Transcriptional regulation of the human proenkephalin gene by conformational switching: implications for decoy design

Molecular chaperones are essential to all living organisms. Their key role consists of mediating protein folding within the cell. Recent functional studies have provided more detailed information about the function and regulation of the chaperone network. Highlights of the past year include the crystal structure determinations of the asymmetric GroEL-GroES complex and of their isolated peptide-binding domains.     

Tobamovirus evolution: gene overlaps, recombination, and taxonomic implications

Tobamoviruses, mostly isolated from solanaceous plants, may represent ancient virus lineages that have codiverged with their hosts. Recently completed nucleotide sequences of six nonsolanaceous tobamoviruses allowed assessment of the codivergence hypothesis and support a third subgroup within tobamoviruses. The genomic sequences of 12 tobamoviruses and the partial sequences of 11 others have been analyzed. Comparisons of the predicted protein sequences revealed three clusters of tobamoviruses, corresponding to those infecting solanaceous species (subgroup 1), those infecting cucurbits and legumes (subgroup 2), and those infecting crucifers. The orchid-infecting odontoglossum ringspot tobamovirus was associated with subgroup 1 genomes by its coat and movement protein sequences, but with the crucifer-pathogenic tobamoviruses by the remainder of its genome, suggesting that it is the progeny of a recombinant. For four of five genomic regions, subgroup 1 and 3 genomes were equidistant from a subgroup 2 genome chosen for comparison, suggesting uniform rates of evolution. A phylogenetic tree of plant families based on the tobamoviruses they harbor was congruent with that based on rubisco sequences but had a different root, suggesting that codivergence was tempered by rare events of viruses of one family colonizing another family. The proposed subgroup 3 viruses probably have an origin of virion assembly in the movement protein gene, a large (25-codon) overlap of movement and coat protein open reading frames, and a comparably shorter genome. Codon-position-dependent base compositions and codon prevalences suggested that the coat protein frame of the overlap region was ancestral. Bootstrapped parsimony analysis of the nucleotides in the overlap region and of the sequences translated from the -1 frame (the subgroup 3 movement protein frame) of this region produced trees inconsistent with those deduced from other regions. The results are consistent with a model in which a no or short overlap organization was ancestral. Despite encoding of subgroup 2 and 3 movement protein C-termini by nonhomologous nucleotides, weak similarities between their amino acid sequences suggested convergent sequence evolution.     

Ascidian actin genes: developmental regulation of gene expression and molecular evolution

Actin is a ubiquitous protein in eukaryotic cells and plays an important role in cell structure, cell motility, and the generation of contractile force in both muscle and nonmuscle cells. Multiple genes encoding muscle or nonmuscle actins have been isolated from several species of ascidians and their expression patterns have been investigated. Sequence and expression analyses of muscle actin genes have shown that ascidians have at least two distinct isoforms of muscle actin, the larval muscle and body-wall isoforms. In the ascidian Halocynthia roretzi, two clusters of actin genes are expressed in the larval muscle cells. The HrMA2/4 cluster contains at least five actin genes and the HrMA1 cluster contains a pair of actin genes whose expression is regulated by a single bidirectional promoter. cis-Regulatory elements essential for muscle-specific expression of a larval muscle actin gene HrMA4a have been identified. The adult body-wall muscle actin is clearly distinguished from the larval muscle actin by diagnostic amino acids. The adult muscle actin genes may be useful tools to investigate the mechanisms of muscle development in ascidian adults. The evolution of chordate actin genes has been inferred by comparing the organization and sequences of actin genes and performing molecular phylogenetic analysis. The results suggest a close relationship between ascidian and vertebrate actins. The chordate ancestor seems to have evolved the "chordate-type" cytoplasmic and muscle actins before its divergence into vertebrates and urochordates. The phylogenetic analysis also suggests that the vertebrate muscle actin isoforms evolved after the separation of the vertebrates and urochordates. Muscle actin genes have been used to investigate the mechanism of muscle cell regression during the evolution of anural development. The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression. Actin genes have provided a unique system to study developmental and evolutionary mechanisms in chordates.     

Networks of gene regulation, neural development and the evolution of general capabilities, such as human empathy

A network of gene regulation organized in a hierarchical and combinatorial manner is crucially involved in the development of the neural network, and has to be considered one of the main substrates of genetic change in its evolution. Though qualitative features may emerge by way of the accumulation of rather unspecific quantitative changes, it is reasonable to assume that at least in some cases specific combinations of regulatory parts of the genome initiated new directions of evolution, leading to novel capabilities of the brain. These notions are applied, in this paper, to the evolution of the capability of cognition-based human empathy. It is suggested that it has evolved as a secondary effect of the evolution of strategic thought. Development of strategies depends on abstract representations of one's own possible future states in one's own brain to allow assessment of their emotional desirability, but also on the representation and emotional evaluation of possible states of others, allowing anticipation of their behaviour. This is best achieved if representations of others are connected to one's own emotional centres in a manner similar to self-representations. For this reason, the evolution of the human brain is assumed to have established representations with such linkages. No group selection is involved, because the quality of strategic thought affects the fitness of the individual. A secondary effect of this linkage is that both the actual states and the future perspectives of others elicit vicarious emotions, which may contribute to the motivations of altruistic behaviour.     

Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118-452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiled-coil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.     

Expression of deletion-containing dystrophins in mdx muscle: implications for gene therapy and dystrophin function

The expression of full-length dystrophin and various dystrophin deletion mutants was monitored in mdx mouse muscle after intramuscular injection of dystrophin-encoding plasmid DNAs. Recombinant dystrophin proteins, including those lacking either the amino terminus, carboxyl terminus, or most of the central rod domain, showed localization to the plasma membrane. This suggests that there are multiple attachment sites for dystrophin to the plasma membrane. Only those constructs containing the carboxyl terminus were able to stabilize dystrophin-associated proteins (DAP) at the membrane, consistent with other studies that suggest that this domain is critical to DAP binding. Colocalization with DAP was not necessary for membrane localization of the various dystrophin molecules. However, stabilization and co-localization of the DAP did seem to be a prerequisite for expression and/or stabilization of mutant dystrophins beyond 1 wk and these same criteria seemed important for mitigating the histopathological consequences of dystrophin deficiency.     

Structure of the gene encoding the alpha subunit of the human interleukin 3 receptor

Interleukin 3 is a cytokine that stimulates proliferation and differentiation of hematopoietic progenitor cells. Its receptor consists of two subunits, an interleukin 3-specific alpha subunit and a beta subunit shared by garanulocyte-macrophage colony stimulating factor and interleukin 5 receptors. In this paper, we determined the genomic structure of the alpha subunit of the human interleukin 3 receptor, which spans approximately 40 kb and has 12 exons. We found that the genomic structures of the alpha subunits of the human interleukin 3 and granulocyte-macrophage colony stimulating factor receptors are very similar. They possess a unique additional intron in the 'C domain', which is absent in the alpha subunit of the interleukin 5 receptor. These results suggest a shared evolutionary pathway of these two genes.     

Cellular heterogeneity of CFTR expression and function in the lung: implications for gene therapy of cystic fibrosis

Cystic fibrosis (CF) has become a paradigm disorder for the clinical testing of gene therapies in the treatment of inherited disease. In recent years, efforts directed at gene therapy of CF have concentrated on improving gene delivery systems to the airway. Surrogate endpoints for complementation of CFTR dysfunction in the lung have been primarily dependent on correction of chloride transport abnormalities. However, it is now clear that the pathophysiology of CF airways disease is far more complex than can be solely attributed to altered chloride permeability. For example, in addition to functioning as a chloride channel, CFTR also has been implicated in the regulation of other apical membrane conductance pathways through interactions with the amiloride sensitive epithelial sodium channel (ENaC) and the outwardly rectifying chloride channel (ORCC). Superimposed on this functional diversity of CFTR is a highly regulated pattern of CFTR expression in the lung. This heterogeneity occurs at both the level of CFTR protein expression within different cell types in the airway and the anatomical location of these cells in the lung. Potential targets for gene therapy of CF include ciliated, non-ciliated, and goblet cells in the surface airway epithelium as well as submucosal glands within the interstitium of the airways. Each of these distinct cellular compartments may have functionally distinct roles in processes which affect the pathogenesis of CF airways disease, such as fluid and electrolyte balance. However, it is presently unclear which of these cellular targets are most pathophysiologic relevant with regard to gene therapy. Elucidation of the underlying mechanisms of CFTR function in the airway will allow for the rational design of gene therapy approaches for CF lung diseases. This review will provide a summary of the field's current knowledge regarding CFTR functional diversity in the airway and the implications of such diversity for gene therapies of CF lung disease.     

Structure and regulation of the murine Clara cell secretory protein gene

The sheep haemophilia A, recently described in a flock of White Alpine sheep, is probably caused by the production of a less potent mutant of factor VIII (F-VIII). Other potential mechanisms, such as drastic reduction (or lack) of F-VIII production, or occurrence of an F-VIII inhibitor, are less likely. This conclusion was based on the analysis of plasma dilution-clotting effect relationships in normal female (anestrous, non-pregnant) sheep, female carriers of haemophilia A, and afflicted male sheep. A response model underlying the analysis was suggested, its validity tested, and a mathematical analysis suitable for routine evaluation procedure recommended.     

A gene fusion at a homeobox locus: alterations in leaf shape and implications for morphological evolution

Compound leaves are seen in many angiosperm genera and are thought to be either fundamentally different from simple leaves or elaborations of simple leaves. The knotted1-like homeobox (knox) genes are known to regulate plant development. When overexpressed in homologous or heterologous species, this family of genes can cause changes in leaf morphology, including excessive leaf compounding in tomato. We describe here an instance of a spontaneously arisen fusion between a gene encoding a metabolic enzyme and a homeodomain protein. We show that the fusion results in overexpression of the homeodomain protein and a change in morphology that approximates the changes caused by overexpression of the same gene under the control of the cauliflower mosaic virus 35S promoter in transgenic plants. Exon-shuffling events can account for the modularity of proteins. If the shuffled exons are associated with altered promoters, changes in gene expression patterns can result. Our results show that gene fusions of this nature can cause changes in expression patterns that lead to altered morphology. We suggest that such phenomena may have played a role in the evolution of form.     

Structure and function of neural plasticity-related gene products

We have isolated novel immediate early genes (IEGs) from the hippocampus by differential cloning techniques. These mRNAs are induced by synaptic activity and translated into proteins that may affect neural function. We have analyzed a variety of "effector" immediate early genes. These mRNAs encode: 1) cytoplasmic proteins, such as cyclooxygenase-2, a small G protein, Rheb, and a cytoskeleton-associated protein, Arc; 2) membrane-bound proteins, such as the cell adhesion protein Arcadlin, and a neurite-outgrowth protein, Neuritin; and 3) a secreted protein, Narp. We hypothesize that physiological stimulation induces "effector" proteins that might strengthen synaptic connections of activated synapses. In contrast, pathological conditions such as epilepsy or drug addiction may accelerate overproduction of these gene products, which cause abnormal synapse formation. Gene targeting and in vivo gene transfer techniques are required to prove this hypothesis.     

The gene for pyruvate, orthophosphate dikinase in C4 plants: structure, regulation and evolution

Pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1) is a key enzyme in photosynthesis in plants that exploit the C4 photosynthetic pathway for the fixation of CO2. This review focuses on the structure, regulation and evolution of the C4-type ppdk gene in the maize genome. The C4-ppdk gene in maize consists of 19 exons spanning about 12 kbp. The gene is transcribed from two different initiation sites under the control of two promoters to produce two mRNAs of different sizes. The larger one contains the exon 1 sequence that encodes the chloroplast transit peptide and its product acts as C4-PPDK in chloroplasts, while the smaller one does not contain the sequence and its product may function as a C3-enzyme in the cytosol. This unusual dual promoter system is not unique to the maize C4-type ppdk gene since the same organization is also observed in the rice (C3 plant) ppdk gene and in Flaveria. Thus, the two-promoter system is common to plant ppdk genes from C3 and C4, monocot and dicot plants. A discussion is also presented of the generation of a system for regulation of the expression of the C4-type ppdk gene. A chimeric gene consisting of a reporter gene under the control of the promoter of maize C4-ppdk is exclusively expressed in photosynthetic tissues and not in roots or stems of transgenic rice. The expression of the introduced gene is also regulated by light: it is low in etiolated leaves and is enhanced by illumination. These results indicate that the regulatory system that controls ppdk expression in maize is not unique to C4 plants.