The diversification of the genus Nesotes (Coleoptera: Tenebrionidae) in the Canary Islands: evidence from mtDNA.
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Animals↗
Biomedical subjects
D J Rees
Publications and source records attributed to D J Rees.
Mitochondrial DNA, ecology and morphology: interpreting the phylogeography of the Nesotes (Coleoptera: Tenebrionidae) of Gran Canaria (Canary Islands).
The genus Nesotes (Coleoptera: Tenebrionidae) is represented in the Canary Islands by 19 endemic species, the majority of which are single island endemics. Nesotes conformis and N. fusculus are described on four and three islands, respectively, but each forms a paraphyletic assemblage between Gran Canaria and the other islands. The other described species for Gran Canaria are N. quadratus, N. lindbergi and N. piliger. Thirty-six individuals representing the five species on Gran Canaria have been sequenced for 675 bp of the mitochondrial DNA (mtDNA) cytochrome oxidase II gene. Neighbour-joining analysis of maximum likelihood distances resulted in five distinct mtDNA lineages for N. quadratus, two of which also include mitotypes of N. conformis. Each of the other three species is found on only one mtDNA lineage. We propose from the molecular data that differentiation in a widespread N. quadratus-type ancestor was followed by morphological adaptation to coastal, pine and laurel forest habitats.
Animals↗
Sequence analysis or rat integrin alphaE1 and alphaE2 subunits: tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph.
The integrin alphaOX-62 subunit is defined by the OX-62 monoclonal antibody that was raised against rat dendritic cells in lymph (veiled cells) and shows properties similar to those of human alphaE that is predominantly expressed on intraepithelial lymphocytes. To clone alphaOX-62, rat probes generated using primers specific for the human alphaE sequence were used to screen rat T cell cDNA libraries. cDNA clones encoding two similar but not identical alpha subunits that are closely related but distinct from human alphaE were isolated. alphaE1 is predicted to be the rat homolog of mouse alphaM290 and alphaE2 corresponds to rat alphaOX-62. Immunofluorescence analysis revealed that mouse alphaE1 and rat alphaE2 are expressed in dendritic epidermal T cells in the skin, intraepithelial lymphocytes in the small intestine and in cells with a dendritic morphology present at sites where gammadelta T cells occur in lymphoid organs. Unexpectedly, in veiled cells alphaE2 is co-expressed with intracellular CD3-delta and a 33-kDa CD3 chain but not the T cell receptor. These findings suggest that veiled cells may be derived from a lymphoid precursor. Furthermore, veiled cells show phenotypic similarities to intraepithelial lymphocytes.
Amino Acid Sequence↗
The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation.
The beta subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to beta integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of beta(1)A, beta(1)D and beta(3) integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin beta tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin alpha(IIb)beta(3); activation was dependent on the presence of both the talin head domain and the integrin beta(3) cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.
Amino Acid Sequence↗
Sequence analysis of rat integrin alpha E1 and alpha E2 subunits: tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph.
The integrin alpha OX-62 subunit is defined by the OX-62 monoclonal antibody that was raised against rat dendritic cells in lymph (veiled cells) and shows properties similar to those of human alpha E2 that is predominantly expressed on intraepithelial lymphocytes. To clone alpha OX-62, rat probes generated using primers specific for the human alpha E sequence were used to screen rat T cell cDNA libraries. cDNA clones encoding two similar but not identical alpha subunits that are closely related to but distinct from human alpha E were isolated. alpha E1 is predicted to be the rat homolog of mouse alpha M290 and alpha E2 corresponds to rat alpha OX-62. Immunofluorescence analysis revealed that mouse alpha E1 and rat alpha E2 are expressed in dendritic epidermal T cells in the skin, intraepithelial lymphocytes in the small intestine and in cells with a dendritic morphology present at sites where gamma delta T cells occur in lymphoid organs. Unexpectedly, alpha E2 is co-expressed with intracellular CD3-delta and a 33-kDa CD3 chain but not the T cell receptor in veiled cells. These findings suggest that veiled cells may be derived from a lymphoid precursor. Furthermore, veiled cells show phenotypic similarities to intraepithelial lymphocytes.
Amino Acid Sequence↗
Ca2+ binding to the first epidermal growth factor-like domain of human blood coagulation factor IX promotes enzyme activity and factor VIII light chain binding.
Ca2+ binding to the first epidermal growth factor (EGF)-like domain of factor IX is known to be required for biological activity, but the mechanism by which Ca2+ contributes to factor IX function has remained unclear. We have studied recombinant factor IX mutants which lack Ca2+ binding to the first EGF-like domain, due to a replacement of Asp64 by Glu, Lys, or Val. The purified mutants (factors IX D64E, D64K, and D64V), were compared to plasma-derived and recombinant wild-type factor IX with regard to a number of metal-ion dependent functional parameters. In the presence of Mg2+, the activated mutants were indistinguishable from normal factor IXa in hydrolyzing the synthetic substrate CH3-SO2-Leu-Gly-Arg-p-nitroanilide. Replacing Mg2+ by Ca2+ further stimulated the activity of normal factor IXa but not of mutant factor IXa. In factor VIII-independent factor X activation, factor IXa D64K and D64E displayed reduced catalytic activity compared to normal factor IXa (apparent kcat/Km approximately 1, 2, and 4 x 10(3) M-1 s-1, respectively). In the presence of factor VIIIa, factor X activation rates by normal and mutant factor IXa were stimulated by factor VIIIa to a different extent ( approximately700- and 200-fold, respectively), indicating that Asp64 replacements affect the interaction with factor VIIIa. This possibility was addressed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy or light chains. Whereas the heavy chain peptide (Ser558-Gln565) inhibited factor VIII-dependent factor X activation by normal and mutant factor IXa with similar efficiency, the light chain peptide (Lys1804-Lys1818) inhibited normal factor IXa 2-3-fold more efficiently than did mutant factor IXa. This indicates that the reduced response to factor VIIIa may be due to impaired binding of mutant factor IXa to the factor VIII light chain. This was further explored in direct binding studies. In the presence of Mg2+, normal and mutant factor IXa were similar in binding to the factor VIII light chain. However, in the presence of Ca2+, factor IXa mutants were less efficient than normal factor IXa, which was illustrated by a 4-5-fold lower affinity than normal factor IXa for factor VIII light chain. Collectively, our data demonstrate that a number of factor IXa functions, including enzymatic activity and assembly into the factor IXa-factor VIIIa complex, are dependent on Ca2+ binding to the first EGF-like domain of factor IX.
Amino Acid Sequence↗
Isolation and sequence analysis of a cDNA encoding the c subunit of a vacuolar-type H(+)-ATPase from the CAM plant Kalanchoë daigremontiana.
We report the sequence of a cDNA clone encoding the c ("16 kDa') subunit of a vacuolar-type H(+)-ATPase (V-ATPase) from Kalanchoë daigremontiana, a plant in which the cell vacuole plays a pivotal role in crassulacean acid metabolism. The clone, pKVA211, was isolated from a K. daigremontiana leaf cDNA library constructed in lambda ZAP II using a homologous PCR-generated cDNA probe for the V-ATPase c subunit. The KVA211 cDNA was 839 nucleotides long and included a 20 bp poly(A)+ tail together with a complete 495 bp coding region for a polypeptide with a predicted molecular mass of 16659 Da. The deduced amino acid sequence was highly conserved across the wide range of eukaryotes (vertebrates, invertebrates, fungi, plants and protozoa) in which this gene has now been identified. Sequence comparison of several PCR products and genomic Southern analysis indicated that the V-ATPase c subunit in K. daigremontiana is encoded by a small multi-gene family. Steady-state levels of the KVA211 mRNA were much higher in leaves than in roots or flowers, and expression of this transcript in leaves was shown to be strongly light-dependent.
Amino Acid Sequence↗
Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site.
We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.
Actins↗
Analysis of repeated motifs in the talin rod.
The amino acid sequence of the rod portion of talin contains strong periodic patterns with a long period of 32 to 34 residues superimposed on short periods of 7 and 7/2 residues. The rod includes 50 to 60 copies of an irregular repeated motif approximately 34 residues long. The motif itself consists of three sections: a short "leader" segment of about six residues, which has a high proportion of the prolines and acidic residues; a relatively well-conserved hydrophobic "core" pattern of approximately 21 residues; and a highly variable "linker" region of seven residues which joins onto the next leader. The core section sequence has many of the characteristics of an amphipathic helix. The extensive hydrophobic side of this postulated helix has a characteristic surface pattern of large and small hydrophobic residues (mainly Leu and Ala), with a strong periodicity of seven residues. It also has a narrow hydrophilic edge with a highly variable sequence. The core sequence is unlike either a normal helical coiled coil or a leucine zipper, because it contains several helical ridges and grooves. The helical cores probably form a tightly packed hydrophobic central strand for the fibrous tail. The leader and linker sections are highly variable in length, so that the spacing between the starting points of adjacent cores varies between 20 and 40 residues. The most common spacing is 34, and many spacings are close to this length.
Amino Acid Sequence↗
The cytoskeletal protein talin contains at least two distinct vinculin binding domains.
We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and 125I-vinculin in both gel-overlay and microtitre well binding assays, we present evidence for three separable binding sites for vinculin. All three are in the tail segment of talin (residues 434-2541) and are recognized by the same fragment of vinculin (residues 1-258). Two sites are adjacent to each other and span residues 498-950, and the third site is more than 700 residues distant in the primary sequence. Scatchard analysis of 125I-vinculin binding to talin also indicates three sites, each with a similar affinity (Kd = 2-6 x 10(-7) M). We also detect a substoichiometric interaction of higher affinity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regions of the chicken talin molecule in heterologous cells, we have shown that the sequences required to target talin to focal contacts overlap those which bind vinculin.
3T3 Cells↗
Determination of blood lead levels.
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Humans↗
Sequence and domain structure of talin.
Talin is a high-molecular-weight cytoskeletal protein concentrated at regions of cell-substratum contact and, in lymphocytes, at cell-cell contacts. Integrin receptors are involved in the attachment of adherent cells to extracellular matrices and of lymphocytes to other cells. In these situations, talin codistributes with concentrations of integrins in the cell surface membrane. Furthermore, in vitro binding studies suggest that integrins bind to talin, although with low affinity. Talin also binds with high affinity to vinculin, another cytoskeletal protein concentrated at points of cell adhesion. Finally, talin is a substrate for the Ca2(+)-activated protease, calpain II, which is also concentrated at points of cell-substratum contact. To learn more about the structure of talin and its involvement in transmembrane connections between extracellular adhesions and the cytoskeleton, we have cloned and sequenced murine talin. We describe a model for the structure of talin based on this sequence and other data. Homologies between talin and other proteins define a novel family of submembranous cytoskeleton-associated proteins all apparently involved in connections to the plasma membrane.
Amino Acid Sequence↗
sevenless: Seven found?
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Amino Acid Sequence↗
An ancient, highly conserved family of cysteine-rich protein domains revealed by cloning type I and type II murine macrophage scavenger receptors.
Scavenger receptors have been implicated in the development of atherosclerosis and other macrophage-associated functions. The bovine type I and type II scavenger receptors are multidomain transmembrane proteins that differ only by the presence in the type I receptor of an additional, extracellular cysteine-rich C-terminal domain. The isolation of type I and type II receptor cDNAs from a murine macrophage cell line, P388D1, establishes the presence of mRNAs encoding both receptor types in a single cell. Their sequences are highly similar to the bovine cDNAs. Receptor type-specific cDNA probes map to a common locus on murine chromosomes 8, suggesting that a single gene encodes both mRNAs. The type I-specific scavenger receptor cysteine-rich (SRCR) domain helps define a previously unrecognized family of remarkably well-conserved domains. Highly homologous SRCR domains (one, three, or four per polypeptide chain) are found in diverse secreted and cell-surface proteins from humans (e.g., CD5, complement factor I), mice (Ly-1), and sea urchins (speract receptor).
Amino Acid Sequence↗
Detection of polymorphisms at cytosine phosphoguanadine dinucleotides and diagnosis of haemophilia B carriers.
The polymerase chain reaction procedure (PCR) was used to detect a polymorphic Hha I site adjacent to the factor IX locus in a panel of 33 phenotypically normal caucasian individuals. This technique was also applied to a haemophilia B family pedigree. The Hha I polymorphic site was located 8 kb 3' to the factor IX gene, and the proportion of female subjects expected to be heterozygous at this site was 0.48. The Hha I locus was in linkage equilibrium with the other polymorphic loci on the factor IX gene. These findings, besides increasing the proportion of caucasian individuals whose haemophilia B carrier state can be diagnosed from 79% to 89%, demonstrate this widely applicable use of PCR for the detection of DNA polymorphism at cytosine phosphoguanadine dinucleotides irrespective of the methylation status.
Alleles↗
Randomly picked cosmid clones overlap the pyrB and oriC gap in the physical map of the E. coli chromosome.
Sets of overlapping cosmid clones generated by random sampling and fingerprinting methods complement data at pyrB (96.5') and oriC (84') in the published physical map of E. coli. A new cloning strategy using sheared DNA, and a low copy, inducible cosmid vector were used in order to reduce bias in libraries, in conjunction with micro-methods for preparing cosmid DNA from a large number of clones. Our results are relevant to the design of the best approach to the physical mapping of large genomes.
Chromosome Mapping↗
The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.
beta-Hydroxyaspartic acid is a post-translationally modified amino acid found in a number of plasma proteins in a domain homologous to epidermal growth factor. Its presence can be correlated with a high affinity Ca2+ binding site, with a dissociation constant of 10-100 microM. We describe a system for the expression of human coagulation factor IX in dog kidney cells in tissue culture, in which the post-translational modifications and the biochemical activity are indistinguishable from factor IX synthesized in vivo. This system has been used to express eight different point mutations of human factor IX in the first epidermal growth factor domain in order to study the role of beta-hydroxyaspartate at residue 64, and the adjacent carboxylate residues at positions 47, 49 and 78. We conclude that this domain is essential for factor IX function and suggest that Ca2+ binds to carboxylate ions in this domain and stabilizes a conformation necessary for the interaction of factor IXa with factor X, factor VIII and phospholipid in the next step of the clotting cascade.
Amino Acid Sequence↗
Defective propeptide processing of blood clotting factor IX caused by mutation of arginine to glutamine at position -4.
Blood clotting factor IX is synthesized as a precursor polypeptide that would be expected to be proteolytically cleaved in at least two positions during maturation to remove the prepeptide and propeptide regions. We show that a point mutation causing hemophilia B changes the amino acid at position -4 in the propeptide region of factor IX from an arginine to a glutamine, which results in the expression of a stable longer protein with 18 additional amino acids of the N-terminal propeptide region still attached. This suggests that in the normal maturation of factor IX the signal peptidase cleaves the peptide bond between amino acid residues -18 and -19, generating an unstable profactor IX intermediate. Further proteolytic processing to the mature factor IX depends on the arginine residue at -4. The significance of the homologous arginine residue in other processed proteins is discussed.
Amino Acid Sequence↗