conserved sequence

Transcription from 54 -dependent bacterial promoters can be regarded as a second paradigm for bacterial gene transcription. The initial 54 -RNA polymerase (RNAP)⅐promoter complex, the closed complex, is transcriptionally silent. The transcriptionally proficient 54 -RNAP⅐promoter complex, the open complex, is formed upon remodeling of the closed complex by actions of a specialized activator protein that belongs to the AAA (ATPases associated with various cellular activities) protein family in an ATP hydrolysis-dependent reaction. The integrity of a highly conserved signature motif in the AAA activator (known as the GAFTGA motif) is important for the remodeling activity of the AAA activator and for open complex formation. We now provide evidence that the invariant threonine residue of the GAFTGA motif plays a role in sensing the DNA downstream of the 54 -RNAP-binding site and in cou- pling this information to 54 -RNAP via the conserved regula- tory Region I domain of 54 during open complex formation. * This work was supported in part by the Biotechnology and Biological Sciences Research Council (to M. B.) and the Direccio ´n General de Asuntos del Personal Acade ´mico (DGAPA) (to E. M.

The dinoflagellates have repeatedly replaced their ancestral peridinin-plastid by plastids derived from a variety of algal lineages ranging from green algae to diatoms. Here, we have characterized the genome of a dinoflagellate plastid of tertiary origin in order to understand the evolutionary processes that have shaped the organelle since it was acquired as a symbiont cell. To address this, the genome of the haptophyte-derived plastid in Karlodinium veneficum was analyzed by Sanger sequencing of library clones and 454 pyrosequencing of plastid enriched DNA fractions. The sequences were assembled into a single contig of 143 kb, encoding 70 proteins, 3 rRNAs and a nearly full set of tRNAs. Comparative genomics revealed massive rearrangements and gene losses compared to the haptophyte plastid; only a small fraction of the gene clusters usually found in haptophytes as well as other types of plastids are present in K. veneficum. Despite the reduced number of genes, the K. veneficum plastid genome has retained a large size due to expanded intergenic regions. Some of the plastid genes are highly diverged and may be pseudogenes or subject to RNA editing. Gene losses and rearrangements are also features of the genomes of the peridinin-containing plastids, apicomplexa and Chromera, suggesting that the evolutionary processes that once shaped these plastids have occurred at multiple independent occasions over the history of the Alveolata.

Haemophilus ducreyi is an extracellular pathogen of human epithelial surfaces that resists human antimicrobial peptides (APs). The organism's genome contains homologs of genes s ensitive to a ntimicrobial p eptides ( sap operon) in nontypeable Haemophilus influenzae . In this study, we characterized the sap -containing loci of H. ducreyi 35000HP and demonstrated that sapA is expressed in broth cultures and H. ducreyi -infected tissue; sapA is also conserved among both class I and class II H. ducreyi strains. We constructed a nonpolar sapA mutant of H. ducreyi 35000HP, designated 35000HP sapA , and compared the percent survival of wild-type 35000HP and 35000HP sapA exposed to several human APs, including α-defensins, β-defensins, and the cathelicidin LL-37. Unlike an H. influenzae sapA mutant, strain 35000HP sapA was not more susceptible to defensins than strain 35000HP was. However, we observed a significant decrease in the survival of strain 35000HP sapA after exposure to LL-37...

The polarity of residues at certain positions in the transmembrane domains of G-protein coupled receptors (GPCR) is found to be conserved, and to indicate the pattern of specific helix-helix packing of the helices. A concept of polarity conserved positions (PCP) is proposed to describe this conserved property, and is applied to obtain insight into the structural features of the transmembrane proteins. The common pattern of PCPs for GPCRs indicates that they share a similar packing arrangement of their transmembrane helix bundles. For proteins in the bacteriorhodopsin family the PCP pattern suggests a common packing arrangement that differs from that of GPCRs, in agreement with experimental data. This difference in the packing arrangement underscores the shortcomings of a BR template for the construction of molecular models of GPCRs.

Despite the critical importance of Ca2+/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1α and CL1β were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2α and CL2β contained a full N-terminal Dx, whereas CLr only posse...

Sex determination has long intrigued evolutionists, geneticists, and developmental biologists in a similar way. Substantial evidence indicates that sex determination evolves rapidly and, therefore, can be used to study how molecular patterning processes evolve. In Caenorhabditis elegans, sex determination relies on a signaling pathway that involves a cascade of negatively acting factors, finally triggering the GLI-family zinc-finger transcription factor TRA-1. We have started to investigate sex determination in the nematode satellite species Pristionchus pacificus that is separated from C. elegans for 200-300 million years. In P. pacificus, animals with two X chromosomes develop as hermaphrodites, whereas XO animals develop as males. We used an unbiased forward genetic approach and isolated several mutants with a hermaphrodite to male transformation of the XX karyotype. We identified one complementation group as representing the P. pacificus ortholog of tra-1, providing the first evidence for the conservation of a global sex determination gene over a time period of at least 200 million years. A Ppa-tra-1 morpholino phenocopies Ppa-tra-1 mutants and establishes the morpholino technology as a reverse genetic approach in P. pacificus.

Adaptor protein-2 (AP2), a central component of clathrin-coated vesicles (CCVs), is pivotal in clathrin-mediated endocytosis which internalises plasma membrane constituents such as G protein-coupled receptors (GPCRs) 1-3 . AP2, a heterotetramer of alpha, beta, mu and sigma subunits, links clathrin to vesicle membranes and binds to tyrosine-based and dileucine-based motifs of membrane-associated cargo proteins . Here, we show that AP2 sigma subunit (AP2S1) missense mutations, which all involved the Arg15 residue (Arg15Cys, Arg15His and Arg15Leu) that forms key contacts with dileucine-based motifs of CCV cargo proteins 4 , result in familial hypocalciuric hypercalcemia type 3 (FHH3), an extracellular-calcium homeostasis disorder

The ciliate Tetrahymena thermophila incorporates sterols from its environment that desaturates at positions C5(6), C7(8), and C22 (23). Phytosterols are additionally modified by removal of the ethyl group at carbon 24 (C24). The enzymes involved are oxygen-, NAD(P)H-, and cytochrome b 5 dependent, reason why they were classified as members of the hydroxylases/desaturases superfamily. The ciliate's genome revealed the presence of seven putative sterol desaturases belonging to this family, two of which we have previously characterized as the C24-de-ethylase and C5(6)-desaturase. A Rieske oxygenase was also identified; this type of enzyme, with sterol C7(8)-desaturase activity, was observed only in animals, called Neverland in insects and DAF-36 in nematodes. They perform the conversion of cholesterol into 7-dehydrocholesterol, first step in the synthesis of the essential hormones ecdysteroids and dafachronic acids. By adapting an RNA interference-by-feeding protocol, we easily screened six of the eight genes described earlier, allowing the characterization of the Rieske-like oxygenase as the ciliate's C7(8)-desaturase (Des7p). This characterization was confirmed by obtaining the corresponding knockout mutant, making Des7p the first nonanimal Rieske-sterol desaturase described. To our knowledge, this is the first time that the feeding-RNAi technique was successfully applied in T. thermophila, enabling to consider such methodology for future reverse genetics high-throughput screenings in this ciliate. Bioinformatics analyses revealed the presence of Des7p orthologs in other Oligohymenophorean ciliates and in nonanimal Opisthokonts, like the protists Salpingoeca rosetta and Capsaspora owczarzaki. A horizontal gene transfer event from a unicellular Opisthokont to an ancient phagotrophic Oligohymenophorean could explain the acquisition of the Rieske oxygenase by Tetrahymena.

The ciliate Tetrahymena thermophila incorporates sterols from its environment that desaturates at positions C5(6), C7(8), and C22 (23). Phytosterols are additionally modified by removal of the ethyl group at carbon 24 (C24). The enzymes involved are oxygen-, NAD(P)H-, and cytochrome b 5 dependent, reason why they were classified as members of the hydroxylases/desaturases superfamily. The ciliate's genome revealed the presence of seven putative sterol desaturases belonging to this family, two of which we have previously characterized as the C24-de-ethylase and C5(6)-desaturase. A Rieske oxygenase was also identified; this type of enzyme, with sterol C7(8)-desaturase activity, was observed only in animals, called Neverland in insects and DAF-36 in nematodes. They perform the conversion of cholesterol into 7-dehydrocholesterol, first step in the synthesis of the essential hormones ecdysteroids and dafachronic acids. By adapting an RNA interference-by-feeding protocol, we easily screened six of the eight genes described earlier, allowing the characterization of the Rieske-like oxygenase as the ciliate's C7(8)-desaturase (Des7p). This characterization was confirmed by obtaining the corresponding knockout mutant, making Des7p the first nonanimal Rieske-sterol desaturase described. To our knowledge, this is the first time that the feeding-RNAi technique was successfully applied in T. thermophila, enabling to consider such methodology for future reverse genetics high-throughput screenings in this ciliate. Bioinformatics analyses revealed the presence of Des7p orthologs in other Oligohymenophorean ciliates and in nonanimal Opisthokonts, like the protists Salpingoeca rosetta and Capsaspora owczarzaki. A horizontal gene transfer event from a unicellular Opisthokont to an ancient phagotrophic Oligohymenophorean could explain the acquisition of the Rieske oxygenase by Tetrahymena.

We have cloned and expressed a mouse brain cDNA, mShal, that encodes a transient, A-type K+ current. mShal, the vertebrate homolog of the Drosophila Shal gene, defines a distinct subfamily of voltage-gated K+ channels. The Shal deduced proteins are more highly conserved between mouse and Drosophila than other presently known K+ chan- nels. mShal carries a "low-threshold" A-type current with a hyperpolarized steady-state inactivation midpoint. Marked similarity was observed between mShal and its Drosophila homolog, fShal, with regard to voltage sensitivity of activation, macroscopic inactivation, steady-state inactivation, and 4-ami- nopyridine sensitivity. Sequence conservation for Shal proteins is unusually hligh at the amino terminus, an area considered important for inactivation. Removal of conserved amino- terminal residues from mShal modifies macroscopic inactivation but the transient nature of the current is preserved.

The accurate detection of genes and the identification of functional regions is still an open issue in the annotation of genomic sequences. This problem affects new genomes but also those of very well studied organisms such as human and mouse where, despite the great efforts, the inventory of genes and regulatory regions is far from complete. Comparative genomics is an effective approach to address this problem. Unfortunately it is limited by the computational requirements needed to perform genome-wide comparisons and by the problem of discriminating between conserved coding and non-coding sequences. This discrimination is often based (thus dependent) on the availability of annotated proteins. In this paper we present the results of a comprehensive comparison of human and mouse genomes performed with a new high throughput grid-based system which allows the rapid detection of conserved sequences and accurate assessment of their coding potential. By detecting clusters of coding conserved sequences the system is also suitable to accurately identify potential gene loci. Following this analysis we created a collection of human-mouse conserved sequence tags and carefully compared our results to reliable annotations in order to benchmark the reliability of our classifications. Strikingly we were able to detect several potential gene loci supported by EST sequences but not corresponding to as yet annotated genes. Here we present a new system which allows comprehensive comparison of genomes to detect conserved coding and non-coding sequences and the identification of potential gene loci. Our system does not require the availability of any annotated sequence thus is suitable for the analysis of new or poorly annotated genomes.

The Msn2/4 transcription factors play crucial roles in the yeast general stress response. Previous studies identified several large functional domains of Msn2 mainly through crude truncations. Here, employing bioinformatics and experimental approaches to examine Msn2 structure-function relationships, we have identified new functional motifs in the Msn2 transcriptional-activating domain (TAD). We found that Msn2 is predicted to adopt an intrinsically disordered structure with two short structural motifs in its TAD. Mutations in these motifs dramatically decreased Msn2 transcriptional activity, yeast stress survival and Msn2 nuclear localization levels. Using the split ubiquitin assay, we found that these motifs are important for the interaction of Msn2 with Gal11, a subunit of the mediator complex. Finally, we show that one of these motifs is functionally conserved in several yeast species, highlighting a common mechanism of Msn2 transcriptional activation throughout yeast evolution.

Fanconi anemia (FA) is a rare recessive disease resulting from mutations in one of at least 16 different genes. Mutation types and phenotypic manifestations of FA are highly heterogeneous and influence the clinical management of the disease. We analyzed 202 FA families for large deletions, using high-resolution comparative genome hybridization arrays, single-nucleotide polymorphism arrays, and DNA sequencing. We found pathogenic deletions in 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. We find 35% of FA families carry large deletions, accounting for 18% of all FA pathogenic variants. Cloning and sequencing across the deletion breakpoints revealed that 52 FANCA deletion ends, and one FANCC deletion end extended beyond the gene boundaries, potentially affecting neighboring genes with phenotypic consequences. Seventy-five percent of the FANCA deletions are Alu-Alu mediated, predominantly by AluY elements, and appear to be caused by nonallelic homologous recombination. Ind...

The terminal DNA restriction fragments (PstI-D and -B) of Pseudomonas aeruginosa bacteriophage D3 were ligated, cloned, and sequenced. Of the nine open reading frames in this 8.3-kb fragment, four were identified as encoding large-subunit terminase, portal, ClpP protease, and major head proteins. The portal and capsid proteins showed significant homology with proteins of the lambdoid coliphage HK97. Phage D3 was purified by CsCl equilibrium gradient centrifugation ( ‫؍‬ 1.533 g/ml), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed six proteins with molecular masses of 186, 91, 79, 70, 45, and 32 kDa. The pattern was unusual, since a major band corresponding to the expected head protein (43 kDa) was missing and a significant amount of the protein was retained in the stacking gel. The amino terminus of the 186-kDa protein was sequenced, revealing that the D3 head is composed of cross-linked 31-kDa protein subunits, resulting from the proteolysis of the 43-kDa precursor. This is identical to the situation observed with coliphage HK97.

The terminal DNA restriction fragments ( Pst I-D and -B) of Pseudomonas aeruginosa bacteriophage D3 were ligated, cloned, and sequenced. Of the nine open reading frames in this 8.3-kb fragment, four were identified as encoding large-subunit terminase, portal, ClpP protease, and major head proteins. The portal and capsid proteins showed significant homology with proteins of the lambdoid coliphage HK97. Phage D3 was purified by CsCl equilibrium gradient centrifugation (ρ = 1.533 g/ml), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed six proteins with molecular masses of 186, 91, 79, 70, 45, and 32 kDa. The pattern was unusual, since a major band corresponding to the expected head protein (43 kDa) was missing and a significant amount of the protein was retained in the stacking gel. The amino terminus of the 186-kDa protein was sequenced, revealing that the D3 head is composed of cross-linked 31-kDa protein subunits, resulting from the proteolysis of the 43-kDa pr...