Cis-acting sequences in the 5'-untranslated region of the ribosomal protein A1 mRNA mediate its translational regulation during early embryogenesis of Drosophila.

The rate of ribosomal (r)-protein synthesis in the early Drosophila embryo is low despite the presence of abundant, maternally supplied r-protein mRNAs. This low rate is due to specific repression of r-protein mRNA translation. In contrast to r-protein mRNAs, most other mRNAs are efficiently translated in the early embryo. Here we report on the identification of cis-acting sequences that mediate translational repression of the r-protein A1 (rpA1) mRNA. Chimeric genes containing sequences from the translationally regulated rpA1 mRNA fused to the constitutively translated alpha-tubulin mRNA were constructed and transformed into the Drosophila germ line. Translation of the corresponding hybrid mRNAs was measured in ovaries and embryos of the transgenic flies. The results indicated that a 89-nucleotide sequence in the untranslated rpA1 mRNA leader is by itself sufficient to confer full translational regulation to a heterologous mRNA.     

Drosophila acidic ribosomal protein rpA2: Sequence and characterization

A cDNA encoding the Drosophila melanogaster acidic ribosomal protein rpA2 was cloned and sequenced. rpA2 is homologous to the Artemia salina acidic ribosomal protein eL12′. In situ hybridization to salivary gland polytene chromosomes localizes the rpA2 gene to band 21C. It is a single copy gene, with an mRNA of 0.8 kb. Two-dimensional gel electrophoresis of Drosophila ribosomal proteins followed by immuno-blotting showed that the rpA2 protein has an apparent relative mobility in SDS of 17 kD and an isoelectric point less than pH 5.0. Although the Drosophila gene rp21C may be the same as rpA2, the reported sequences differ. Comparisons of the aligned nucleotide sequences coding for the acidic ribosomal proteins rpA1 and rpA2 of Drosophila with those of other eukaryotes support the view of two separate, though closely related, groups of acidic proteins. Comparison with the Artemia homologues suggests that nucleotide identity may have been conserved by some constraint that acts in addition to the requirement for substantial similarity of amino acid sequences. © 1993 Wiley-Liss, Inc.     

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.     

Molecular cloning and characterization of the genes encoding the two subunits of Drosophila melanogaster calcineurin.

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila melanogaster genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is 67 and 65% identical to human calcineurin A alpha and beta cDNAs, respectively. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurins A, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man.     

The Drosophila ninaE gene encodes an opsin

The Drosophila ninaE gene was isolated by a multistep protocol on the basis of its homology to bovine opsin cDNA. The gene encodes the major visual pigment protein (opsin) contained in Drosophila photoreceptor cells R1-R6. The coding sequence is interrupted by four short introns. The positions of three introns are conserved with respect to positions in mammalian opsin genes. The nucleotide sequence has intermittent regions of homology to bovine opsin coding sequences. The deduced amino acid sequence reveals significant homology to vertebrate opsins; there is strong conservation of the retinal binding site and two other regions. The predicted protein secondary structure strikingly resembles that of mammalian opsins. We conclude the Drosophila and vertebrate opsin genes are derived from a common ancestor.     

Xenopus laevis ribosomal protein genes: isolation of recombinant cDNA clones and study of the genomic organization.

Poly-A+ mRNA from Xenopus laevis oocytes, partially enriched for r-protein coding capacity has been used as starting material for preparing a cDNA bank in plasmid pBR322. The clones containing sequences specific for r-proteins have been selected by translation of the complementary mRNAs. Clones for six different r-proteins have been identified and utilized as probes for studying their genomic organization. Two gene copies per haploid genome were found for r-proteins L1, L14, S19, and four-five for protein S1, S8 and L32. Moreover a population polymorphism has been observed for the genomic regions containing sequences for r-protein S1, S8 and L14.     

Sequence analysis of a processed gene coding for mouse ribosomal protein L32

The nucleotide sequence of a mouse ribosomal protein gene, identified by hybridization with the gene encoding the Drosophila ribosomal (r-) protein 49, was determined by cloning in the phage M13 and dideoxy sequencing. The mouse gene, L32', is a member of the multigene family encoding mammalian r-protein L32. L32' is a processed gene that could encode a 135 amino acid protein similar to that of mouse L32 and Drosophila r-protein 49.     

Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster

We (C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and others (N. Birgül et al. (1999) EMBO J. 18, 5892-5900) have recently cloned a Drosophila receptor that was structurally related to the mammalian galanin receptors, but turned out to be a receptor for a Drosophila peptide belonging to the insect allatostatin neuropeptide family. In the present paper, we screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to the conserved regions of the four rat (delta, kappa, mu, nociceptin/orphanin FQ) opioid receptors. This yielded alignment with a Drosophila genomic database clone that contained a DNA sequence coding for a protein having, again, structural similarities with the rat galanin receptors. Using PCR with primers coding for the presumed exons of this second Drosophila receptor gene, 5'- and 3'-RACE, and Drosophila cDNA as template, we subsequently cloned the cDNA of this receptor. The receptor cDNA codes for a protein that is strongly related to the first Drosophila receptor (60% amino acid sequence identity in the transmembrane region; 47% identity in the overall sequence) and that is, therefore, most likely to be a second Drosophila allatostatin receptor (named DAR-2). The DAR-2 gene has three introns and four exons. Two of these introns coincide with two introns in the first Drosophila receptor (DAR-1) gene, and have the same intron phasing, showing that the two receptor genes are clearly evolutionarily related. The DAR-2 gene is located at the right arm of the third chromosome, position 98 D-E. This is the first report on the existence of two different allatostatin receptors in an animal.     

Neuronal expression of a newly identified Drosophila melanogaster G protein alpha 0 subunit.

Guanine nucleotide-binding proteins (G proteins) mediate signals between activated cell-surface receptors and cellular effectors. A bovine G-protein alpha-subunit cDNA has been used to isolate similar sequences from Drosophila genomic and cDNA libraries. One class, which we call DG alpha 0, hybridized to position 47A on the second chromosome of Drosophila. The nucleotide sequence of the protein coding region of one cDNA has been determined, revealing an alpha subunit that is 81% identical with rat alpha 0. The cDNA hybridizes strongly to a 3.8 kb mRNA and weakly with a 5.3 kb message. Antibodies raised against a trp-E-DG alpha 0 fusion protein recognized a 39,000 Da protein in Drosophila extracts. In situ hybridization to adult Drosophila sections combined with immunohistochemical studies revealed expression throughout the optic lobes and central brain and in the thoracic and abdominal ganglia. DG alpha 0 message and protein were also detected in the antennae, oocytes, and ovarian nurse cells. The neuronal expression of this gene is similar to mammalian alpha 0, which is most abundantly expressed in the brain.     

cDNA sequence of a Drosophila melanogaster gene, Dfur1, encoding a protein structurally related to the subtilisin-like proprotein processing enzyme furin

Screening a genomic library of Drosophila melanogaster DNA with a human fur cDNA probe resulted in the isolation of DNA clones that apparently belonged to two different DNA regions of the Drosophila genome. Subsequently, corresponding Drosophila cDNA clones were isolated. Nucleotide sequence analysis indicated that these cDNA clones originated from two different genes, which were called Dfur1 and Dfur2. From overlapping Dfur1 cDNA clones, a composite cDNA could be constructed and analysis of its nucleotide sequence revealed the coding sequence for a protein of 899 amino acid residues. This protein, designated Dfurin1, exhibited striking sequence homology to human furin and contained the same protein domains except for the cysteine-rich region. Furthermore, unlike human furin, Dfurin1 possessed an extended amino-terminal region in which a potential transmembrane anchor was present.     

果蝇程序化死亡基因5(PDCD5)同源cDNA的克隆和序列分析

 为了解人类白血病细胞凋亡相关新基因 TFAR1 9(PDCD5,programmed cell death5)在不同种属间的序列同源性 ,利用 EST(expression sequence tag)拼接、RT- PCR、DNA序列测定技术及计算机分析技术 ,首次成功地进行了果蝇 PDCD5同源 c DNA编码区基因克隆和序列分析 .发现果蝇与小鼠及果蝇与人 PDCD5在核苷酸水平上分别有 57.5%和 57.1 %的同源性 ,在氨基酸水平上分别有 46.8%和 46.4%的同源性 .功能区分析发现 ,果蝇 PDCD5c DNA编码 1 33个氨基酸 ,计算机预测可能是一种核蛋白 ,含 5个可能的酪蛋白激酶 (casein kinase )磷酸化位点 ,2个可能的 PKC磷酸化位点 ,与人 PDCD5的功能区类似 .因而果蝇 PDCD5是与人 PDCD5同源的新基因 ,可能都与细胞程序化死亡相关 .     

Isolation and nucleotide sequence of the ribosomal protein S16-encoding gene from Aspergillus nidulans.

A genomic clone has been isolated from Aspergillus nidulans which is homologous to the ribosomal (r) protein S16-encoding gene of Saccharomyces cerevisiae (S16A) and the r-protein S19-encoding gene of rat (S19). The amino acid (aa) sequences, deduced from nucleotide (nt) sequence analysis, show that in both cases more than 63% of the aa are conserved. The proposed A. nidulans r-protein S16 gene (rps16) differs from that of S. cerevisiae in that it occurs as a single copy in the haploid genome (rather than two copies as in yeast) and contains two putative introns (rather than one). The mRNA leader is long compared to many Aspergillus genes, commencing 293 nt upstream from the coding region, and contains an open reading frame of 13 codons.     

Molecular cloning and chromosomal localization of a novel Drosophila protein phosphatase

A 1.0 kilobase cDNA coding for the complete amino acid sequence of a putative protein phosphatase (314 amino acid residues, molecular mass 36 kDa) has been isolated from a Drosophila head cDNA library. The cDNA hybridises to a single site on the right arm of the second chromosome at cytological position 55A1-3. The deduced sequence of the protein, designated protein phosphatase-Y, is homologous to the catalytic subunits of Drosophila and rabbit protein phosphatase-1 alpha (64 and 59% identity, respectively) and rabbit protein phosphatase-2A (39% identity). These and other comparisons demonstrate that this novel enzyme is not the Drosophila counterpart of mammalian protein phosphatases 1, 2A, 2B, 2C or X.     

The DNA sequence of Bombyx mori fibroin gene including the 5' flanking, mRNA coding, entire intervening and fibroin protein coding regions.

Sequence analysis of the cloned genomic fibroin gene and cDNA containing the sequence complementary to fibroin mRNA has been carried out for the regions covering the 5′ flanking, mRNA coding, entire intervening sequence and its borders, and fibroin coding sequences. The sequences determined on the gene extend from nucleotide ?552 to +1497 (assigning +1 to the cap locus); sequence analysis of the cDNA has confirmed our previous mapping of the cap locus (Tsujimoto and Suzuki, 1979). Comparison of the nucleotide sequence of the genomic gene with that of cDNA has confirmed the existence of an intervening sequence 970 bases long. The sequence comparison also pinpointed the 5′ coding-intervening junction at +64?66 and the 3′ intervening-coding junction at +1034?1036. Both the 5′ and 3′ junctions of the fibroin gene (insect) share homologous segments of about 10 nucleotides each with the published sequences of β-globin (mammal), immunoglobulin (mammal) and ovalbumin (avian) genes. A long inverted repeat sequence (17 of 23 base match) has been found next to the junctions within the intervening sequence of the fibroin gene. The repetitious sequence that codes for the Gly-Ala peptide characteristic of fibroin protein begins at position +1448. The characteristics of the N terminal portion of fibroin protein (or its precursor) are discussed, as are the features of the 5′ flanking sequence of the gene and the mRNA sequence (with special attention to the putative promoter sequence for the gene), the possible secondary structure and a sequence complementary to the 3′ end of 18S ribosomal RNA at the 5′ proximal region of fibroin mRNA.