Biomedical subjects

P J Lockhart

Publications and source records attributed to P J Lockhart.

15 recordsLinked to original sources

PubMed1998

Correction of the copper transport defect of Menkes patient fibroblasts by expression of the Menkes and Wilson ATPases.

Menkes' disease is a fatal, X-linked, copper deficiency disorder that results from defective copper efflux from intestinal cells and inadequate copper delivery to other tissues, leading to deficiencies of critical copper-dependent enzymes. Wilson's disease is an autosomally inherited, copper toxicosis disorder resulting from defective biliary excretion of copper, which leads to copper accumulation in the liver. The ATP7A and ATP7B genes that are defective in patients with Menkes' and Wilson's diseases, respectively, encode transmembrane, P-type ATPase proteins (ATP7A or MNK and ATP7B or WND, respectively) that function to translocate copper across cellular membranes. In this study, the cDNAs derived from a normal human ATP7A gene and the murine ATP7B homologue, Atp7b, were separately transfected into an immortalized fibroblast cell line obtained from a Menkes' disease patient. Both MNK and WND expressed from plasmid constructs were able to correct the copper accumulation and copper retention phenotype of these cells. However, the two proteins responded differently to elevated extracellular copper levels. Although MNK showed copper-induced trafficking from the trans-Golgi network to the plasma membrane, in the same cell line the intracellular location of WND did not appear to be affected by elevated copper.

Adenosine Triphosphatases↗

PubMed1998

Eukaryotic expression vectors that replicate to low copy number in bacteria: transient expression of the Menkes protein.

A set of low copy number plasmid vectors for mammalian gene expression has been constructed. These vectors are derived from the previously described bacterial low copy number expression vectors, pWSK29 and pWKS30, which are present at six to eight copies per cell. The new plasmids also have the following useful properties: (1) they contain antibiotic resistance markers for the selection of stable mammalian cell lines; (2) they have either constitutive or inducible promoters; (3) a chimeric intron, for enhancing gene expression, is present; (4) they contain unique cloning sites; (5) they have an SV40 polyadenylation signal, and a subset of the vectors have an SV40 origin of replication for episomal replication and transient gene expression. A cDNA encoding the Menkes disease protein was cloned into two of these vectors, and transient expression studies in COS-7 cells showed that both constitutive and inducible expression was possible. This set of expression vectors will provide a useful tool for the manipulation, in Escherichia coli, of mammalian genes or cDNAs that are unstable in the high copy number vectors that are currently available.

Adenosine Triphosphatases↗

PubMed1998

Phylogenetic analysis of plastid origins based on secA sequences.

We have generated secA sequence data from a number of photosynthetic prokaryotes and carried out a phylogenetic analysis using secA sequences from prokaryotes, green plants, and red and brown algae. We have studied the substitution patterns that give rise to the apparent phylogenetic structure. We show that the high AT content of the plastid sequences significantly affects the amino-acid composition. We also show that most of the apparent evidence for an edge separating red and brown plastids from green plants within the phylogenetic tree is due to differences in nucleotide composition. The remaining apparent evidence is likely to be due, at least in part, to differences in the distribution of sites free to vary. We discuss the implications of this study for hypotheses of plastid origins.

Adenosine Triphosphatases↗

PubMed1998

Functional analysis and intracellular localization of the human menkes protein (MNK) stably expressed from a cDNA construct in Chinese hamster ovary cells (CHO-K1).

The Menkes protein (MNK or ATP7A) is an important component of the mammalian copper transport pathway and is defective in Menkes disease, a fatal X-linked disorder of copper transport. To study the structure and function of this protein and to elucidate its role in cellular copper homeostasis, a cDNA construct encoding the full-length MNK protein was cloned into a mammalian expression vector under the control of the CMV promoter. Transfection of this plasmid construct into CHO-K1 cells yielded clones that expressed MNK at varying levels. Detailed characterization of four clones showed that an increase in MNK protein expression led to a corresponding increase in the level of copper resistance of the cells. Subcellular localization studies showed that in the parental CHO-K1 and the transfected cell lines, MNK was located in a post-Golgi compartment which, based on immunogold electron microscopic analyses, most likely represented the trans -Golgi network (TGN). When the extracellular copper concentration was increased, MNK in the clones as well as in CHO-K1 cells was redistributed to the cytoplasm and plasma membrane, but returned to the TGN under basal, low copper conditions. This report presents the first ultrastructural evidence for the association of MNK with vesicles within the cell and with the TGN and plasma membrane. It also demonstrates the stable expression of a functional MNK protein from a cDNA construct in mammalian cells, as well as the copper-induced redistribution of MNK in a cell line (CHO-K1) that was not selected for copper resistance or overexpression of MNK.

Adenosine Triphosphatases↗

PubMed1998

A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages.

The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms. (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed. The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number. Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism. The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns. This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution. We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution. We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.

Markov Chains↗

PubMed1997

Identification of soil bacteria expressing a symbiotic plasmid from Rhizobium leguminosarum bv. trofolii.

A hundred strains of non-nodulating, Gram-negative, rod-shaped bacteria were isolated from clover-ryegrass pastures on three different soil types and from a sandy loam under lupins. When crossed with Escherichia coli PN200 containing the cointegrate plasmid pPN1, 11 transconjugants gained the ability to form nodules on the roots of white clover (Trifolium repens cv. Grasslands Huia). A nodA probe indicated that they had gained nodulation genes. The identities of these 11 strains and 4 others derived from earlier work on non-nodulating root nodule bacteria, were determined by ribotyping, DNA-DNA hybridization, and partial 16S rRNA sequencing. Good agreement was obtained between the three methods, and 11 of the strains were identified as Rhizobium leguminosarum (6), Rhizobium loti (2), Rhizobium etli (1), Rhizobium tropici (1), and Sinorhizobium meliloti (1). DNA-DNA hybridization indicated that the remaining four strains were related to the Rhizobium leguminosarum reference strains. The existence of several species of non-nodulating rhizobia in pasture soil, including species for which the normal host plant was absent, is discussed in relation to the fate of symbiotic plasmids from Rhizobium seed inoculants. It is also suggested that new species should be named for the geographical region from which they are first isolated rather than the host plant.

Base Sequence↗

PubMed1996

Gene duplication and the evolution of photosynthetic reaction center proteins.

We investigate the evolutionary relationships between photosynthetic reaction center proteins (D1, D2, L and M) and demonstrate that the pattern of nucleotide substitution in these is more complicated than has been assumed in previous phylogenetic analyses. We show that there are serious violations of methodological assumptions in previous published studies. We conclude that there is equal support for hypotheses indicating (i) a single gene duplication of an ancestral reaction center protein followed by diversification and (ii) two independent gene duplications giving rise to proteins in oxygenic and anoxygenic systems.

Amino Acid Sequence↗

PubMed1996

Evolution of chlorophyll and bacteriochlorophyll: the problem of invariant sites in sequence analysis.

Competing hypotheses seek to explain the evolution of oxygenic and anoxygenic processes of photosynthesis. Since chlorophyll is less reduced and precedes bacteriochlorophyll on the modern biosynthetic pathway, it has been proposed that chlorophyll preceded bacteriochlorophyll in its evolution. However, recent analyses of nucleotide sequences that encode chlorophyll and bacteriochlorophyll biosynthetic enzymes appear to provide support for an alternative hypothesis. This is that the evolution of bacteriochlorophyll occurred earlier than the evolution of chlorophyll. Here we demonstrate that the presence of invariant sites in sequence datasets leads to inconsistency in tree building (including maximum-likelihood methods). Homologous sequences with different biological functions often share invariant sites at the same nucleotide positions. However, different constraints can also result in additional invariant sites unique to the genes, which have specific and different biological functions. Consequently, the distribution of these sites can be uneven between the different types of homologous genes. The presence of invariant sites, shared by related biosynthetic genes as well as those unique to only some of these genes, has misled the recent evolutionary analysis of oxygenic and anoxygenic photosynthetic pigments. We evaluate an alternative scheme for the evolution of chlorophyll and bacteriochlorophyll.

Bacteriochlorophylls↗

PubMed1996

Evolutionary analysis of the multigene pregnancy-specific beta 1-glycoprotein family: separation of historical and nonhistorical signals.

The pregnancy-specific beta 1-glycoproteins (PSG) form a large family of closely related proteins. Using newly developed methods of sequence analysis, in combination with protein modeling, we provide a framework for investigating the evolution and biological function of genes like the PSG. Evolutionary trees, based on C-terminal sequence, group PSG genes in a manner consistent with their genomic organization. Trees constructed using the N-terminal domain sequences are unreliable as an indicator of phylogeny because of non-neutral processes of sequence change. During duplication of the PSG genes, evolutionary pressures have resulted in a gradient of constrained change across each gene. The N-terminal domains show a nonrandom pattern of amino acid substitutions clustered in the immunoglobulin complementarity-determining region (CDR)-like regions, which appear to be important in the function of the protein.

Carcinoembryonic Antigen↗

PubMed1996

Cloning of novel kinectin splice variants with alternative C-termini: structure, distribution and evolution of mouse kinectin.

The analysis of cDNA clones encoding novel variant forms of mouse kinectin, an endoplasmic reticulum (ER)-bound receptor for the motor protein kinesin, is reported. Kinesin and cytoplasmic dynein are involved in mediating the anterograde and retrograde movements of intracellular vesicles along the microtubule network. The amino acid sequence deduced from kinectin cDNA isolated from mouse spleen cell and testis libraries revealed a long signal peptide or transmembrane sequence, and a 328 amino acid residue globular N-terminal domain adjacent to a much larger 858-999-residue C-terminal coiled-coil rod domain. The C-terminal domain was composed of 18 coiled-coil regions formed from multiple contiguous heptad repeats which undergo alternative splicing as evidenced by the presence of at least five small (23-33 amino acid residue) insertion sequences scattered throughout. The inserts are present in any one of a number of combinations, generating an array of novel kinectin variants. Insert 5 contains a termination codon, producing a C-terminus that is highly homologous to that of human kinectin. Three out of five mouse kinectin clones lack insert 5, generating a novel eleven amino acid C-terminus encoded by sequence that extends past the insertion site. The existence of alternative C-termini may have functional relevance given that the C-termini are exposed for interaction with kinesin, whereas the globular N-terminus is embedded in the ER membrane. Alternative C-termini represent candidate modifications that could determine specificity of binding to kinesin or cytoplasmic dynein, and the switching of directionality of movement. The cDNA hybridized to 4.5 kb transcripts expressed in all mouse cell lines and tissues examined, which provides the first indication that the kinectins are very widely distributed. Mouse kinectin is 42% similar over a 203 amino acid region to the chicken extracellular cardiac morphogen ES/130, whose canine homologue containing an inserted sequence of 10 amino acids repeated 54 times in tandem, is a ribosome receptor expressed on the ER. Mouse kinectin shares 64 and 83% identity, respectively, with its M(r) 160000 chicken and human kinectin homologues. There is a two-fold molar excess of kinectin over kinesin in unextracted vesicles, suggesting that kinectin might be a dimer. The electrostatic properties of the coiled-coil region of mouse kinectin, together with the relative frequencies of residues in particular positions within the heptad repeats support this notion.

Alternative Splicing↗

PubMed1995

A frequency-dependent significance test for parsimony.

We describe techniques for assessing evolutionary trees constructed by the parsimony criteria, when sequences exhibit irregular base compositions. In particular, we extend a recently described frequency-dependent significance test to handle any number of taxa and describe a modification of the Kishino-Hasegawa sites test. These modifications are useful for detecting historical signals beyond those patterns which arise purely from irregular base compositions between the compared sequences. We apply the test to extend our earlier studies on chloroplast origins using 16S rDNA sequences, where a failure to compensate for irregular base compositions between the compared sequences provides statistically significant support for unjustified phylogenetic inferences. We also describe how the techniques can be modified to determine how "tree-like" data are, given independent variation in the base frequencies.

Models, Statistical↗

PubMed1993

Confidence in evolutionary trees from biological sequence data.

The reliable construction of evolutionary trees from nucleotide sequences often depends on randomization tests such as the bootstrap and PTP (cladistic permutation tail probability) tests. The genomes of bacteria, viruses, animals and plants, however, vary widely in their nucleotide frequencies. Where genomes have independently acquired similar G+C base compositions, signals in the data arise that cause methods of evolutionary tree reconstruction to estimate the wrong tree by grouping together sequences with similar G+C content. Under these conditions randomization tests can lead to both the rejection of the correct evolutionary hypothesis and acceptance of an incorrect hypothesis (such as with the contradictory inferences from the photosynthetic rbcS and rbcL sequences). We have proposed one approach to testing for G+C content problem. Here we present a formalization of this method, a frequency-dependent significance test, which has general application.

Base Sequence↗

PubMed1992

Controversy on chloroplast origins.

Controversy exists over the origins of photosynthetic organelles in that contradictory trees arise from different sequence, biochemical and ultrastructural data sets. We propose a testable hypothesis which explains this inconsistency as a result of the differing GC contents of sequences. We report that current methods of tree reconstruction tend to group sequences with similar GC contents irrespective of whether the similar GC content is due to common ancestry or is independently acquired. Nuclear encoded sequences (high GC) give different trees from chloroplast encoded sequences (low GC). We find that current data is consistent with the hypothesis of multiple origins for photosynthetic organelles and single origins for each type of light harvesting complex.

Base Composition↗

PubMed1992

Sequence of Prochloron didemni atpBE and the inference of chloroplast origins.

The prochlorophytes, oxygenic photosynthetic prokaryotes containing chlorophylls a and b, have been put forward as descended from the organisms that gave rise to chloroplasts of green plants and algae by endosymbiosis, although this has always been controversial. To assess the phylogenetic position of the prochlorophyte Prochloron didemni, we have cloned and sequenced its atpBE genes. Phylogenetic inference under a range of models gives moderate to strong support for a cyanobacterial grouping rather than a chloroplast one. Possible systematic errors in this and previous analyses of prochlorophyte sequences are discussed.

Amino Acid Sequence↗

PubMed1992

Substitutional bias confounds inference of cyanelle origins from sequence data.

Available molecular and biochemical data offer conflicting evidence for the origin of the cyanelle of Cyanophora paradoxa. We show that the similarity of cyanelle and green chloroplast sequences is probably a result of these two lineages independently developing the same pattern of directional nucleotide change (substitutional bias). This finding suggests caution should be exercised in the interpretation of nucleotide sequence analyses that appear to favor the view of a common endosymbiont for the cyanelle and chlorophyll-b-containing chloroplasts. The data and approaches needed to resolve the issue of cyanelle origins are discussed. Our findings also have general implications for phylogenetic inference under conditions where the base compositions (compositional bias) of the sequences analyzed differ.

Base Composition↗