Molecular cloning and genomic organization of a novel receptor from Drosophila melanogaster structurally related to mammalian galanin receptors

We screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to conserved amino acid sequences from the five known rat somatostatin receptors. This yielded alignment with a Drosophila genomic clone that contained a DNA sequence coding for a protein, having amino acid sequence identities with the rat galanin receptors. Using PCR with Drosophila cDNA as a template, and oligonucleotide probes coding for the exons of the presumed Drosophila gene, we were able to clone the cDNA for this receptor. The Drosophila receptor has most amino acid sequence identity with the three mammalian galanin receptors (37% identity with the rat galanin receptor type-1, 32% identity with type-2, and 29% identity with type-3). Less sequence identity exists with the mammalian opioid/nociceptin-orphanin FQ receptors (26% identity with the rat micro opioid receptor), and mammalian somatostatin receptors (25% identity with the rat somatostatin receptor type-2). The novel Drosophila receptor gene contains ten introns and eleven exons and is located at the distal end of the X chromosome.     

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.     

A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes.

The murine gene lambda 5 is selectively expressed in pre-B lymphocytes. Of the three exons encoding lambda 5, exons II and III show strong homologies to immunoglobulin lambda light (L) chain gene segments, i.e. to J lambda intron and exon, and C lambda exon sequences respectively. We have now found, 4.6 kb upstream of lambda 5, another gene composed of two exons which is selectively expressed in pre-B cell lines as a 0.85 kb mRNA potentially coding for a protein of 142 amino acids including a 19 amino acid-long signal peptide. The 5' sequences of this gene show homologies to sequences encoding the variable regions of kappa and lambda L chains and of heavy (H) chains. The deduced amino acid sequence contains the consensus cysteine residues as well as other consensus amino acids at positions which characterize immunoglobulin (Ig) domains. We call the second gene VpreB. The 3' end of VpreB encoding the 26 carboxyl terminal amino acids shows no homology to any known nucleotide sequence. The putative protein encoded by VpreB is a potential candidate for association with the putative protein encoded by lambda 5, and thereby a candidate for association with H chains in pre-B cells. Southern blot analysis of DNA from liver (germ line) and 70Z/3 pre-B cell lines reveals two genes which hybridize to the VpreB gene. We call VpreB1 the gene which is found 5' of lambda 5. The other gene, called VpreB2, which has not yet been located within the genome, shows 97% nucleotide sequence homology to VpreB1 in an area of 1 kb which covers the coding region of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and expression pattern of Bmlark, a homolog of the Drosophila gene lark in Bombyx mori.

Two Bombyx mori isoforms of the gene lark, which is shown to play an important role in Drosophila circadian rhythms, were identified and named Bmlark-PA and Bmlark-PB, respectively. Bmlark-PA consists of 5 exons and encodes a protein of 343 amino acid residues which contains 3 functional domains: two RRM (RNA recognization motif) domains and an RTZF (retroviral-type zinc finger) and shares 72% identity with the Drosophila gene lark at the amino acid level. Bmlark-PB lacks the sequence between 118 and 791 nt of Bmlark-PA and codes for a protein of 68 amino acid residues, which contains no distinct functional domains. Alignments of the cDNAs of Bmlark to the genomic draft sequence of B. mori showed that the gene Bmlark had a single copy in the genome, suggesting that an alternative splicing mechanism occurs in the gene Bmlark. RT-PCR analysis indicated that Bmlark-PA was expressed only in late pupae and adult but Bmlark-PB was broadly expressed in many tissues and throughout the developmental stages from embryo to adult.     

Concerted and divergent evolution within the rat gamma-crystallin gene family

The nucleotide sequences of six rat gamma-crystallin genes have been determined. All genes have the same mosaic structure: the first exons contain a relatively short (25 to 44 base-pair) 5' non-coding region and the first nine base-pairs of the coding sequence, the second exons encode protein motifs I and II, while protein motifs III and IV are encoded by the third exons. The third exons also contain a 60 to 67-base-pair long 3' non-coding region. In the gamma 1-2 gene, the splice acceptor site of the third exon has been shifted three base-pairs upstream. Hence, the protein product of this gene is one amino acid residue longer. The first introns, though varying in length from 85 to 100 base-pairs, are conserved in sequence. The second introns vary considerably in length (0.9 X 10(3) to 1.9 X 10(3) base-pairs) and sequence. The second exons of the genes show concerted evolution and have undergone multiple gene conversions. In contrast, the third exons show divergent evolution. From the sequences of the third exons, an evolutionary tree of the gene family was constructed. This tree suggests that three of the present genes derive directly from the genes that originated from a tandem duplication of a two-gene cluster. Two duplications of the last gene of the four-gene cluster then yielded the other three genes. Region a' of the third exon, encoding protein motif III, is variable, while the region encoding protein motif IV (b') is constant. We postulate that this variability in region a' is due to a period of radiation after each gene duplication. A comparison of the rat sequences with those of orthologous sequences from other species shows that the variation in region a' is now preserved. Hence, it might specify the specific functional property of each gamma-crystallin protein within the lens.     

Analysis of apolipoprotein E5 gene from a patient with hyperlipoproteinemia

We have isolated and analyzed apolipoprotein E5 gene from a patient with hyperlipoproteinemia. Apolipoprotein E5 is a variant of apolipoprotein E with two additional units of positive charge and smaller apparent molecular weight than apolipoprotein E3, which is the major isoform of apolipoprotein E. The heterozygous gene of apolipoprotein E5/3 from the patient was cloned into lambda phage. The cloned apolipoprotein E genes were subcloned into a murine retrovirus shuttle vector and were expressed. One out of four clones expressed apolipoprotein E5. The analysis of the nucleotide sequence of the exons and exon-intron boundary regions has shown a G to A substitution in the 18th nucleotide from the 5'-end of the third exon. This single base substitution changes the amino acid residue Glu to Lys at the third position from the amino-terminus of the mature protein, and gives two additional units of positive charge to the molecule.     

Isolation of the gene encoding the Drosophila melanogaster homolog of the Saccharomyces cerevisiae GCN2 eIF-2alpha kinase.

Genomic and cDNA clones homologous to the yeast GCN2 eIF-2alpha kinase (yGCN2) were isolated from Drosophila melanogaster. The identity of the Drosophila GCN2 (dGCN2) gene is supported by the unique combination of sequence encoding a protein kinase catalytic domain and a domain homologous to histidyl-tRNA synthetase and by the ability of dGCN2 to complement a deletion mutant of the yeast GCN2 gene. Complementation of Deltagcn2 in yeast by dGCN2 depends on the presence of the critical regulatory phosphorylation site (serine 51) of eIF-2alpha. dGCN2 is composed of 10 exons encoding a protein of 1589 amino acids. dGCN2 mRNA is expressed throughout Drosophila development and is particularly abundant at the earliest stages of embryogenesis. The dGCN2 gene was cytogenetically and physically mapped to the right arm of the third chromosome at 100C3 in STS Dm2514. The discovery of GCN2 in higher eukaryotes is somewhat unexpected given the marked differences between the amino acid biosynthetic pathways of yeast vs. Drosophila and other higher eukaryotes. Despite these differences, the presence of GCN2 in Drosophila suggests at least partial conservation from yeast to multicellular organisms of the mechanisms responding to amino acid deprivation.     

Isolation and characterization of the Drosophila translational elongation factor 2 gene.

We have isolated a cDNA clone that encodes the Drosophila melanogaster elongation factor 2 (EF2), a protein involved in the elongation step of protein synthesis. This identification was based on the high degree of its amino acid sequence identity (greater than 80%) to that of hamster EF2. The gene encoding Drosophila EF2 is found at position 39E-F of the 2L chromosomal arm and maybe identical to the M(2)H locus, which produces a Minute phenotype when mutated. The genomic organization of the locus includes four exons. Conserved sequence segments shared with a variety of GTP binding proteins are found in the amino terminal third of the protein, and segments unique to EF2 and its prokaryotic functional homolog, EF-G, are in the carboxy terminal half; these two regions are segregated in two respective exons.     

Structure of the RESA gene of Plasmodium falciparum.

We have determined the nucleotide sequence of the gene encoding the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum, an antigen that has been shown to confer protective immunity on monkeys. The sequence has enabled us to predict the structure of the RESA gene and the amino acid sequence of its protein product. The gene consists of two exons with a short intron located near the 5' end of the coding region. A hydrophobic amino acid segment predicted for the 3' end of exon 1 is consistent with the possibility that exon 1 encodes trafficking signal sequences. We show that restriction fragment length polymorphisms can be used to define two different alleles of RESA, represented by isolates FC27 and NF7, and compare the FC27 sequence with that of a long cDNA clone from NF7 described previously.