Immune-associated nucleotide-1 (IAN-1) is a thymic selection marker and defines a novel gene family conserved in plants.

Positive selection of thymocytes is a complex and crucial event in T cell development that is characterized by cell death rescue, commitment toward the helper or cytotoxic lineage, and functional maturation of thymocytes bearing an appropriate TCR. To search for novel genes involved in this process, we compared gene expression patterns in positively selected thymocytes and their immediate progenitors in mice using the differential display technique. This approach lead to the identification of a novel gene, mIAN-1 (murine immune-associated nucleotide-1), that is switched on upon positive selection and predominantly expressed in the lymphoid system. We show that mIAN-1 encodes a 42-kDa protein sharing sequence homology with the pathogen-induced plant protein aig1 and that it defines a novel family of at least three putative GTP-binding proteins. Analysis of protein expression at various stages of thymocyte development links mIAN-1 to CD3-mediated selection events, suggesting that it represents a key player of thymocyte development and that it participates to peripheral specific immune responses. The evolutionary conservation of the IAN family provides a unique example of a plant pathogen response gene conserved in animals.     

The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins

Background  

The NCOA7 gene product is an estrogen receptor associated protein that is highly similar to the human OXR1 gene product, which functions in oxidation resistance. OXR genes are conserved among all sequenced eukaryotes from yeast to humans. In this study we examine if NCOA7 has an oxidation resistance function similar to that demonstrated for OXR1. We also examine NCOA7 expression in response to oxidative stress and its subcellular localization in human cells, comparing these properties with those of OXR1.     

The conserved Candida albicans CA3427 gene product defines a new family of proteins exhibiting the generic periplasmic binding protein structural fold

Nosocomial diseases due to Candida albicans infections are in constant rise in hospitals, where they cause serious complications to already fragile intensive care patients. Antifungal drug resistance is fast becoming a serious issue due to the emergence of strains resistant to currently available antifungal agents. Thus the urgency to identify new potential protein targets, the function and structure of which may guide the development of new antifungal drugs. In this context, we initiated a comparative genomics study in search of promising protein coding genes among the most conserved ones in reference fungal genomes. The CA3427 gene was selected on the basis of its presence among pathogenic fungi contrasting with its absence in the non pathogenic Saccharomyces cerevisiae. We report the crystal 3D-structure of the Candida albicans CA3427 protein at 2.1 Å resolution. The combined analysis of its sequence and structure reveals a structural fold originally associated with periplasmic binding proteins. The CA3427 structure highlights a binding site located between the two protein domains, corresponding to a sequence segment conserved among fungi. Two crystal forms of CA3427 were found, suggesting that the presence or absence of a ligand at the proposed binding site might trigger a “Venus flytrap” motion, coupled to the previously described activity of bacterial periplasmic binding proteins. The conserved binding site defines a new subfamily of periplasmic binding proteins also found in many bacteria of the bacteroidetes division, in a choanoflagellate (a free-living unicellular and colonial flagellate eukaryote) and in a placozoan (the closest multicellular relative of animals). A phylogenetic analysis suggests that this gene family originated in bacteria before its horizontal transfer to an ancestral eukaryote prior to the radiation of fungi. It was then lost by the Saccharomycetales which include Saccharomyces cerevisiae.     

The wingless gene is required for embryonic brain development in Drosophila

 We have studied the role of the wingless gene in embryonic brain development of Drosophila. wingless is expressed in a large domain in the anlage of the protocerebrum and also transiently in smaller domains in the anlagen of the deutocerebrum and tritocerebrum. Elimination of the wingless gene in null mutants has dramatic effects on the developing protocerebrum; although initially generated, approximately one half of the protocerebrum is deleted in wingless null mutants by apoptotic cell death at late embryonic stages. Using temperature sensitive mutants, a rescue of the mutant phenotype can be achieved by stage-specific expression of functional wingless protein during embryonic stages 9–10. This time period correlates with that of neuroblast specification but preceeds the generation and subsequent loss of protocerebral neurons. Ectopic wingless over-expression in gain-of-function mutants results in dramatically oversized CNS. We conclude that wingless is required for the development of the anterior protocerebral brain region in Drosophila. We propose that an important role of wingless in this part of the developing brain is the determination of neural cell fate. Received: 7 October 1997 / Accepted: 30 December 1997     

alpha-Endosulfine is a conserved protein required for oocyte meiotic maturation in Drosophila

Meiosis is coupled to gamete development and must be well regulated to prevent aneuploidy. During meiotic maturation, Drosophila oocytes progress from prophase I to metaphase I. The molecular factors controlling meiotic maturation timing, however, are poorly understood. We show that Drosophila alpha-endosulfine (endos) plays a key role in this process. endos mutant oocytes have a prolonged prophase I and fail to progress to metaphase I. This phenotype is similar to that of mutants of cdc2 (synonymous with cdk1) and of twine, the meiotic homolog of cdc25, which is required for Cdk1 activation. We found that Twine and Polo kinase levels are reduced in endos mutants, and identified Early girl (Elgi), a predicted E3 ubiquitin ligase, as a strong Endos-binding protein. In elgi mutant oocytes, the transition into metaphase I occurs prematurely, but Polo and Twine levels are unaffected. These results suggest that Endos controls meiotic maturation by regulating Twine and Polo levels, and, independently, by antagonizing Elgi. Finally, germline-specific expression of the human alpha-endosulfine ENSA rescues the endos mutant meiotic defects and infertility, and alpha-endosulfine is expressed in mouse oocytes, suggesting potential conservation of its meiotic function.     

ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins

Background  

Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms.     

Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases.

The polytopic membrane protein Rhomboid-1 promotes the cleavage of the membrane-anchored TGFalpha-like growth factor Spitz, allowing it to activate the Drosophila EGF receptor. Until now, the mechanism of this key signaling regulator has been obscure, but our analysis suggests that Rhomboid-1 is a novel intramembrane serine protease that directly cleaves Spitz. In accordance with the putative Rhomboid active site being in the membrane bilayer, Spitz is cleaved within its transmembrane domain, and thus is, to our knowledge, the first example of a growth factor activated by regulated intramembrane proteolysis. Rhomboid-1 is conserved throughout evolution from archaea to humans, and our results show that a human Rhomboid promotes Spitz cleavage by a similar mechanism. This growth factor activation mechanism may therefore be widespread.     

The egghead gene is required for compartmentalization in Drosophila optic lobe development

The correct targeting of photoreceptor neurons (R-cells) in the developing Drosophila visual system requires multiple guidance systems in the eye-brain complex as well as the precise organization of the target area. Here, we report that the egghead (egh) gene, encoding a glycosyltransferase, is required for a compartment boundary between lamina glia and lobula cortex, which is necessary for appropriate R1-R6 innervation of the lamina. In the absence of egh, R1-R6 axons form a disorganized lamina plexus and some R1-R6 axons project abnormally to the medulla instead of the lamina. Mosaic analysis demonstrates that this is not due to a loss of egh function in the eye or in the neurons and glia of the lamina. Rather, as indicated by clonal analysis and cell-specific genetic rescue experiments, egh is required in cells of the lobula complex primordium which transiently abuts the lamina and medulla in the developing larval brain. In the absence of egh, perturbation of sheath-like glial processes occurs at the boundary region delimiting lamina glia and lobula cortex, and inappropriate invasion of lobula cortex cells across this boundary region disrupts the pattern of lamina glia resulting in inappropriate R1-R6 innervation. This finding underscores the importance of the lamina/lobula compartment boundary in R1-R6 axon targeting.     

extramacrochaetae, a negative regulator of sensory organ development in Drosophila, defines a new class of helix-loop-helix proteins.

The function of the extramacrochaetae (emc) gene is required to establish the normal spatial pattern of adult sensory organs in Drosophila. emc acts to suppress sensory organ development in certain regions of the body surface, apparently by antagonizing the function of the achaete and scute genes of the achaetescute complex (AS.C). We have found that emc encodes a novel member of the helix-loop-helix (HLH) family of proteins. The emc protein shares the dimerization domain of other HLH proteins but lacks their DNA binding motif. We propose a model in which the emc protein negatively regulates sensory organ determination by forming heterodimers with the HLH proteins encoded by the AS-C and/or daughterless, thereby altering or interfering with their activity.     

A new member of the NY-ESO-1 gene family is ubiquitously expressed in somatic tissues and evolutionarily conserved

Three single copy ATP-binding cassette (ABC) transporter encoding genes, designated MgAtr3, MgAtr4, and MgAtr5, were cloned and sequenced from the plant pathogenic fungus Mycosphaerella graminicola. The encoded ABC proteins all exhibit the [NBD-TMS(6)](2) configuration and can be classified as novel members of the pleiotropic drug resistance (PDR) class of ABC transporters. The three proteins are highly homologous to other fungal and yeast, ABC proteins involved in multidrug resistance or plant pathogenesis. MgAtr4 and MgAtr5 possess a conserved ABC motif at both the N- and C-terminal domain of the protein. In contrast, the Walker A motif in the N-terminal and the ABC signature in the C-terminal domain of MgAtr3, deviate significantly from the consensus sequence found in other members of the PDR class of ABC transporters. Expression of MgAtr3 could not be detected under any of the conditions tested. However, MgAtr4 and MgAtr5 displayed distinct expression profiles when treated with a range of compounds known to be either substrates or inducers of ABC transporters. These included synthetic fungitoxic compounds, such as imazalil and cyproconazole, natural toxic compounds, such as the plant defence compounds eugenol and psoralen, and the antibiotics cycloheximide and neomycin. The expression pattern of the genes was also dependent on the morphological state of the fungus. The findings suggest a role for MgAtr4 and MgAtr5 during plant pathogenesis and in protection against toxic compounds.     

The yan gene is highly conserved in Drosophila and its expression suggests a complex role throughout development

 Competence for cell fate determination and cellular differentiation is under tight control of regulatory genes. Yan, a nuclear target of receptor tyrosine kinase (RTK) signaling, is an E twenty six (ETS) DNA-binding protein that functions as a negative regulator of cell differentiation and proliferation in Drosophila. Most members of RTK signaling pathways are highly conserved through evolution, yet no yan orthologues have been identified to date in vertebrates. To investigate the degree of yan conservation during evolution, we have characterized a yan homologue from a sibling species of D. melanogaster, D. virilis. Our results show that the organization, primary structure and expression pattern of yan are highly conserved. Both genes span over 20 kb and contain four exons with introns at identical positions. The areas with highest amino acid similarity include the Pointed and ETS domain but there are other discrete regions with a high degree of similarity. Phylogenetic analysis reveals that yan’s closest relative is the human tel gene, a negative regulator of differentiation in hematopoetic precursors. In both species, Yan is dynamically expressed beginning as early as stage 4/5 and persisting throughout embryogenesis. In third instar larvae, Yan is expressed in and behind the morphogenetic furrow of the eye imaginal disc as well as in the laminar precursor cells of the brain. Ovarian follicle cells also contain Yan protein. Conservation of the structure and expression patterns of yan genes strongly suggests that regulatory mechanisms for their expression are also conserved in these two species. Received: 3 November 1998 / Accepted: 9 December 1998     

The Drosophila segmentation gene runt has an extended cis-regulatory region that is required for vital expression at other stages of development.

The Drosophila runt gene functions in several developmental pathways during embryogenesis. This gene was initially characterized due to the pivotal role that it plays in the genetic regulatory network that establishes the segmented body pattern. Recently it was found that this X-chromosome-linked gene is one of several dosage-sensitive, X-linked components that is involved in activating the Sex-lethal gene in blastoderm stage female embryos. Finally, this gene is also extensively re-expressed in later stages of embryogenesis in the developing nervous system where it plays an important role in the development of specific neural lineages. We have initiated an analysis of the runt cis-regulatory region in order to investigate runt's roles in these (and other) developmental pathways. Analysis of both the function and the expression patterns of runt genes with truncated cis-regulatory regions indicates that there are multiple elements that make quantitative contributions to runt regulation during segmentation. We find that sequences that are more than 8.5 kb upstream of the runt promoter are necessary for normal expression during the post-blastoderm stages of embryogenesis. Genetic experiments indicate that the post-blastoderm expression of runt is vital to the organism.     

RASSF7 is a member of a new family of RAS association domain-containing proteins and is required for completing mitosis

Mitosis is a fundamental feature of all cellular organisms. It must be tightly regulated to allow normal tissue growth and to prevent cancer formation. Here, we identify a new protein that is required for mitosis. We show that the Ras association (RA) domain-containing protein, RASSF7, is part of an evolutionarily conserved group of four proteins. These are RASSF7, RASSF8, and two new RASSF proteins P-CIP1/RASSF9 and RASSF10. We call this group the N-terminal RASSF family. We analyzed the function of Xenopus RASSF7. RASSF7 was found to be expressed in several embryonic tissues including the skin, eyes, and neural tube. Knocking down its function led to cells failing to form a mitotic spindle and arresting in mitosis. This caused nuclear breakdown, apoptosis, and a striking loss of tissue architecture in the neural tube. Consistent with a role in spindle formation, RASSF7 protein was found to localize to the centrosome. This localization occurred in a microtubule-dependent manner, demonstrating that there is a mutually dependant relationship between RASSF7 localization and spindle formation. Thus RASSF7, the first member of the N-terminal RASSF family to be functionally analyzed, is a centrosome-associated protein required to form a spindle and complete mitosis in the neural tube.     

The fat facets gene is required for Drosophila eye and embryo development.

In a screen for mutations affecting Drosophila eye development, we have identified a gene called fat facets (faf) which is required for cell interactions that prevent particular cells in the developing eye from becoming photoreceptors. Analysis of eyes mosaic for faf+ and faf- cells shows that faf is required in cells near to, but outside, normal developing photoreceptors and also outside of the ectopic photoreceptors in mutant facets. faf is also essential during oogenesis, and we show that a faf-lacZ hybrid protein is localized via the first 392 amino acids of faf to the posterior pole of oocytes. Posterior localization of faf-lacZ depends on oskar. oskar encodes a key organizer of the pole plasm, a specialized cytoplasm at the posterior pole of embryos. The pole plasm is required for germ cell formation and contains the determinant of posterior polarity, encoded by nanos. Although other pole plasm components are required for localization of nanos RNA or for nanos protein function, faf is not. We have cloned the faf gene, and have shown that it encodes two similar large (approximately 300 x 10(3) M(r)) proteins that are unique with respect to other known proteins.     

A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis

We have identified and characterized a novel, repeating 34 amino acid motif (the TPR motif) that is reiterated several times within the CDC23 gene product of S. cerevisiae. Multiple copies of this motif were discovered in five other proteins, three encoded by cell division cycle genes required to complete mitosis and two involved in RNA synthesis. Quantitative sequence analyses suggest the existence of a common underlying structure in each TPR unit that consists of amphipathic alpha-helical regions punctuated by proline-induced turns. The TPR motif defines a new family of genes and an important structural unit common to several proteins whose functions are required for mitosis and RNA synthesis.