Cloning and functional characterization of a putative sodium channel auxiliary subunit gene from the house fly (Musca domestica)

The functional expression of cloned Drosophila melanogaster and house fly (Musca domestica) voltage-sensitive sodium channels in Xenopus oocytes is enhanced, and the inactivation kinetics of the expressed channels are accelerated, by coexpression with the tipE protein, a putative sodium channel auxiliary subunit encoded by the tipE gene of D. melanogaster. These results predict the existence of a tipE ortholog in the house fly. Using a PCR-based homology probing approach, we isolated cDNA clones encoding an ortholog of tipE (designated Vssc beta) from adult house fly heads. Clones comprising 3444 bp of cDNA sequence contained a 1317 bp open-reading frame encoding a 438 amino acid protein. The predicted Vssc beta protein exhibited 72% amino acid sequence identity to the entire D. melanogaster tipE protein sequence and 97% identity within the two hydrophobic segments identified as probable transmembrane domains. Coexpression of Vssc beta with the house fly sodium channel alpha subunit (Vssc1) in oocytes enhanced the level of sodium current expression five-fold and accelerated the rate of sodium current inactivation 2.2-fold. Both of these effects were significantly larger in magnitude than the corresponding effects of the D. melanogaster tipE protein on the expression and kinetics of Vssc1 sodium channels. These results identify a second example of a putative sodium channel auxiliary subunit from an insect having functional but not structural homology to vertebrate sodium channel beta subunits.     

Characterization of recombinant and native Ih-channels from Apis mellifera

Recently, a novel class of genes coding for Ih-channels has been identified in several vertebrates and invertebrates. We isolated a cDNA (AMIH) encoding a putative member of these ion channels from Apis mellifera heads by means of polymerase chain reaction and homology screening. High similarity (88% identical amino acids) to the putative Drosophila melanogaster Ih-channel suggests that the Apis cDNA codes for a hyperpolarization-activated and cyclic nucleotide-gated channel. Functional expression of recombinant AMIH in HEK293 cells gave unitary currents that were preferentially selective for potassium over sodium ions and were activated by hyperpolarizing voltage steps. Cyclic nucleotides shifted the voltage activation curve to more positive membrane potentials. The current kinetics, activation by hyperpolarizing voltage steps and modulatory influence of cyclic nucleotides properties closely resemble those of mammalian Ih-channels. RT-PCR analysis showed pronounced mRNA expression in the antennae, head and body of Apis mellifera. Investigation of hyperpolarization-activated currents in olfactory receptor neurons (ORNs) in a primary cell culture of Apis mellifera antennal cells revealed activation properties similar to the heterologously expressed Ih-channel. By in-situ hybridization and immunohistochemistry, expression of AMIH was seen in olfactory receptor neurons of the bee antennae. We conclude that AMIH is the ion channel responsible for the hyperpolarization-activated currents in olfactory receptor neurons of bee.     

Novel odorant-binding proteins expressed in the taste tissue of the fly

A taste tissue cDNA library of the fleshfly Boettcherisca peregrina was screened with a subtracted cDNA probe enriched with taste-receptor-tissue-specific cDNA. Seven genes were identified with sequence similarity to insect odorant-binding protein (OBP) genes. The predicted amino acid sequences of the genes contain the putative signal peptide sequence at the N-terminal and most of them conserve the six cysteines common to known insect OBPs. These genes show a high degree of sequence divergence with approximately 20% amino acid identity. The most striking feature was that all seven of these genes are expressed mainly in the taste tissues, such as the labellum and tarsus, unlike the known insect OBP genes expressed in olfactory tissue. The predicted amino acid sequences had the highest degree of sequence similarity to the Drosophila melanogaster OBPs named pheromone binding protein-related proteins (PBPRPs). These gene products are here referred to as gustatory PBP-related proteins (GPBPRPs) 1-7. Homologous GPBPRP genes were found also in D. melanogaster by database search and are shown to be expressed in Drosophila taste tissues.     

Isolation and characterization of a Drosophila neuropeptide gene

We have purified a 9 amino acid amidated neuropeptide, DPKQDFMRFamide, from whole adult D. melanogaster. This peptide exhibits sequence homology to the molluscan bioactive tetrapeptide FMRFamide and is a novel member of the FMRFamide peptide family. The gene encoding DPKQDFMRFamide has been cloned and characterized. It is present in a single copy per haploid genome, is expressed as a unique 1.7 kb mRNA species, and cytologically maps to 46C on the right arm of chromosome 2. Characterization of a cDNA clone indicates that the precursor protein is 347 amino acids in length and contains 5 copies of DPKQDFMRFamide, as well as 10 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides.     

Cloning of a putative γ-aminobutyric acid (GABA) receptor subunit ϱ3 cDNA

Cloned cDNA encoding a putative member of GABA receptor ϱ-subunit class was isolated from rat-retina-mRNA-derived libraries. The cDNA encodes a signal peptide of 21 amino acids followed by the mature ϱ3 subunit sequence of 443 amino acids. The proposed amino acid sequence exhibits 63 and 61% homology to the previously-reported human ϱ1 and rat ϱ2 sequences, respectively. Northern blot analysis demonstrated the expression of mRNA for ϱ3 subunit in retina.     

Drosophila and vertebrate myb proteins share two conserved regions, one of which functions as a DNA-binding domain

We report the nucleotide sequence of a cDNA clone of the Drosophila melanogaster homologue of c-myb, a member of the class of vertebrate transforming genes encoding nuclear proteins. We predict the mol. wt of the Drosophila myb (D-myb) protein to be 74,000. The D-myb protein contains two clusters of sequences homologous to vertebrate myb proteins, surrounded by sequences lacking homology. These results extend previous evidence for the existence of a D. melanogaster homologue of c-myb and identify two highly conserved and therefore presumably functionally important domains of c-myb proteins. DNA-binding experiments indicate that the NH2-proximal of the two homology regions functions as a DNA-binding domain. Based on the absence of the COOH-proximal homology region in truncated oncogenic derivatives of c-myb it is likely that this homology region encodes a function whose loss is involved in activating the oncogenic potential of c-myb.     

Molecular characterization of a putative peroxidase gene of Drosophila melanogaster.

We have identified genomic clones and corresponding cDNAs that encode a putative peroxidase of Drosophila melanogaster. The gene (DmPO) appears as a single copy gene located on the third chromosome at position 89 D/E. It is interrupted by seven small introns and one unusually large 5' intron (about 11 kb). Sequence analysis of the cDNA showed an open reading frame of 690 amino acids resulting in a protein of 77 kDa. The deduced amino acid sequence reveals an overall homology to myeloeosinophil and thyroid peroxidase, a human superfamily of peroxidases.     

Drosophila melanogaster has only one myosin alkali light-chain gene which encodes a protein with considerable amino acid sequence homology to chicken myosin alkali light chains.

A chimeric lambda DNA molecule containing the myosin alkali light-chain gene of Drosophila melanogaster was isolated. The encoded amino acid sequence was determined from the nucleic acid sequence of a cDNA homologous to the genomic clone. The identity of the encoded protein was established by two criteria: (i) sequence homology with the chicken alkali light-chain proteins and (ii) comparison of the two-dimensional gel electrophoretic pattern of the peptides synthesized by in vitro translation of hybrid-selected RNA to that of myosin alkali light-chain peptides extracted from Drosophila myofibrils. There is only one myosin alkali light-chain in D. melanogaster; its chromosomal location is region 98B . This gene is abundantly expressed during the development of larval as well as adult muscles. The Drosophila protein appears to contain one putative divalent cation-binding domain (an EF hand) as compared with the three EF hands present in chicken alkali light chains.     

Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis.

A cDNA expression library prepared from mRNA of Haemaphysalis longicornis (H. longicornis) was screened with a H. longicornis-infested rabbit serum. A cDNA encoding 27/30kDa proteins was cloned and designated P27/30 gene. The predicted amino acid sequence of the P27/30 gene shows a rather high homology (58% amino acid identities and 11% amino acid similarity) with Drosophila melanogaster troponin I clone E2. H. longicornis P27/30 possesses amino acid sequence of actin-binding domains of troponin I at the amino acid residues 128-148, suggesting that H. longicornis P27/30 is a troponin I-like protein. By immunoblot analysis, mouse anti-recombinant P27/30 serum reacted with major constituent protein bands in extracts of adult ticks, and also immunoreacted with muscle, cuticle, gut, and salivary gland in H. longicornis ticks. Moreover, immunohistochemistry using the anti-P27/30 serum showed a strong reactivity in muscle, suggesting that native P27/30 is expressed abundantly in that tissue.     

A steroid/thyroid hormone receptor superfamily member in Drosophila melanogaster that shares extensive sequence similarity with a mammalian homologue

A gene in Drosophila melanogaster that maps cytologically to 2C1-3 on the distal portion of the X-chromosome encodes a member of the steroid/thyroid hormone receptor superfamily. The gene was isolated from an embryonic cDNA library using an oligonucleotide probe that specifies the consensus amino acid sequence in the DNA-binding domain of several human receptors. The conceptual amino acid sequence of 2C reveals at least four regions of homology that are shared with all identified vertebrate receptors. Region I includes the two cysteine-cysteine zinc fingers that comprise a DNA-binding domain which typifies all members of the superfamily. In addition, three regions (Regions II-IV) in the carboxy-terminal portion of the protein that encode the putative hormone-binding domain of the 2C gene product resemble similar sequences in vertebrate steroid/thyroid hormone receptors. The similarity suggests that this Drosophila receptor possesses many of the regulatory functions attributed to these regions in vertebrate counterparts. A portion of Region II also resembles part of the human c-jun oncoprotein's leucine zipper, which in turn, has been demonstrated to be the heterodimerization site between the jun and fos oncoproteins. The 2C receptor-like protein most resembles the mouse H2RII binding protein, a member of the superfamily which has been implicated in the regulation of major histocompatibility complex (MHC) class I gene expression. These two gene products are 83% identical in the DNA-binding domain and 50% identical in the putative hormone-binding domain, although no ligand has been identified for either protein. The high degree of similarity in the hormone-binding domain between the 2C protein and the H2RII binding protein outside regions II-IV suggests specific functional roles which are not shared by other members of the superfamily.     

Identification and complete cDNA sequence of the missing Drosophila MCMs: DmMCM3, DmMCM6 and DmMCM7

The minichromosome maintenance (MCM) gene family consists of six members (MCM2, 3, 4, 5, 6 and 7) in Saccharomyces cerevisiae as well as in humans. Each family member plays an essential role in the replication of DNA. In Drosophila melanogaster only three members, DmMCM2, DmMCM4/dpa and DmMCM5/DmCDC46, have been studied. In addition, two other partial sequences were recently reported. Using degenerate primers and low stringency PCR conditions six different DNA sequences were identified with highest sequence similarity to MCM2, 3, 4, 5, 6 and 7. Sequence analysis of full length cDNA clones corresponding to the MCM3, 6 and 7 fragment proves the existence of six MCM genes in Drosophila melanogaster. Strong homology to the human counterparts, mRNA expression analysis and physico-chemical properties suggest a conserved function in DNA replication for DmMCM3, 6 and 7.     

Isolation and characterization of cDNA encoding Argonaute, a component of RNA silencing in shrimp (Penaeus monodon)

We have identified a cDNA clone that encodes a protein with high sequence homology to Argonaute proteins of mammals and Drosophila melanogaster. The cDNA of Penaeus monodon (Pm Ago) consisted of 3178 nucleotides encoding 939-amino acid residues with a calculated molecular weight of 104 kDa. The primary structure of Pm Ago showed the presence of two signature domains, PAZ and PIWI domains that exhibit highest homology to their counterparts in D. melanogaster. The inferred protein sequence of Pm Ago was 80.8% identical with D. melanogaster and 82.1% identical with Anopheles gambiae Ago proteins. Phylogenetic analysis of Pm Ago with other invertebrate and vertebrate Argonaute proteins suggested that Pm Ago belongs to the Ago1 subfamily that plays crucial roles in stem cell differentiation or RNA interference (RNAi). Semi-quantitative RT-PCR analysis showed that the gene is highly expressed in the lymphoid organ and moderately expressed in intestine, muscle, pleopods and hemocytes. The expression of Pm Ago1 mRNA was 2-3-fold increased during the early period of viral infection but declined rapidly at 30 hour post infection. By contrast, infection of shrimp by a bacterial pathogen, Vibrio harveyi did not induce a reduction of Pm Ago1 mRNA suggesting that its expression is associated with virus infection.     

Cloning and chromosomal mapping of the human DNA polymerase theta (POLQ), the eighth human DNA polymerase.

We have cloned the cDNA for the eighth human DNA polymerase, DNA polymerase θ. The human cDNA encodes a putative DNA polymerase of 1762 amino acids with a calculated molecular mass of 198 kDa. The derived protein sequence is homologous to the Drosophila melanogaster mus308 protein product, a putative DNA polymerase-helicase involved in repair of interstrand crosslinks. The C-terminal region contains the canonical DNA polymerase motifs A, B, and C found in the family A type of DNA polymerases, which includes Escherichia coli polymerase I. The N-terminal region contains a putative ATP binding domain but not motifs for a helicase. The gene was mapped by radiation hybrid analysis to chromosome 3q within an interval flanked by proximal marker D3S1303 and distal marker D3S3576 and, based on proximity to a gene that has been mapped cytogenetically, within band 3q13.31.     

Cloning and expression of Drosophila melanogaster UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I

A TBLASTN search of the Drosophila melanogaster expressed sequence tag (EST) database with the amino acid sequence of human UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) as probe yielded a clone (GM01211) with 56% identity over 36 carboxy-terminal amino acids. A 550 base pair (bp) probe derived from the EST clone was used to screen a Drosophila cDNA library in lambda-ZAP II and two cDNAs lacking a start ATG codon were obtained. 5'-Rapid amplification of cDNA ends (5'-RACE) yielded a 2828 bp cDNA containing a full-length 1368 bp open reading frame encoding a 456 amino acid protein with putative N-terminal cytoplasmic (5 residues) and hydrophobic transmembrane (20 residues) domains. The protein showed 52% amino acid sequence identity to human GnT I. This cDNA, truncated to remove the N-terminal hydrophobic domain, was expressed in the baculovirus/Sf9 system as a secreted protein containing an N-terminal (His)6 tag. Protein purified by adsorption to and elution from nickel beads converted Man alpha1-6(Man alpha1-3)Man beta-octyl (M3-octyl) to Man alpha1-6(GlcNAc beta1-2Man alpha1-3)Man beta-octyl. The Km values (0.7 and 0.03 mM for M3-octyl and UDP-GlcNAc respectively), temperature optimum (37 degrees C), pH optimum (pH 5 to 6) and divalent cation requirements (Mn > Fe, Mg, Ni > Ba, Ca, Cd, Cu) were similar to mammalian GnT I. TBLASTN searches of the Berkeley Drosophila Genome Project database with the Drosophila GnT I cDNA sequence as probe allowed localization of the gene to chromosomal region 2R; 57A9. Comparison of the cDNA and genomic DNA sequences allowed the assignment of seven exons and six introns; all introns showed GT-AG splice site consensus sequences. This is the first insect GnT I gene to be cloned and expressed.     

The chicken IL-1 receptor: differential evolution of the cytoplasmic and extracellular domains.

The evolutionary conservation of a sequence or part of it can help to identify the essential functional and structural domains within a protein. We have cloned and characterised a cDNA coding for the type-I interleukin-1 receptor (IL-1R) of chick (ch) embryo fibroblasts. The comparison of the amino acid (aa) sequences of the avian with that of murine (m) and human (h) IL-1Rs shows a 60% homology. The intracellular domain is the most conserved region of the chIL-1R, showing 76-79% homology to the murine and human sequences, respectively. The striking conservation of the cytoplasmic region of the receptor is confirmed by its homology with the Toll receptor protein of Drosophila melanogaster. The alignment between the chicken and D. melanogaster proteins shows the presence of four aa blocks with more than 80% homology. The possible functional significance of this homology is discussed. The extracellular binding region of the receptor has a clearly recognisable immunoglobulin-like structure although the sequence divergence is higher than in the cytoplasmic domain.     

Cloning the Drosophila homolog of the xeroderma pigmentosum complementation group C gene reveals homology between the predicted human and Drosophila polypeptides and that encoded by the yeast RAD4 gene.

A human xeroderma pigmentosum group C (XPC) cDNA has been previously isolated by functional complementation (Legerski and Peterson, Nature, 359, 70-73, 1992). Sequence analysis did not reveal protein motifs which might suggest a possible biochemical function for the putative XPC protein. In order to identify functional domains in the translated XPC sequence the homologous gene from Drosophila melanogaster, designated XPCDM, was cloned by DNA hybridization. Sequence analysis of an apparently full-length cDNA revealed an open reading frame which can encode a predicted polypeptide of 1293 amino acids. Significant homology of the C-terminal 346 amino acids with both the human XPC and Saccharomyces cerevisiae Rad4 protein sequences is observed, suggesting that these proteins are functional homologs.