Direct evidence for secondary loss of mitochondria in Entamoeba histolytica.

Archezoan protists are though to represent lineages that diverged from other eukaryotes before acquisition of the mitochondrion and other organelles. The parasite Entamoeba histolytica was originally included in this group. Ribosomal RNA based phylogenies, however, place E. histolytica on a comparatively recent branch of the eukaryotic tree, implying that its ancestors had these structures. In this study, direct evidence for secondary loss of mitochondrial function was obtained by isolating two E. histolytica genes encoding proteins that in other eukaryotes are localized in the mitochondrion: the enzyme pyridine nucleotide transhydrogenase and the chaperonin cpn60. Phylogenetic analysis of the E. histolytica homolog of cpn60 confirmed that it is specifically related to the mitochondrial lineage. The data suggest that a mitochondrial relic may persist in this organism. Similar studies are needed in archezoan protists to ascertain which, if any, eukaryotic lineages primitively lack mitochondria.

PXA1, a possible Saccharomyces cerevisiae ortholog of the human adrenoleukodystrophy gene.

The adrenoleukodystrophy protein (ALDp) is an ATP-binding cassette (ABC) transporter in the human peroxisome membrane. It is defective in X chromosome-linked adrenoleukodystrophy (ALD), a neurodegenerative disorder with impaired peroxisomal oxidation of very long chain fatty acids. We report cloning and characterization of PXA1, a yeast gene encoding a protein (Pxa1p) exhibiting high similarity to ALDp. Disruption of PXA1 results in impaired growth on oleic acid and reduced ability to oxidize oleate. Pxa1p is peroxisome associated; however, in the PXA1 mutant yeast, as in ALD cells, peroxisomes are morphologically intact. Disruption of a second yeast gene, YKL741, which encodes a more distantly related ALDp homolog (Yk174p), in either wild-type or PXA1 mutant yeast, results in a growth phenotype identical to that of the PXA1 mutant. This result suggests that Yk1741p and Pxa1p may be subunits of the same transporter. Sequence analysis of Pxa1p, ALDp, and related ABC transporters reveals a possible fatty acid binding domain and a 14-amino acid EAA-like motif, previously described only in prokaryotes. Because of the similarities in sequence and function, we propose that Pxa1p is the Saccharomyces cerevisiae ortholog of ALDp.

Isolation of a yeast protein kinase that is activated by the protein encoded by SRP1 (Srp1p) and phosphorylates Srp1p complexed with nuclear localization signal peptides.

Srp1p, the protein encoded by SRP1 of Saccharomyces cerevisiae, is a nuclear-pore-associated protein. Its Xenopus homolog, importin, was recently shown to be an essential component required for nuclear localization signal (NLS)-dependent binding of karyophilic proteins to the nuclear envelope [Gorlich, D., Prehn, S., Laskey, R. A. & Hartman, E. (1994) Cell 79, 767-778]. We have discovered a protein kinase whose activity is stimulated by Srp1p (Srp1p fused to glutathione S-transferase and expressed in Escherichia coli) and is detected by phosphorylation of Srp1p and of a 36-kDa protein, a component of the protein kinase complex. The enzyme, called Srp1p kinase, is a protein-serine kinase and was found in extracts in two related complexes of approximately 180 kDa and 220 kDa. The second complex, when purified, contained four protein components including the 36-kDa protein. We observed that, upon purification of the kinase, phosphorylation of Srp1p became very weak, while activation of phosphorylation of the 36-kDa protein by Srp1p remained unaltered. Significantly, NLS peptides and the nuclear proteins we have tested greatly stimulated phosphorylation of Srp1p, suggesting that Srp1p, complexed with karyophilic proteins carrying an NLS, is the in vivo substrate of this protein kinase.

The ethylene hormone response in Arabidopsis: a eukaryotic two-component signaling system.

The simple gas ethylene affects numerous physiological processes in the growth and development of higher plants. With the use of molecular genetic approaches, we are beginning to learn how plants perceive ethylene and how this signal is transduced. Components of ethylene signal transduction are defined by ethylene response mutants in Arabidopsis thaliana. The genes corresponding to two of these mutants, etr1 and etr1, have been cloned. The ETR1 gene encodes a homolog of two-component regulators that are known almost exclusively in prokaryotes. The two-component regulators in prokaryotes are involved in the perception and transduction of a wide range of environmental signals leading to adaptive responses. The CTR1 gene encodes a homolog of the Raf family of serine/threonine protein kinases. Raf is part of a mitogen-activated protein kinase cascade known to regulate cell growth and development in mammals, worms, and flies. The ethylene response pathway may, therefore, exemplify a conserved protein kinase cascade regulated by a two-component system. The dominance of all known mutant alleles of ETR1 may be due to either constitutive activation of the ETR1 protein or dominant interference of wild-type activity. The discovery of Arabidopsis genes encoding proteins related to ETR1 suggests that the failure to recover recessive etr1 mutant alleles may be due to the presence of redundant genes.

Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.

An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive plaques. These clones were classified into 40 groups by hybridization analysis and 5'- and 3'-terminal sequencing. By searching nucleic acid and protein sequence data bases, 11 groups of cDNAs were identified, among which valosin-containing protein (VCP), clathrin heavy chain, phospholipase C, and S-adenosylmethionine:delta 24-sterol-C-methyltransferase have not to date been cloned from plants. The remaining 29 groups did not match any current data base entries and may, therefore, represent additional or yet uncharacterized genes. A full-length cDNA encoding the soybean VCP was sequenced. The high level of amino acid identity with vertebrate VCP and yeast CDC48 protein indicates that the soybean protein is a plant homolog of vertebrate VCP and yeast CDC48 protein.

Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast.

The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UME5. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL1 promoter.

Déficience dans la réparation par excision de nucléotides en phase S induite par la séquestration du facteur de réplication RPA

Introduction Les lésions induites par les rayons UV peuvent causer des blocages dans la réplication de l'ADN. Ces dommages sont éliminés par le processus moléculaire très conservé de réparation par excision de nucléotides (NER). Nous avons précédemment démontré que la protéine ATR, une kinase majeure impliquée dans le stress réplicatif, est requise pour une NER efficace, et ce exclusivement durant la phase S. Des résultats subséquents ont suggéré que ce prérequis n’était pas lié à la réponse induite par ATR, mais plutôt d’une conséquence globale causée par la présence de stress réplicatif. En ce sens, nous mettons l’emphase qu’après irradiation UV, le complexe RPA joue un rôle crucial dans l'activation des mécanismes de NER ainsi que dans le redémarrage des fourches de réplication bloquées. Hypothèses: En général, les mutations qui confèrent une augmentation du stress réplicatif engendrent une séquestration excessive du facteur RPA aux fourches de réplication bloquées ce qui réduit son accessibilité pour le NER. Méthodes et résultats: Le modèle de la levure a été choisi pour vérifier cette hypothèse. Nous avons développé un essai de NER spécifique à chacune des phases du cycle cellulaire pour démontrer que les cellules déficientes en Mec1, l’homologue d’ATR, sont défectives dans la réparation par excision de nucléotides spécifiquement en phase S. De plus, plusieurs autres mutants de levure, caractérisés par un niveau de dommages spontanés élevé, ont aussi exhibé un défaut similaire. Ces mutants ont démontré une fréquence et une intensité de formation de foyers de RPA plus élevée. Finalement, une diminution partielle de RPA dans les levures a induit un défaut significatif dans le NER spécifiquement durant la phase S. Conclusion: Nos résultats supportent la notion que la séquestration de RPA aux fourches de réplication endommagées durant la phase S prévient son utilisation pour la réparation par excision de nucléotides ce qui inhibe fortement l'efficacité de réparation. Cette étude chez la levure facilite l’élucidation du phénomène analogue chez l’humain et, ultimement, comprend des implications majeures dans la compréhension du mécanisme de développement des cancers UV-dépendants.

Expression at the Imprinted Dlk1-Gtl2 Locus Is Regulated by Proneural Genes in the Developing Telencephalon

Imprinting is an epigenetic mechanism that restrains the expression of about 100 genes to one allele depending on its parental origin. Several imprinted genes are implicated in neurodevelopmental brain disorders, such as autism, Angelman, and Prader-Willi syndromes. However, how expression of these imprinted genes is regulated during neural development is poorly understood. Here, using single and double KO animals for the transcription factors Neurogenin2 (Ngn2) and Achaete-scute homolog 1 (Ascl1), we found that the expression of a specific subset of imprinted genes is controlled by these proneural genes. Using in situ hybridization and quantitative PCR, we determined that five imprinted transcripts situated at the Dlk1-Gtl2 locus (Dlk1, Gtl2, Mirg, Rian, Rtl1) are upregulated in the dorsal telencephalon of Ngn2 KO mice. This suggests that Ngn2 influences the expression of the entire Dlk1-Gtl2 locus, independently of the parental origin of the transcripts. Interestingly 14 other imprinted genes situated at other imprinted loci were not affected by the loss of Ngn2. Finally, using Ngn2/Ascl1 double KO mice, we show that the upregulation of genes at the Dlk1-Gtl2 locus in Ngn2 KO animals requires a functional copy of Ascl1. Our data suggest a complex interplay between proneural genes in the developing forebrain that control the level of expression at the imprinted Dlk1-Gtl2 locus (but not of other imprinted genes). This raises the possibility that the transcripts of this selective locus participate in the biological effects of proneural genes in the developing telencephalon.

Transient Activation of Meox1 Is an Early Component of the Gene Regulatory Network Downstream of Hoxa2

Hox genes encode transcription factors that regulate morphogenesis in all animals with bilateral symmetry. Although Hox genes have been extensively studied, their molecular function is not clear in vertebrates, and only a limited number of genes regulated by Hox transcription factors have been identified. Hoxa2 is required for correct development of the second branchial arch, its major domain of expression. We now show that Meox1 is genetically downstream from Hoxa2 and is a direct target. Meox1 expression is downregulated in the second arch of Hoxa2 mouse mutant embryos. In chromatin immunoprecipitation (ChIP), Hoxa2 binds to the Meox1 proximal promoter. Two highly conserved binding sites contained in this sequence are required for Hoxa2-dependent activation of the Meox1 promoter. Remarkably, in the absence of Meox1 and its close homolog Meox2, the second branchial arch develops abnormally and two of the three skeletal elements patterned by Hoxa2 are malformed. Finally, we show that Meox1 can specifically bind the DNA sequences recognized by Hoxa2 on its functional target genes. These results provide new insight into the Hoxa2 regulatory network that controls branchial arch identity.

Mechanisms of Thermal Adaptation Revealed From the Genomes of the Antarctic Archaea Methanogenium frigidum and Methanococcoides burtonii

We generated draft genome sequences for two cold-adapted Archaea, Methanogenium frigidum and Methanococcoides burtonii, to identify genotypic characteristics that distinguish them from Archaea with a higher optimal growth temperature (OGT). Comparative genomics revealed trends in amino acid and tRNA composition, and structural features of proteins. Proteins from the cold-adapted Archaea are characterized by a higher content of noncharged polar amino acids, particularly Gin and Thr and a lower content of hydrophobic amino acids, particularly Leu. Sequence data from nine methanogen genomes (OGT 15degrees-98degreesC) were used to generate IIII modeled protein structures. Analysis of the models from the cold-adapted Archaea showed a strong tendency in the solvent-accessible area for more Gin, Thr, and hydrophobic residues and fewer charged residues. A cold shock domain (CSD) protein (CspA homolog) was identified in M. frigidum, two hypothetical proteins with CSD-folds in M. burtonii, and a unique winged helix DNA-binding domain protein in M. burtonii. This suggests that these types of nucleic acid binding proteins have a critical role in cold-adapted Archaea. Structural analysis of tRNA sequences from the Archaea indicated that GC content is the major factor influencing tRNA stability in hyperthermophiles, but not in the psychrophiles, mesophiles or moderate thermophiles. Below an OGT of 60degreesC, the GC content in tRNA was largely unchanged, indicating that any requirement for flexibility of tRNA in psychrophiles is mediated by other means. This is the first time that comparisons have been performed with genome data from Archaea spanning the growth temperature extremes. from psychrophiles to hyperthermophiles

The 2.2 A crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation

Reef-building corals contain host pigments, termed pocilloporins, that function to regulate the light environment of their resident microalgae by acting as a photoprotectant in excessive sunlight. We have determined the crystal structure of an intensely blue, non-fluorescent pocilloporin to 2.2 Angstrom resolution and a genetically engineered fluorescent variant to 2.4 Angstrom resolution. The pocilloporin chromophore structure adopts a markedly different conformation in comparison with the DsRed chromophore, despite the chromophore sequences (Gin-Tyr-Gly) being identical; the tyrosine ring of the pocilloporin chromophore is noncoplanar and in the trans configuration. Furthermore, the fluorescent variant adopted a noncoplanar chromophore conformation. The data presented here demonstrates that the conformation of the chromophore is highly dependent on its immediate environment.

Characterizing embryonic gene expression patterns in the mouse using nonredundant sequence-based selection

This article investigates the expression patterns of 160 genes that are expressed during early mouse development. The cDNAs were isolated from 7.5 d postcoitum (dpc) encloderm, a region that comprises visceral encloderm (VE), definitive encloderm, and the node-tissues that are required for the initial steps of axial specification and tissue patterning in the mouse. To avoid examining the same gene more than once, and to exclude potentially ubiquitously expressed housekeeping genes, cDNA sequence was derived from 1978 clones of the Endoderm library. These yielded 1440 distinct cDNAs, of which 123 proved to be novel in the mouse. In situ hybridization analysis was carried out on 160 of the cDNAs, and of these, 29 (18%) proved to have restricted expression patterns.

Overexpression of sonic hedgehog suppresses embryonic hair follicle morphogenesis

The Sonic Hedgehog (Shh) signalling pathway plays a central role in the development of the skin and hair follicle and is a major determinant of skin tumorigenesis, most notably of basal cell carcinoma (BCC). Various mouse models involving either ablation or overexpression of key members of the Shh signalling pathway display a range of skin tumours. To further examine the role of Shh in skin development. we have overexpressed Shh in a subset of interfollicular basal cells from 12.5 dpc under the control of the human keratin 1 (HK1) promoter. The HK1-Shh transgenic mice display a range of skin anomalies, including highly pigmented inguinal lesions and regions of alopecia. The most striking hair follicle phenotype is a suppression in embryonic follicle development between 14.0 and 19.0 dpc, resulting in a complete absence of guard, awl, and auchene hair fibres. These data indicate that alternative signals are responsible for the development of different hair follicles and point to a major role of Shh signalling in the morphogenesis of guard, awl, and auchene hair fibres. Through a comparison with other mouse models, the characteristics of the HK1-Shh transgenic mice suggest that the precise timing and site of Shh expression are key in dictating the resultant skin and tumour phenotype. 2003 Elsevier Inc. All rights reserved.

Patched 1 conditional null allele in mice

The patched gene (Ptc) is a member of the hedgehog signaling pathway which plays a central role in the development of many invertebrate and vertebrate tissues. In addition, Ptc and a number of other pathway members are mutated in some common human cancers. Patched is the receptor for the hedgehog ligand and in the mouse ablation of the Ptc gene leads to developmental defects and an embryonic lethal phenotype. Here we describe a conditional Ptc allele in mice which will have utility for the temporospatial ablation of Ptc function. genesis 36:158-161, 2003. (C) 2003 Wiley-Liss, Inc.

The production, purification and crystallization of a pocilloporin pigment from a reef-forming coral

Reef-building corals contain fluorescent pigments termed pocilloporins that function by regulating the light environment of coral and acting as a photoprotectant in excessive sunlight. These pocilloporins are related to the monomeric green fluorescent protein and the tetrameric DsRed fluorescent proteins, which have widespread use as biotechnological tools. An intensely blue-coloured pocilloporin, termed Rtms5, was expressed in Escherichia coli, purified and crystallized. Rtms5 was shown to be tetrameric, with deep blue crystals that diffract to 2.2 Angstrom resolution and belong to space group I4(1)22. The colour of this pocilloporin was observed to be sensitive to pH and a yellow (pH 3.5) and a red form (pH 4.5) of Rtms5 were also crystallized. These crystals belong to space group P4(2)22 and diffract to 2.4 Angstrom resolution or better.