A panel of bovine, ovine and caprine polymorphic microsatellites

We report a set of six new bovine microsatellite polymorphisms based on (CA)_n repeats. They are highly polymorphic and thus represent valuable markers for genome mapping. Four of the six are polymorphic in sheep and two are polymorphic in goats. One, which is polymorphic in cattle and sheep and apparently monomorphic in goats, is X-chromosome specific and has potential value in, for example, sex determination and detection of chimaerism.     

The disulfide bridges of the immunoglobulin-like domains of FcγRIIIB are essential for efficient expression and biological activity

The immunoglobulin G receptor FcRIIIB belongs to the immunoglobulin superfamily as two extracellular domains show homology to the immunoglobulin domains. Since some residues in these domains, such as the two cysteines, are supposed to form an intrachain disulfide bridge are so commonly conserved, they may be of importance for correct folding. Site-directed mutagenesis and expression in BHK21 confirmed this supposition for the FcRIIIB. Replacing both cysteines in the first and/or second domain by serines reduced the surface expression level by 50%, whereas the ligand binding capability was 20–30% of that seen in cells expressing the wild-type receptor. Replacing one of the four cysteines resulted in the loss of surface expression. Exchanging the conserved tryptophan in the first domain by phenylalanine only slightly affected the ligand binding (25%), whereas the surface expression remained unchanged.     

Woody plant encroachment in granite barrens on the Frontenac Arch,eastern Ontario,Canada

Aims

Woody plant encroachment is a widespread phenomenon affecting treeless or sparsely treed habitats. We aimed to determine the extent and timing of tree and shrub encroachment into rock barrens of eastern Ontario over the last century, and to assess implications for their ongoing management.

Location

Queen's University Biological Station in the Frontenac Arch ecoregion.

Methods

We quantified the extent of change in woody vegetation in 290 rock barrens using aerial photography from 1925, 1965, and 2008. Composition and structure of woody plant communities in 10 barrens was subsequently quantified in the field using plot-based sampling. Cores or cross-sections were obtained from individuals >1.5 m height and dendrochronological techniques were used to determine their age and identify temporal patterns of any woody encroachment.

Results

Aerial photography indicated that the mean proportion of woody plant cover in barrens increased 22.5% from 1925 to 2008. Dendroecological analysis supported this. Few trees were present prior to 1900 and most established since 1960. Fraxinus americana, Juniperus virginiana, and Juniperus communis were the most common woody species colonizing the barrens. Remnants of large Pinus strobus stumps with extensive charring were found in 90% of the sampled barrens at a mean density of 22.6 stumps ha⁻¹.

Conclusions

Rock barrens on the Frontenac Arch have changed substantially over the past century; gradually being colonized by trees and shrubs and losing their distinctly open character. Active management — including prescribed fire and mechanical thinning — may be necessary if there is a desire to maintain these barrens and the rare species they support as components of the region's biodiversity. However, identification of a reference state for restoration is complicated by the fact that the structure and composition of these habitats were undoubtedly altered by European land clearance in the 19th century, and that some of these areas likely existed as pine woodlands before that.     

The Gene Encoding the Heat-Stable Enterotoxin of Vibrio cholerae Is Flanked by 123-Base Pair Direct Repeats

The heat-stable enterotoxin (O1-ST) gene (sto) was cloned from chromosome of the strain GP156 of Vibrio cholerae O1 (Inaba, El Tor) in Escherichia coli K-12, and its nucleotide seqence was determined. The nucleotide sequence of sto was very similar to that of NAG-ST gene (stn) of V. cholerae non-O1. Both sto and stn were flanked by 123-base pair direct repeats which had at least 93% homology to one another and included some inverted repeats. All the strains of V. cholerae, V. mimicus, V. metschnikovii, V. hollisae and Yersinia enterocolitica examined by colony hybridization had the direct repeat sequence regardless of ST-gene possession.     

Ribosomal DNA variation and its phylogenetic implication in the genusBrachypodium (Poaceae)

The structure of ribosomal DNA ofBrachypodium and several other grass species was investigated using a heterologous rDNA probe from wheat. Several different rDNA families were present among perennial and annual species within the genus. In contrast to the annual species the perennial species exhibited a very low degree of repeat length variation. An extra Eco RI site and a Hin dIII site were observed in the IGS, which distinguishedBrachypodium from other grass genera. The restriction fragment length polymorphism and length variation of the repeat units have taxonomic value withinBrachypodium and are correlated with the classification ofBrachypodium derived from other data.     

Peptide-protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase.

Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.     

Zinc-mediated interaction of copper chaperones through their heavy-metal associated domains

BackgroundA copper chaperone CCS is a multi-domain protein that supplies a copper ion to Cu/Zn-superoxide dismutase (SOD1). Among the domains of CCS, the N-terminal domain (CCS^dI) belongs to a heavy metal-associated (HMA) domain, in which a Cys-x-x-Cys (CxxC) motif binds a heavy metal ion. It has hence been expected that the HMA domain in CCS has a role in the metal trafficking; however, the CxxC motif in the domain is dispensable for supplying a copper ion to SOD1, leaving an open question on roles of CCS^dI in CCS.MethodsTo evaluate protein-protein interactions of CCS through CCS^dI, yeast two-hybrid assay, a pull-down assay using recombinant proteins, and the analysis with fluorescence resonance energy transfer were performed.ResultsWe found that CCS specifically interacted with another copper chaperone HAH1, a HMA domain protein, through CCS^dI. The interaction between CCS^dI and HAH1 was not involved in the copper supply from CCS to SOD1 but was mediated by a zinc ion ligated with Cys residues of the CxxC motifs in CCS^dI and HAH1.ConclusionWhile physiological significance of the interaction between copper chaperones awaits further investigation, we propose that CCS^dI would have a role in the metal-mediated interaction with other proteins including heterologous copper chaperones.     

Higher-order interactions of Bcr-Abl can broaden chronic myeloid leukemia (CML) drug repertoire

Bcr-Abl, a nonreceptor tyrosine kinase, is associated with leukemias, especially chronic myeloid leukemia (CML). Deletion of Abl's N-terminal region, to which myristoyl is linked, renders the Bcr-Abl fusion oncoprotein constitutively active. The substitution of Abl's N-terminal region by Bcr enables Bcr-Abl oligomerization. Oligomerization is critical: it promotes clustering on the membrane, which is essential for potent MAPK signaling and cell proliferation. Here we decipher the Bcr-Abl specific, step-by-step oligomerization process, identify a specific packing surface, determine exactly how the process is structured and identify its key elements. Bcr's coiled coil (CC) domain at the N-terminal controls Bcr-Abl oligomerization. Crystallography validated oligomerization via Bcr-Abl dimerization between two Bcr CC domains, with tetramerization via tight packing between two binary assemblies. However, the structural principles guiding Bcr CC domain oligomerization are unknown, hindering mechanistic understanding and drugs exploiting it. Using molecular dynamics (MD) simulations, we determine that the binary complex of the Bcr CC domain serves as a basic unit in the quaternary complex providing a specific surface for dimer–dimer packing and higher-order oligomerization. We discover that the small α1-helix is the key. In the binary assembly, the helix forms interchain aromatic dimeric packing, and in the quaternary assembly, it contributes to the specific dimer–dimer packing. Our mechanism is supported by the experimental literature. It offers the key elements controlling this process which can expand the drug discovery strategy, including by Bcr CC-derived peptides, and candidate residues for small covalent drugs, toward quenching oligomerization, supplementing competitive and allosteric tyrosine kinase inhibitors.     

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)_n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.     

The role of local tight packing of hydrophobic groups in β-structure

An analysis of the tendency of hydrophobic groups to tight packing on the surface of β-sheets based on well-known parameters of β-sheets and hydrophobic groups was conducted. This analysis shows the existence of very limited numbers and clearly outlined architecture families of regular parts for the majority of β-structure-containing domains. Each family of architecture strongly depends on the number of β-strands in the pure β-domains and on the existence and number of additional α-helixes and on the mutual arrangements β-strands and α-helixes along the chain in mixed α/β-domains. This paper demonstrates that the tendency of hydrophobic groups to the local tight packing on the surface of β-sheets is probably the main reason for the twist of β-sheets. © 1993 Wiley-Liss, Inc.