DNA methylation of fly genes and transposons

The use of anti-5-methylcytosine antibodies in affinity columns allowed the identification of methylated sequences in the genome of Drosophila melanogaster adults. In view of the presence of transposable elements amongst the identified sequences, it has been suggested that DNA methylation is involved in transposon control in the fly genome. On the contrary, a reanalysis of these data furnishes several intriguing elements that could raise new questions about the role that DNA methylation plays in the fly genome. The aim of the present paper is to discuss some features that emerge from the analysis of the identified methylated sequences. Received 26 January 2006; received after revision 8 May 2006; accepted 2 June 2006     

Chimeras of human lysozyme and α-lactalbumin: an interesting tool for studying partially folded states during protein folding

Protein folding is an extremely active field of research where biology, chemistry, computer science and physics meet. Although the study of protein-folding intermediates in general and equilibrium intermediates in particular has grown considerably in recent years, many questions regarding the conformational state and the structural features of the various partially folded intermediate states remain unanswered. Performing kinetic measurements on proteins that have had their structures modified by site-directed mutagenesis, the so-called protein-engineering method, is an obvious way to gain fine structural information. In the present review, this method has been applied to a variety of proteins belonging to the lysozyme/α-lactalbumin family. Besides recombinants obtained by point mutations of individual critical residues, chimeric proteins in which whole structural elements (10 – 25 residues) from α-lactalbumin were inserted into a human lysozyme matrix are examined. The conformational properties of the equilibrium intermediate states are discussed together with the structural characterization of the partially unfolded states encountered in the kinetic folding pathway. Received 28 May 1998; received after revision 6 July 1998; accepted 6 July 1998     

Germline development in vertebrates and invertebrates

In all animals information is passed from parent to offspring via the germline, which segregates from the soma early in development and undergoes a complex developmental program to give rise to the adult gametes. Many aspects of germline development have been conserved throughout the animal kingdom. Here we review the unique properties of germ cells, the initial determination of germ cell fates, the maintenance of germ cell identity, the migration of germ cells to the somatic gonadal primordia and the proliferation of germ cells during development in vertebrates and invertebrates. Similarities in germline development in such diverse organisms as Drosophila melanogaster, Caenorhabditis elegans, Xenopus laevis and Mus musculus will be highlighted. Received 11 December 1998; received after revision 25 January 1999; accepted 25 January 1999     

Central IL-1 differentially regulates peripheral IL-6 and TNF synthesis

Centrally given interleukin (IL)-1 is known to induce a rapid rises in blood IL-6. To extend this and to examine the mechanism by which this occurs, the effects of intracerebroventricular (icv) injection of human recombinant IL-1β on mRNA expression of IL-6 and tumour necrosis factor (TNF) in the spleen and liver were examined in rats. Icv injection of IL-1 produced a rapid rise of the tissue mRNA levels of IL-6 and TNF in both organs, prior to and/or in parallel with an increase in their serum levels. Pretreatment with chlorisondamine, a ganglionic blocking agent, inhibited the IL-6 responses, while it had little influence on the TNF responses. The results suggest that brain IL-1 induces peripheral production of IL-6, but not of TNF, through autonomic nervous system activation. Received 27 October 1997; received after revision 15 December 1997; accepted 12 January 1998     

Dictyostelium mobile elements: strategies to amplify in a compact genome

Dictyostelium discoideum is a eukaryotic microorganism that is attractive for the study of fundamental biological phenomena such as cell-cell communication, formation of multicellularity, cell differentiation and morphogenesis. Large-scale sequencing of the D. discoideum genome has provided new insights into evolutionary strategies evolved by transposable elements (TEs) to settle in compact microbial genomes and to maintain active populations over evolutionary time. The high gene density (about 1 gene/2.6 kb) of the D. discoideum genome leaves limited space for selfish molecular invaders to move and amplify without causing deleterious mutations that eradicate their host. Targeting of transfer RNA (tRNA) gene loci appears to be a generally successful strategy for TEs residing in compact genomes to insert away from coding regions. In D. discoideum, tRNA gene-targeted retrotransposition has evolved independently at least three times by both non-long termina l repeat (LTR) retrotransposons and retrovirus-like LTR retrotransposons. Unlike the nonspecifically inserting D. discoideum TEs, which have a strong tendency to insert into preexisting TE copies and form large and complex clusters near the ends of chromosomes, the tRNA gene-targeted retrotransposons have managed to occupy 75% of the tRNA gene loci spread on chromosome 2 and represent 80% of the TEs recognized on the assembled central 6.5-Mb part of chromosome 2. In this review we update the available information about D. discoideum TEs which emerges both from previous work and current large-scale genome sequencing, with special emphasis on the fact that tRNA genes are principal determinants of retrotransposon insertions into the D. discoideum genome. Received 10 May 2002; received after revision 10 June 2002; accepted 12 June 2002 RID="*" ID="*"Corresponding author.     

A common ancestor for a subunit in the mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) and short-chain dehydrogenases/reductases

The proton-translocating NADH:ubiquinone oxidoreductase or complex I is located in the inner membranes of mitochondria, where it catalyzes the transfer of electrons from NADH to ubiquinone. Here we report that one of the subunits in complex I is homologous to short-chain dehydrogenases and reductases, a family of enzymes with diverse activities that include metabolizing steroids, prostaglandins and nucleotide sugars. We discovered that a subunit of complex I in human, cow, Neurospora crassa and Aquifex aeolius is homologous to nucleotide-sugar epimerases and hydroxysteroid dehydrogenases while seeking distant homologs of these enzymes with a hidden Markov model-based search of Genpept. This homology allows us to use information from the solved three-dimensional structures of nucleotide-sugar epimerases and hydroxysteroid dehydrogenases and our motif analysis of these enzymes to predict functional domains on their homologs in complex I. Received 26 November 1998; received after revision 12 January 1999; accepted 12 January 1999     

Production of functional rat liver PSP protein in Escherichia coli

An efficient Escherichia coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. Complementary DNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E. coli was expressed by isopropyl 1-thio-β-D-galactopyranoside (IPTG), the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The purified product showed the expected NH₂-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver. Received 8 October 1998; received after revision 6 November 1998; accepted 6 November 1998     

Protein kinase-dependent overexpression of the nuclear protein pirin in c-JUN and RAS transformed fibroblasts

Signalling via the protein kinase Raf-MEK-ERK pathway is of major importance for transformation by oncogenes. To identify genes affected by inhibition of this pathway, c-JUN transformed rat fibroblasts were treated with a MEK1 inhibitor (PD98059) and subjected to two-dimensional gel electrophoresis after cell lysis. Gene products with expression influenced by MEK1 inhibition were determined by mass spectrometry of fragments from in-gel tryptic digestions. The expression of pirin, a nuclear factor I-interacting protein, was lowered after inhibition of MEK1. Western blot analysis revealed increased expression of pirin in RAS and c-JUN transformed cells in the absence of PD98059. Inhibition of MEK1 also led to reduced expression of α-enolase, phosphoglycerate kinase, elongation factor 2 and heterogeneous nuclear ribonucleoprotein A3, the latter two being detected as truncated proteins. In contrast, the level of ornithine aminotransferase was increased. We conclude that inhibition of MEK1 results in major alterations of protein expression in c-JUN transformed cells, suggesting that this pathway is important for oncogene-induced phenotypic changes. Received 30 December 1998; accepted 12 January 1999     

Retroelements and their impact on genome evolution and functioning

Retroelements comprise a considerable fraction of eukaryotic genomes. Since their initial discovery by Barbara McClintock in maize DNA, retroelements have been found in genomes of almost all organisms. First considered as a “junk DNA” or genomic parasites, they were shown to influence genome functioning and to promote genetic innovations. For this reason, they were suggested as an important creative force in the genome evolution and adaptation of an organism to altered environmental conditions. In this review, we summarize the up-to-date knowledge of different ways of retroelement involvement in structural and functional evolution of genes and genomes, as well as the mechanisms generated by cells to control their retrotransposition.     

Long-term changes in tyrosine phosphorylation of the abundant nuclear proteins during granulocytic differentiation of HL-60 cells

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N ⁶,O ²-dibutyryl adenosine 3′5′-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12–72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35–110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation. Analysis of the nuclear proteins differentially extracted by salt and detergents indicates that changes in their tyrosine phosphorylation during the maturation stage of differentiating granulocytes occur mainly in proteins which are abundant in nucleoplasm, chromatin and residual nuclear structures. The abundance of these proteins, residing in the nuclear structures, and their long-term modification in phosphorylation during the maturation stages of differentiation strongly suggest that tyrosine phosphorylation of these proteins is involved in reorganization of the differentiating cell nucleus. Received 21 September 1998; received after revision 24 November 1998; accepted 3 December 1998     

Homing endonucleases: structure, function and evolution

‘Homing’ is the lateral transfer of an intervening genetic sequence, either an intron or an intein, to a cognate allele that lacks that element. The end result of homing is the duplication of the intervening sequence. The process is initiated by site-specific endonucleases that are encoded by open reading frames within the mobile elements. Several features of these proteins make them attractive subjects for structural and functional studies. First, these endonucleases, while unique, may be contrasted with a variety of enzymes involved in nucleic acid strand breakage and rearrangement, particularly restriction endonucleases. Second, because they are encoded within the intervening sequence, there are interesting limitations on the position and length of their open reading frames, and therefore on their structures. Third, these enzymes display a unique strategy of flexible recognition of very long DNA target sites. This strategy allows these sequences to minimize nonspecific cleavage within the host genome, while maximizing the ability of the endonuclease to cleave closely related variants of the homing site. Recent studies explain a great deal about the biochemical and genetic mechanisms of homing, and also about the structure and function of several representative members of the homing endonuclease families. Received 6 January 1999; received after revision 24 February 1999; accepted 24 February 1999     

Screening of conformationally constrained random polypeptide libraries displayed on a protein scaffold

The selection of novel proteins or enzymes from random protein libraries has come to be a major objective in current biology, and these enzymes should prove useful in various biological and biomedical fields. New technologies such as in vitro selection of proteins in cell-free systems have high potential to realize evolu tionary molecular engineering of proteins. This review highlights an application of insertional mutagenesis of proteins to evolutionary molecular engineering. Random sequence proteins are inserted into the surface of a host enzyme which serves as a scaffold to display random protein libraries. Constraints on random polypeptide conformations owing to the proximity of N- and C-termini on the scaffold would result in greater screening efficiency of libraries. The scaffold enzyme is also used as a probe for monitoring the hill climbing of random sequence proteins on a fitness landscape and navigating rapid protein folding in the sequence space. Received 9 October 1997; received after revision 6 January 1998; accepted 19 January 1998     

Stomatal guard cell responses to kinetin and natriuretic peptides are cGMP-dependent

Immunological evidence suggests that plants contain natriuretic peptides (NPs) and furthermore (3- [¹²⁵I]iodotyrosol²⁸) rat atrial NP (rANP) binds specifically to plant membranes. rANP and immunoaffinity-purified plant NP analogues also promote concentration-dependent stomatal opening. Here we report that kinetin, a synthetic cytokinin, and rANP induce stomatal opening in Tradescantia albiflora and that the effect of rANP is critically dependent on the secondary structure of the peptide hormone. The native circular molecule is active, whereas the linearized molecule shows no biological activity. Furthermore, kinetin- and rANP-induced stomatal opening is reversibly inhibited by two in hibitors of guanylate cyclase, LY 83583 and methylene blue. Stomatal opening is also induced in a concentration-dependent manner by the cell-permeant cyclic guanosine-3′,5′-monophosphate (cGMP) analogue 8-Br-cGMP, and this effect is prevented by the stomatal closure promoting plant hormone abscisic acid (ABA). We conclude that in guard cells kinetin and rANP pathways operate via guanylate cyclase upregulation, and we propose that ABA-induced closure is not cGMP-dependent. Received 1 October 1997; received after revision 2 December 1997; accepted 6 January 1998     

G proteins as drug targets

The structure and function of heterotrimeric G protein subunits is known in considerable detail. Upon stimulation of a heptahelical receptor by the appropriate agonists, the cognate G proteins undergo a cycle of activation and deactivation; the α-subunits and the βγ-dimers interact sequentially with several reaction partners (receptor, guanine nucleotides and effectors as well as regulatory proteins) by exposing appropriate binding sites. For most of these domains, low molecular weight ligands have been identified that either activate or inhibit signal transduction. These ligands include short peptides derived from receptors, G protein subunits and effectors, mastoparan and related insect venoms, modified guanine nucleotides, suramin analogues and amphiphilic cations. Because compounds that act on G proteins may be endowed with new forms of selectivity, we propose that G protein subunits may therefore be considered as potential drug targets. Received 18 September 1998; received after revision 6 November 1998; accepted 11 November 1998     

Visual pigment: G-protein-coupled receptor for light signals

The visual pigment present in photoreceptor cells is a prototypical G-protein-coupled receptor (GPCR) that receives a light signal from the outer environment using a light-absorbing chromophore, 11-cis-retinal. Through cis-trans isomerization of the chromophore, light energy is transduced into chemical free energy, which is in turn utilized for conformational changes in the protein to activate the retinal G-protein. In combination with site-directed mutagenesis, various spectroscopic and biochemical studies identified functional residues responsible for chromophore binding, color regulation, intramolecular signal transduction and G-protein coupling. Extensive studies reveal that these residues are localized into specific domains of visual pigments, suggesting a highly manipulated molecular architecture in visual pigments. In addition to the recent findings on dysfunctional mutations in patients with retinitis pigmentosa or congenital night blindness, the mechanism of intramolecular signal transduction in visual pigments and their evolutionary relationship are discussed. Received 20 July 1998; received after revision 9 September 1998; accepted 23 September 1998     

The role of conjugative transposons in the <Emphasis Type="Italic">Enterobacteriaceae</Emphasis>

Although widely studied in Gram-positive Streptococci and in the Gram-negative Bacteroides, there is a scarcity of information on the occurrence and nature of conjugative transposon-like elements in the well-studied Enterobacteriaceae. In fact, some of the major reviews on conjugative transposons prior to 1996 failed to mention their occurrence in this group. Recently, their presence has been reported in Salmonella, Vibrio and Proteus species, and in some cases such as the SXT element in Vibrio and the IncJ group element CTnR391, there has been some molecular characterization. The elements thus far examined appear to be larger than the common Gram-positive conjugative transposons and to be mosaic in structure, with genes derived from several sources. Recent evidence suggests that in the Enterobacteriaceae the elements may be related to enteric pathogenicity islands. The evolution, distribution and role of these elements in the Enterobacteriaceae is discussed.     

Phylogeny and rapid Northern and Southern Hemisphere speciation of goldfinches during the Miocene and Pliocene Epochs

Mitochondrial cytochrome b (cyt b) from 25 out of 31 extant goldfinches, siskins, greenfinches and redpolls (genus Carduelis) has been sequenced from living samples taken around the world, specimens have also been photographed. Phylogenetic analysis consistently gave the same groups of birds, and this grouping was generally related to geographical proximity. It has been supposed that Pleistocene glaciations played a crucial role in the origin of extant diversity and distribution of Northern Hemisphere vertebrates. Molecular comparison of most extant songbird species belonging to the genus Carduelis does not support this assertion. The fossil record of chicken and pheasant divergence time has been used to calibrate the molecular clock; cyt b DNA dendrograms suggest that speciation in Carduelinae birds occurred during the Miocene and Pliocene Epochs (9 – 2 million years ago) in both the Northern and Southern Hemispheres. Only about 4% average amount of nucleotide substitution per lineage is found between the most distant Carduelis species; this suggests a remarkably rapid radiation when compared with the radiation of other passerine songbird genera. In addition, a continuum of small songbird speciation may be found during the Miocene Epoch in parallel with speciation of other orders (i.e. Galliformes, chicken/pheasant). Pleistocene glaciations may have been important in subspeciation (i.e. Eastern European grey-headed goldfinches/Western European black-headed goldfinches) and also in ice-induced vicariance (isolation) (i.e. siskin in Western Europe vs. siskin in Far East Asia) around the world. European isolated Serinus citrinella (citril finch) is not a canary, but a true goldfinch. South American siskins have quickly radiated in the last 4 million years coinciding with the emergence of the Isthmus of Panama; probably, a North American siskin related to C. notata invaded a suitable and varied biotope (the South American island) for Carduelis birds. North American goldfinches may be renamed as siskins, because they have a distant genetic relationship with European goldfinches. Genus Acanthis could be dropped, and thus redpolls should be separated from twite and linnet, the latter (Europeans) probably being related to American goldfinches. Also, reproductive barriers are observed between closely related species and not between other more distant ones. Finally, a tentative classification for genus Carduelis species is suggested. Received 6 March 1998; received after revision 3 July 1998; accepted 7 July 1998     

Some cellular and molecular properties of abscisic acid: its particular involvement in growing plant roots

Several characteristics of the plant hormone abscisic acid (ABA) are critically discussed, more or less directly, in relation to the extension of root cells. A few topics have been selected some biochemical characteristics of ABA (chemical structure, metabolism), inhibiting-β complex, inhibiting regulators from root caps, endogenous ABA in growing roots (ABA gradients, microsurgical experiments, light effects), applied ABA on elongating roots, ABA and indol-3yl acetic acid (IAA) interactions (root growth, proton extrusion, hormone transport, auxin herbicides), ABA effect on the root cell cycle, ABA and drought cells of elongating roots [water deficit conditions, IAA and jasmonic acid (JA) as ‘stress hormones’ other than ABA, gene expression]. Received 28 January 1998; received after revision 20 April 1998; accepted 21 April 1998