Drosophila Ribosomal Protein Research Articles

Drosophila ribosomal protein L18a: cDNA sequence, expression and chromosomal localization of the gene

We describe the cDNA sequence of the Drosophila homologue of the rat ribosomal protein L18a. The protein sequence predicted has identical or conservatively substituted amino acids in 80% of positions. It is distinctly basic in character with an overall net positive charge of + 20. Analysis of L18a RNA with the Northern blot technique shows it to be expressed both during embryonic development and in the adult fly. In situ hybridisation to polytene chromosomes reveals that the L18a gene(s) is located at 54B on the second chromosome.

Suppression of a temperature-sensitive cdc33 mutation of yeast by a multicopy plasmid expressing a Drosophila ribosomal protein.

The Saccharomyces cerevisiae cdc33ts4-2 mutant produces a temperature-sensitive allele of the cap-binding subunit of eukaryotic initiation factor-4F (also termed eIF-4E). From a Drosophila cDNA library constructed in a multicopy yeast shuttle vector, a clone was isolated which restored the ability to grow at elevated temperature to cdc33ts4-2 cells. The rescuing Drosophila clone encodes a small ribosomal subunit protein, which we name S15a based on its molecular weight and similarity with the Brassica napus S15a ribosomal protein. Transcription of the Drosophila gene, RpS15a, occurs at all developmental stages and is enhanced during oogenesis. The ribosomal protein gene is capable of suppressing other alleles of cdc33 but not an inactivation mutation, suggesting that suppression is dependent upon the presence of the temperature-sensitive eIF-4E protein. Supporting this, Western blot analysis shows that far more eIF-4E protein is present in cdc33 yeast cells expressing the RpS15a gene than lacking it. Levels of other unrelated proteins are unaffected. We propose therefore that the expression of high levels of the Drosophila S15a ribosomal protein in the cdc33 yeast cells leads to a selective stabilization of the temperature-sensitive eIF-4E protein, which accounts for the suppression phenomenon.

The Drosophila melanogaster Homolog of Ribosomal-Protein L27a

The amino acid sequence of the Drosophila melanogaster 60S ribosomal sub-unit protein, L27a, was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L27a has 149 amino acids with a molecular weight of 17,123 Daltons. Hybridisation of the cDNA to polytene chromosomes indicates the presence of a single gene on chromosome 3R at 87F/88A. There is a single mRNA for the protein of around 650 nucleotides in length. Drosophila ribosomal protein L27a is homologous to the rat and yeast L27a and to the other members of the L15 ribosomal protein family. There is also significant homology with an invertebrate motor protein and a Drosophila photoreceptor morphogenesis protein.

The Drosophila ribosomal protein S6 gene includes a 3' triplication that arose by unequal crossing-over.

Ribosomal protein S6 (rpS6) is the major phosphoprotein of the small ribosomal subunit of eukaryotes and is phosphorylated in response to treatment with mitogens and other stimuli. We have examined the organization of the rpS6 gene of Drosophila melanogaster. Comparisons of a cDNA with genomic DNA identify a transcription unit including three exons. Two tandem repeats downstream of this transcription unit reiterate divergent copies of the third exon and flanking regions. Comparisons of these three repeats with respect to nucleotide base substitutions and deletions or insertions show clearly that they arose via a duplication and subsequent crossing-over between misaligned copies. Although no direct evidence exists that the downstream exons are transcribed, the maintenance of open reading frames in spite of extensive genetic changes is consistent with a protein-coding function.

Type XIV collagen is encoded by alternative transcripts with distinct 5' regions and is a multidomain protein with homologies to von Willebrand's factor, fibronectin, and other matrix proteins

The combined nucleotide sequences of several overlapping cDNAs provide the first complete amino acid sequence of type XIV collagen. Independent confirmation of the deduced sequence is provided by amino acid sequencing of several tryptic peptides isolated from purified chicken skin type XIV collagen. Comparative analyses show that the amino-terminal non-triple-helical region of alpha 1(XIV) chains contains sequence motifs that are similar to alpha 1(IX) collagen, fibronectin type III repeats, and von Willebrand's factor A-domains. The results also strongly suggest that the alpha 1(XIV) collagen gene is identical to the gene encoding the matrix component previously named undulin. cDNAs covering the 5' region of alpha 1(XIV) mRNA fall into two classes with distinct sequences in their 5'-untranslated regions. We believe the two alternative sequences result from differential splicing of the primary transcript. Interestingly, one of the untranslated sequences shows a high degree of identity with the cis-regulatory translational control sequence in the 5'-untranslated region of a Drosophila ribosomal protein mRNA. We hypothesize therefore that the sequence in alpha 1(XIV) collagen may play a role in the control of alpha 1(XIV) protein synthesis.

A Drosophila ribosomal protein functions in mammalian cells.

A cDNA expression vector encoding Drosophila ribosomal protein S14 was transfected into cultured Chinese hamster ovary (CHO) cells that harbor a recessive RPS14 emetine resistance mutation. Transformants synthesized the insect mRNA and polypeptide and consequently displayed an emetine-sensitive phenotype. These observations indicate that the insect protein was accurately expressed and correctly assembled into functional mammalian 40S ribosomal subunits.

A Drosophila Ribosomal Protein Functions in Mammalian Cells

A cDNA expression vector encoding Drosophila ribosomal protein S14 was transfected into cultured Chinese hamster ovary (CHO) cells that harbor a recessive RPS14 emetine resistance mutation. Transformants synthesized the insect mRNA and polypeptide and consequently displayed an emetine-sensitive phenotype. These observations indicate that the insect protein was accurately expressed and correctly assembled into functional mammalian 40S ribosomal subunits.

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 M 13 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.

Ribosomal protein S14 is encoded by a pair of highly conserved, adjacent genes on the X chromosome of Drosophila melanogaster.

We describe a Drosophila DNA clone of tandemly duplicated genes encoding an amino acid sequence nearly identical to human ribosomal protein S14 and yeast rp59. Despite their remarkably similar exons, the locations and sizes of introns differ radically among the Drosophila, human, and yeast (Saccharomyces cerevisiae) ribosomal protein genes. Transcripts of both Drosophila RPS14 genes were detected in embryonic and adult tissues and are the same length as mammalian S14 message. Drosophila RPS14 was mapped to region 7C5-9 on the X chromosome. This interval also encodes a previously characterized Minute locus, M(1)7C.

Ribosomal protein S14 is encoded by a pair of highly conserved, adjacent genes on the X chromosome of Drosophila melanogaster.

We describe a Drosophila DNA clone of tandemly duplicated genes encoding an amino acid sequence nearly identical to human ribosomal protein S14 and yeast rp59. Despite their remarkably similar exons, the locations and sizes of introns differ radically among the Drosophila, human, and yeast (Saccharomyces cerevisiae) ribosomal protein genes. Transcripts of both Drosophila RPS14 genes were detected in embryonic and adult tissues and are the same length as mammalian S14 message. Drosophila RPS14 was mapped to region 7C5-9 on the X chromosome. This interval also encodes a previously characterized Minute locus, M(1)7C.

Isolation of a mouse DNA fragment with homology to a Drosophila ribosomal protein gene

A mouse genomic library in λCharon 4A was screened for putative ribosomal protein genes using a fragment of the gene encoding Drosophila ribosomal protein 49 as a hybridization probe under nonstringent hybridization conditions. A recombinant phage was selected and its restriction enzyme map determined. The major species of mouse poly(A) + mRNA homologous to the putative gene is about 740 nucleotides long.

A Drosophila ribosomal protein gene is located near repeated sequences including rDNA sequences.

We have isolated a cloned segment of Drosophila genomic DNA containing a ribosomal protein gene. Hybridization analysis of the DNA in this clone indicates a complex organization of repeated elements within this cloned segment. At least one of these repeated elements is homologous to regions of rDNA. Restriction analysis of the clone shows that some of the repeated elements are present as tandem duplications and in scattered locations within the cloned DNA segment. There are also three non-ribosomal protein genes contained in this clone, each of which is expressed along with the ribosomal protein gene into RNA species present in Drosophila embryos.

The in vivo expression of pseudo ribosomal RNA genes in Drosophila melanogaster.

Electron microscopic examination of chromatin has indicated that there are two major classes of ribosomal transcription units (rTUs) in Drosophila melanogaster. (a) a standard class which is about 8 kb in length and (b) a longer class (up to 15 kb) which has been hypothesized as having an intron in the 28S region of the gene. On rare occasions, we have observed a third class of TUs, which we term pseudo rTUs (or pseudo ribosomal RNA genes), distributed among the standard rTUs. Pseudo rTUs are composed of short fiber arrays with clear gradients in their RNP fiber lengths. The pseudo rTUs observed in egg chambers and blastoderm embryos are 4.1 +/- 0.58 kb in length and are found in tandem with non-transcribed spacer regions. The lengths of the non-transcribed spacer regions are found in 2 classes: one at 4.94 +/- 1.78 kb and the other at 19.28 +/- 2.11 kb. The present information suggests that these pseudo rTU fiber arrays are ribosomal in origin as their RNP fibers cross-react with antibodies raised against Drosophila ribosomal proteins. The pattern of distribution of D. melanogaster ribosomal protein L4 on RNP fibers in standard rTUs is compared with that in pseudo rTUs. This was determined by a novel approach involving the use of electron microscopic spreads of rTUs to which had been added IgGs labelled with polymethacrylate spheres. Pseudo ribosomal RNA genes are present in both the X (6%) and in the Y (6%) ribosomal chromatin as is indicated by their existence in nurse cells of both Oregon-R females, and females of the genotype sc4sc8/sc4sc8/y+Y.

Purification of Drosophila ribosomal proteins. Isolation of proteins S8, S13, S14, S16, S19, S20/L24, S22/L26, S24, S25/S27, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 7/8, 9, and 11.

The proteins of Drosophila melanogaster embryonic ribosomes were separated into seven groups (A80 through G80) by stepwise elution from carboxymethylcellulose with lithium chloride at pH 6.5 by procedures previously described [Chooi, W. Y., Sabatini, L. M., MacKlin, M. D., & Fraser, W. (1980) Biochemistry 19, 1425-1433]. Three relatively acidic proteins, S14, S25/S27, and 7/8, have now been isolated from group A80 by ion-exchange chromatog raphy on carboxymethylcellulose eluted with a linear gradient of lithium chloride at pH 4.2. Fractions containing the relatively basic proteins (groups B80 through G80) were furher combined into a total of 24 "pools". The criterion for combination was the migration patterns in one-dimensional polyacrylamide gels containing sodium dodecyl sulfate (NaDodS04) of every fifth fraction from the carboxymethylcellulose column. Each pool contained between 1 and 12 major proteins. Proteins S8, S13, S16, S19, S20/L24, S22/L26, S24, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 9, and 11 have now been isolated from selected pools by gel filtration through Sephadix G-100. The amount of each protein recovered from a starting amount of 1.8 g of total 80S proteins varied form 0.2 to 10.8 mg. Five proteins had no detectable contamination, and in each of the others the impurities were no greater than 9%. The amino acid composition of the individual purified proteins was determined. The molecular weights of the proteins were estimated by polyacrylamide gel electrophoresis in NaDodSO4.