vimentin peptides, produced by the cleavage of vimentin to completion by HIV-1 PR in vitro. Similar experiments with 16 purified peptides derived from wild-type or mutant vimentin proteins and five synthetic peptides demonstrated that exclusively N-terminal peptides were capable of altering chromatin distribution. Furthermore, two separate regions of the N-terminal head domain are primarily responsible for perturbing nuclear architecture. The ability of HIV-1 to affect nuclear organization via the liberation of vimentin peptides may play an important role in HIV-1-associated cytopathogenesis and carcinogenesis.     

Deletion mutagenesis of the amino-terminal head domain of vimentin reveals dispensability of large internal regions for intermediate filament assembly and stability

Previous studies have shown that the non-alpha-helical head domain of vimentin is required for polymerization of intermediate filaments (IFs) and, furthermore, a nonapeptide highly conserved among type III IF subunit proteins at their extreme amino-terminus is essential for this process. Recombinant DNA technology was employed to produce specific vimentin deletion mutant proteins (for in vitro studies) or vimentin protein expression plasmids (for in vivo studies), which were used to identify other regions of the vimentin head domain important for polymerization. Various vimentin proteins lacking either residues 25-38, 44-95, or 40-95 polymerized into wild-type or largely normal IFs, both in vitro and in vivo. Vimentin proteins lacking residues 44-69 or 25-63 failed to form IFs in vitro, but assembled into IFs in vivo. Vimentin proteins lacking residues 25-68, 44-103, or 88-103 failed to form IFs in vitro or in vivo. Taken together with previous results, these data demonstrate that the middle of the vimentin non-alpha-helical head domain, which is known to be the site of nucleic acid binding, is completely dispensable for IF formation, whereas both ends of the vimentin non-alpha-helical head domain are required for IF formation. The simplest explanation for these results is that the middle of the vimentin non-alpha-helical head domain loops out, thereby permitting the juxtaposition of the ends of the head domain and their productive interaction with other protein domains (probably the C-terminus of the rod domain) during IF polymerization. The ability of some of the mutant proteins to form IFs in vivo, but not in vitro, suggests that as-yet-unknown cellular proteins may interact with and, in some cases, enable polymerization of IFs, even though they are not absolutely required for IF formation by wild-type vimentin.     

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.