Molecular cloning and characterization of three cDNAs encoding putative mitogen-activated protein kinase kinases (MAPKKs) in Arabidopsis thaliana.
(41/52004)
We isolated three Arabidopsis thaliana cDNA clones (ATMKK3, ATMKK4 and ATMKK5) encoding protein kinases with extensive homology to the mitogen-activated protein kinase kinases (MAPKKs) of various organisms in the catalytic domain. ATMKK3 shows high homology (85% identity) to NPK2, a tobacco MAPKK homologue. ATMKK4 and 5 are closely related to each other (84% identity). Phylogenetic analysis showed that the plant MAPKKs constitute at least three subgroups. The recombinant ATMKK3 and ATMKK4 were expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Affinity purified GST-ATMKK3 and GST-ATMKK4 proteins contained phosphorylation activity, which shows that both the ATMKK3 and ATMKK4 genes encode functional protein kinases. Northern blot analysis revealed that the ATMKK3 gene expressed in all the organs. The levels of ATMKK4 and 5 mRNAs were relatively higher in steins and leaves than in flowers and roots. We determined the map positions of the ATMKK3, 4 and 5 genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones. (+info)
Characterization of a Caenorhabditis elegans recA-like gene Ce-rdh-1 involved in meiotic recombination.
(42/52004)
A recA-like gene was identified in the Caenorhabditis elegans genome project database. The putative product of the gene, termed Ce-rdh-1 (C. elegans RAD51 and DMC1/LIM15 homolog 1), consists of 357 amino acid residues. The predicted amino acid sequence of Ce-rdh-1 showed 46-60% identity to both RAD51 type and DMC1/LIM15 type genes in several eukaryote species. The results of RNAi (RNA-mediated interference) indicated that repression of Ce-rdh-1 blocked chromosome condensation of six bivalents and dissociation of chiasmata in oocytes of F1 progeny. Oogenesis did not proceed to the diakinesis stage. Accordingly, all the eggs produced (F2) died in early stages. These results suggest that Ce-rdh-1 participates in meiotic recombination. (+info)
Unusual ribulose 1,5-bisphosphate carboxylase/oxygenase of anoxic Archaea.
(43/52004)
The predominant pool of organic matter on earth is derived from the biological reduction and assimilation of carbon dioxide gas, catalyzed primarily by the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). By virtue of its capacity to use molecular oxygen as an alternative and competing gaseous substrate, the catalytic efficiency of RubisCO and the enzyme's ability to assimilate CO2 may be severely limited, with consequent environmental and agricultural effects. Recent genomic sequencing projects, however, have identified putative RubisCO genes from anoxic Archaea. In the present study, these potential RubisCO sequences, from Methanococcus jannaschii and Archaeoglobus fulgidus, were analyzed in order to ascertain whether such sequences might encode functional proteins. We also report the isolation and properties of recombinant RubisCO using sequences obtained from the obligately anaerobic hyperthermophilic methanogen M. jannaschii. This is the first description of an archaeal RubisCO sequence; this study also represents the initial characterization of a RubisCO molecule that has evolved in the absence of molecular oxygen. The enzyme was shown to be a homodimer whose deduced sequence, along with other recently obtained archaeal RubisCO sequences, differs substantially from those of known RubisCO molecules. The recombinant M. jannaschii enzyme has a somewhat low, but reasonable kcat, however, unlike previously isolated RubisCO molecules, this enzyme is very oxygen sensitive yet it is stable to hyperthermal temperatures and catalyzes the formation of the expected carboxylation product. Despite inhibition by oxygen, this unusual RubisCO still catalyzes a weak yet demonstrable oxygenase activity, with perhaps the lowest capacity for CO2/O2 discrimination ever encountered for any RubisCO. (+info)
alphaSU2, an epithelial integrin that binds laminin in the sea urchin embryo.
(44/52004)
At gastrulation in the sea urchin embryo dramatic cell adhesion changes contribute to primary mesenchyme cell ingression movements and to cell rearrangements during archenteron invagination. At ingression, quantitative adhesion assays demonstrated previously that primary mesenchyme cells (PMCs) change their affinity for neighboring cells, for a fibronectin-like substrate, and for the hyaline layer. To investigate the molecular basis for these and other differential cell affinities at gastrulation, we have identified an integrin that appears to be responsible for specific alterations in cell-substrate adhesion to laminin. During early cleavage stages blastomeres adhere poorly to laminin substrates. Around hatching there is a large increase in the ability of blastomeres to bind to laminin and this increase correlates temporally with the expression of an integrin on the basal surface all blastomeres. PMCs, after undergoing their epithelial-mesenchymal transition, have a strongly reduced affinity for laminin relative to ectoderm cells and, correspondingly, do not stain for the presence of the integrin. We identified the alpha integrin cDNA from Lytechinus variegatus by RT-PCR. Overlapping clones were obtained from a midgastrula cDNA library to provide a complete sequence for the integrin. The composite cDNA encoded a protein that was most similar to the alpha5 subgroup of vertebrate integrins, but there was not a definitive vertebrate integrin homolog. Northern blots and Western immunoblots showed that the sea urchin integrin, which we have named alphaSU2, is present in eggs and during all stages of development. Immunolocalization with specific polyclonal antibodies showed that alphaSU2 first appears on the basal cell surface of epithelia at the midblastula stage, at a time correlating with the increase in adhesive affinity for laminin. The protein remains at high levels on the basal surface of ectoderm cells but is temporarily reduced or eliminated from endoderm cells during their convergent-extension movements. To confirm integrin binding specificity, alphaSU2 was transfected into an alpha-integrin-deficient CHO cell line. alphaSU2-expressing CHO cells bound well to isolated sea urchin basal lamina and to purified laminin. The transfected cells bound weakly or not at all to fibronectin, type I collagen, and type IV collagen. This is consistent with the hypothesis that alphaSU2 integrin functions by binding epithelial cells to laminin in the basal lamina. In vivo, modulation of alphaSU2 integrin expression correlates with critical adhesive changes during cleavage and gastrulation. Thus, this protein appears to be an important contributor to the morphogenetic rearrangements that characterize gastrulation in the sea urchin embryo. (+info)
Otx expression during lamprey embryogenesis provides insights into the evolution of the vertebrate head and jaw.
(45/52004)
Agnathan or jawless vertebrates, such as lampreys, occupy a critical phylogenetic position between the gnathostome or jawed vertebrates and the cephalochordates, represented by amphioxus. In order to gain insight into the evolution of the vertebrate head, we have cloned and characterized a homolog of the head-specific gene Otx from the lamprey Petromyzon marinus. This lamprey Otx gene is a clear phylogenetic outgroup to both the gnathostome Otx1 and Otx2 genes. Like its gnathostome counterparts, lamprey Otx is expressed throughout the presumptive forebrain and midbrain. Together, these results indicate that the divergence of Otx1 and Otx2 took place after the gnathostome/agnathan divergence and does not correlate with the origin of the vertebrate brain. Intriguingly, Otx is also expressed in the cephalic neural crest cells as well as mesenchymal and endodermal components of the first pharyngeal arch in lampreys, providing molecular evidence of homology with the gnathostome mandibular arch and insights into the evolution of the gnathostome jaw. (+info)
Isolation and characterization of drosocrystallin, a lens crystallin gene of Drosophila melanogaster.
(46/52004)
We have cloned the drosocrystallin gene (dcy) of Drosophila melanogaster, which encodes a major protein of the corneal lens, previously described in part by Komori et al. (1992, J. Cell Sci. 102, 191-201). Synthesis of the DCY protein starts weakly in 2-day-old pupae, reaches a peak at day 3 and day 4 of pupal development, and decreases very fast in young adults. The dcy mRNA is detected in the compound eyes as well as in the ocelli. The presence of a putative signal peptide and the extracellular location of DCY suggest that DCY is a secreted protein. Interestingly, the dcy gene shows sequence similarities to some insect cuticular proteins and is detected as well in two closely related Drosophila species, D. sechellia and D. simulans, and in one more distantly related species, D. virilis. This finding supports the hypothesis that Drosophila used the same strategy as vertebrates and mollusks, namely, recruiting a multifunctional protein for refraction in the lens, by a gene-sharing mechanism. Furthermore, it supports our intercalary evolution hypothesis, which suggests that the development of an elaborate structure (for example, a compound eye) from an original primitive form (an ancestral photoreceptor organ) can be achieved by recruiting novel genes into the original developmental pathway. (+info)
Molecular characterization of the genes pcaG and pcaH, encoding protocatechuate 3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp. strain HR199.
(47/52004)
Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional beta subunit of the protocatechuate 3, 4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the ortho cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed for cis, cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through the ortho-cleavage pathway in Pseudomonas sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants. (+info)
Anaerobic oxidation of o-xylene, m-xylene, and homologous alkylbenzenes by new types of sulfate-reducing bacteria.
(48/52004)
Various alkylbenzenes were depleted during growth of an anaerobic, sulfate-reducing enrichment culture with crude oil as the only source of organic substrates. From this culture, two new types of mesophilic, rod-shaped sulfate-reducing bacteria, strains oXyS1 and mXyS1, were isolated with o-xylene and m-xylene, respectively, as organic substrates. Sequence analyses of 16S rRNA genes revealed that the isolates affiliated with known completely oxidizing sulfate-reducing bacteria of the delta subclass of the class Proteobacteria. Strain oXyS1 showed the highest similarities to Desulfobacterium cetonicum and Desulfosarcina variabilis (similarity values, 98.4 and 98.7%, respectively). Strain mXyS1 was less closely related to known species, the closest relative being Desulfococcus multivorans (similarity value, 86.9%). Complete mineralization of o-xylene and m-xylene was demonstrated in quantitative growth experiments. Strain oXyS1 was able to utilize toluene, o-ethyltoluene, benzoate, and o-methylbenzoate in addition to o-xylene. Strain mXyS1 oxidized toluene, m-ethyltoluene, m-isoproyltoluene, benzoate, and m-methylbenzoate in addition to m-xylene. Strain oXyS1 did not utilize m-alkyltoluenes, whereas strain mXyS1 did not utilize o-alkyltoluenes. Like the enrichment culture, both isolates grew anaerobically on crude oil with concomitant reduction of sulfate to sulfide. (+info)