The insect SNMP gene family

SNMPs are membrane proteins observed to associate with chemosensory neurons in insects; in Drosophila melanogaster, SNMP1 has been shown to be essential for the detection of the pheromone cis-vaccenyl acetate (CVA). SNMPs are one of three insect gene clades related to the human fatty acid transporter CD36. We previously characterized the CD36 gene family in 4 insect Orders that effectively cover the Holometabola, or some 80% of known insect species and the 300 million years of evolution since this lineage emerged: Lepidoptera (e.g. Bombyx mori, Antheraea polyphemus, Manduca sexta, Heliothis virescens, Helicoverpa assulta, Helicoverpa armigera, Mamestra brassicae); Diptera (D. melanogaster, Drosophila pseudoobscura, Aedes aegypti, Anopheles gambiae, Culex pipiens quinquefasciatus); Hymenoptera (Apis mellifera); and Coleoptera (Tribolium castaneum). This previous study suggested a complex topography within the SNMP clade including a strongly supported SNMP1 sub-clade plus additional SNMP genes. To further resolve the SNMP clade here, we used cDNA sequences of SNMP1 and SNMP2 from various Lepidoptera species, D. melanogaster and Ae. aegypti, as well as BAC derived genomic sequences from Ae. aegypti as models for proposing corrected sequences of orthologues in the D. pseudoobscura and An. gambiae genomes, and for identifying orthologues in the B. mori and C. pipiens q. genomes. We then used these sequences to analyze the SNMP clade of the insect CD36 gene family, supporting the existence of two well supported sub-clades, SNMP1 and SNMP2, throughout the dipteran and lepidopteran lineages, and plausibly throughout the Holometabola and across a broad evolutionary time scale. We present indirect evidence based on evolutionary selection (dN/dS) that the dipteran SNMPs are expressed as functional proteins. We observed expansions of the SNMP1 sub-clade in C. pipiens q. and T. castaneum suggesting that the SNMP1s may have an expanded functional role in these species.     

Antennal SNMPs (sensory neuron membrane proteins) of Lepidoptera define a unique family of invertebrate CD36-like proteins

SNMP1-Apol is an antennal-specific protein of the wild silk moth Antheraea polyphemus; the protein is abundantly expressed and localized to the receptor membranes of sex-pheromone specific olfactory sensory neurons (OSNs). SNMP1-Apol is thought to function in odor detection based on its olfactory-specific expression, localization within OSNs, developmental time of expression, and apparent homology to the CD36 family of membrane-bound receptor proteins. In the current study, SNMP1-Apol homologues were identified from the moths Bombyx mori, Heliothis virescens, and Manduca sexta. These species posses antennal mRNAs encoding proteins with amino acid sequence identities ranging from 75-80%; these proteins are collectively designated SNMP1. A second M. sexta SNMP homologue, previously identified and partially sequenced [Robertson et al.: Insect Mol Biol 8:501-518, 1999] was fully sequenced and characterized. The encoded protein shares only 26-27% sequence identity with the SNMP1 proteins, and is thus designated SNMP2-Msex. The SNMP sequences were used to identify 14 and four possible homologues in Drosophila melanogaster and Caenorhabditis elegans genome databases, respectively; thus, greatly expanding CD36 family membership among the invertebrate lineages. Despite their sequence difference, SNMP1-Msex and SNMP2-Msex expression is localized to OSNs and occurs simultaneously with the onset of olfactory function. These findings suggest that SNMPs play a central role in odor detection in insects, and that the CD36 gene family is widely represented among animal phyla. The SNMPs are the only identified neuronal members of the CD36 family, and as such expand the activities of this gene family into roles influencing brain function and behavioral action.     

The cytochrome P450 gene superfamily in Drosophila melanogaster: annotation, intron-exon organization and phylogeny

The cytochrome P450 gene superfamily is represented by 90 sequences in the Drosophila melanogaster genome. Of these 90 P450 sequences, 83 code for apparently functional genes whereas seven are apparent pseudogenes. More than half of the genes belong to only two families, CYP4 and CYP6. The CYP6 family is insect specific whereas the CYP4 family includes sequences from vertebrates. There are eight genes coding for mitochondrial P450s as deduced from their homology to CYP12A1 from the house fly. The genetic map of the distribution of D. melanogaster P450 genes shows (a) the absence of P450 genes on the chromosome 4 and Y, (b) more than half of the P450 genes are found on chromosome 2, and (c) the largest cluster contains nine genes. Sequence alignments were used to draw phylogenetic trees and to analyze the intron-exon organization of each functional P450 gene. Only five P450 genes are intronless. We found 57 unique intron positions, of which 23 were phase zero, 19 were phase one and 15 were phase two. There was a relatively good correlation between intron conservation and phylogenetic relationship between members of the P450 subfamilies. Although the function of many P450 proteins from vertebrates, fungi, plants and bacteria is known, only a single P450 from D. melanogaster, CYP6A2, has been functionally characterized. Gene organization appears to be a useful tool in the study of the regulation, the physiological role and the function of these P450s.     

Identification and localization of two sensory neuron membrane proteins from Spodoptera litura (Lepidoptera: Noctuidae)

Sensory neuron membrane proteins (SNMPs), which are located on the dendritic membrane of olfactory sensory neurons (OSNs), are proposed to be associated with odor reception in insects. Recent studies have demonstrated that SNMP1 is essential for electrophysiological responses of OSNs to the sex pheromone, cis‐vaccenyl acetate (cVA) in Drosophila melanogaster. To investigate the function of Lepidoptera SNMPs, we cloned two SNMP genes, SlituSNMP1 and SltiuSNMP2, from Spodoptera litura (Lepidoptera: Noctuidae). Sequence alignment and phylogenetic analysis showed that both genes bear the general characteristics of SNMPs, including six conserved cysteine residues and two transmembrane domains. Further expression profile experiments showed that SlituSNMP1 is mainly expressed in the antenna, while SlituSNMP2 is broadly expressed in various tissues. By in situ hybridization experiments, it was found that SlituSNMP1 expressing cells are surrounded by the SlituSNMP2 expressing cells in the pheromone sensitive sensilla, suggesting different functions of the two SNMPs in insect olfaction.     

A cluster of candidate odorant receptors from the malaria vector mosquito,Anopheles gambiae

Olfaction is critical to the host preference selection behavior of many disease-transmitting insects, including the mosquito Anopheles gambiae sensu stricto (hereafter A. gambiae), one of the major vectors for human malaria. In order to more fully understand the molecular biology of olfaction in this insect, we have previously identified several members member of a family of candidate odorant receptor proteins from A. gambiae (AgORs). Here we report the cloning and characterization of an additional AgOR gene, denoted as AgOr5, which shows significant similarity to putative odorant receptors in A. gambiae and Drosophila melanogaster and which is selectively expressed in olfactory organs. AgOr5 is tightly clustered within the A. gambiae genome to two other highly homologous candidate odorant receptors, suggesting that these genes are derived from a common ancestor. Analysis of the developmental expression within members of this AgOR gene cluster reveals considerable variation between these AgORs as compared to candidate odorant receptors from D. melanogaster.     

Evolution of the RECQ family of helicases: A drosophila homolog, Dmblm, is similar to the human bloom syndrome gene

Several eukaryotic homologs of the Escherichia coli RecQ DNA helicase have been found. These include the human BLM gene, whose mutation results in Bloom syndrome, and the human WRN gene, whose mutation leads to Werner syndrome resembling premature aging. We cloned a Drosophila melanogaster homolog of the RECQ helicase family, Dmblm (Drosophila melanogaster Bloom), which encodes a putative 1487-amino-acid protein. Phylogenetic and dot plot analyses for the RECQ family, including 10 eukaryotic and 3 prokaryotic genes, indicate Dmblm is most closely related to the Homo sapiens BLM gene, suggesting functional similarity. Also, we found that Dmblm cDNA partially rescued the sensitivity to methyl methanesulfonate of Saccharomyces cerevisiae sgs1 mutant, demonstrating the presence of a functional similarity between Dmblm and SGS1. Our analyses identify four possible subfamilies in the RECQ family: (1) the BLM subgroup (H. sapiens Bloom, D. melanogaster Dmblm, and Caenorhabditis elegans T04A11.6); (2) the yeast RECQ subgroup (S. cerevisiae SGS1 and Schizosaccharomyces pombe rqh1/rad12); (3) the RECQL/Q1 subgroup (H. sapiens RECQL/Q1 and C. elegans K02F3.1); and (4) the WRN subgroup (H. sapiens Werner and C. elegans F18C5.2). This result may indicate that metazoans hold at least three RECQ genes, each of which may have a different function, and that multiple RECQ genes diverged with the generation of multicellular organisms. We propose that invertebrates such as nematodes and insects are useful as model systems of human genetic diseases.     

EXPRESSION OF SNMP1 AND SNMP2 GENES IN ANTENNAL SENSILLA OF Spodoptera exigua (HÜBNER)

Sensory neuron membrane proteins (SNMPs) are olfactory‐specific, two‐transmembrane proteins. Previous publications reported that SNMP1 is expressed on the dendrite membrane of pheromone‐sensitive neurons in Heliothis virescens and is an essential cofactor for pheromone detection in Drosophila. In this study, we cloned two SNMP genes (GenBank accession nos. JX469106 and JX469107) from the antenna of the beet armyworm moth Spodoptera exigua (Lepidoptera: Hübner). These SNMP genes are classified into two highly conserved subclades, indicating their importance in physiological activity of lepidopteran insects. SexiSNMP1 is antenna‐specific in male and female adults, while SexiSNMP2 is antenna‐abundant but also expressed in other chemosensory tissues, particularly proboscises and maxillary palps of adults both sexes. In situ hybridization revealed that both SNMPs are broadly expressed in long and short trichoid and basiconic sensilla. We infer that SNMP1 and SNMP2 act in the detection of the sex pheromone and general odorants.     

Identification and comparative analysis of a second runx3 promoter

Adenosine deaminase (ADA) catalyzes the hydrolysis of adenosine to inosine. Its lack determines severe combined immunodeficiency in mammals, possibly due to accumulation of extracellular adenosine, which induces apoptosis in lymphocytes (Franco et al., 1998). Thus, presence of normal levels of ADA leads to normal growth and proliferation of lymphocytes. Several vertebrate and microbial ADA amino-acid sequences are known, with substantial similarity to each other. On the other hand, there are invertebrate growth factors as well as a candidate gene for the human cat eye syndrome (CECR1) (Riazi et al., 2000. Genomics 64, 277-285), which share substantial similarity to each other, and also to ADA. In this study, we report the expression and ADA enzymatic activity of a cDNA from the salivary glands of Lutzomyia longipalpis, a blood-sucking insect, with substantial similarity to insect growth factors and to human CECR1. We also demonstrate the existence of a subfamily of the adenosine deaminase family characterized by their unique amino-terminal region. Both Drosophila melanogaster and humans have both types of adenosine deaminases. Results indicate that these invertebrate proteins previously annotated as growth factors, as well as the human CECR1 gene product, may exert their actions through adenosine depletion. The different roles played by each type of adenosine deaminase in humans and Drosophila remains to be fully investigated.     

Drosophila melanogaster Methoprene-tolerant (Met) gene homologs from three mosquito species: Members of PAS transcriptional factor family

The Methoprene-tolerant (Met) gene in Drosophila melanogaster has been shown to function in juvenile hormone (JH) action. Met homologs were isolated from three mosquito species, Culex pipiens, Aedes aegypti and Anopheles gambiae. Sequence similarity was found to be high in bHLH and PAS conserved domains, and the majority of the 7-9 introns in AaMet and AgMet are located in either identical or similar positions, indicating evolutionary relatedness. Sequence comparison with Met and the similar germ-cell expressed (gce) gene in D. melanogaster showed that the mosquito genes are more similar to gce than to Met. Moreover, the multiple introns in AgMet and AaMet are more similar in number with the 7 introns in Dmgce than to the single intron in DmMet; in fact, six intron positions in AaMet and AgMet are similar to those in Dmgce. Efforts to identify a second homologous gene in mosquitoes were unsuccessful, suggesting a single gene in lower Diptera, consistent with the single gene uncovered in genomic sequencing of Ae. aegypti and An. gambiae. These results suggest that a gene duplication occurred during the evolution of higher Diptera, resulting in Met and gce.     

Molecular evolution of odorant-binding protein genes OS-E and OS-F in Drosophila

The Drosophila olfactory genes OS-E and OS-F are members of a family of genes that encode insect odorant-binding proteins (OBPs). OBPs are believed to transport hydrophobic odorants through the aqueous fluid within olfactory sensilla to the underlying receptor proteins. The recent discovery of a large family of olfactory receptor genes in Drosophila raises new questions about the function, diversity, regulation, and evolution of the OBP family. We have investigated the OS-E and OS-F genes in a variety of Drosophila species. These studies highlight potential regions of functional significance in the OS-E and OS-F proteins, which may include a region required for interaction with receptor proteins. Our results suggest that the two genes arose by an ancient gene duplication, and that in some lineages, one or the other gene has been lost. In D. virilis, the OS-F gene shows a different spatial pattern of expression than in D. melanogaster. One of the OS-F introns shows a striking degree of conservation between the two species, and we identify a putative regulatory sequence within this intron. Finally, a phylogenetic analysis places both OS-E and OS-F within a large family of insect OBPs and OBP-like proteins.     

棉铃虫感觉神经元膜蛋白基因克隆和表达

从棉铃虫Helicoverpa armigera触角中克隆了一条全长1 690 bp的cDNA序列,该序列阅读框全长1 572 bp,编码523个氨基酸残基,序列中有2个跨膜区,具有昆虫感觉神经元膜蛋白（sensory neuron membrane protein, SNMP）的典型特征。SNMP与已报道的其他昆虫的感觉神经元蛋白的氨基酸序列有很高的同源性。半定量RT-PCR研究结果显示,SNMP在棉铃虫中不仅在触角中表达,也在去掉触角的头、足中表达。但是在触角中的表达量最高,在雌雄触角中的表达量差异不显著。在喙、下颚须和下唇须中也有表达。SNMP在卵、蛹和成虫体内也都有表达,但在卵中表达量相对较低。将SNMP编码区克隆到表达载体pET21b中,成功地进行了原核表达,表达出带有6个组氨酸标签的重组蛋白。