The primary structure of rat ribosomal protein L37a

The amino acid sequence of rat ribosomal protein L37a was deduced from the sequence of nucleotides in recombinant cDNAs isolated in Yamagata and in Chicago and confirmed from the NH2-terminal amino acid sequence of the protein. Ribosomal protein L37a contains 91 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular mass of 10143 Da.     

A green fluorescent protein enhancer trap screen in Drosophila photoreceptor cells

The Drosophila ommatidia contain two classes of photoreceptor cells (PR's), the outer and the inner PR's. We performed an enhancer trap screen in order to target genes specifically expressed in PR's. Using the UAS/GAL4 method with enhanced green fluorescent protein (eGFP) as a vital marker, we screened 180000 flies. Out of 2730 lines exhibiting new eGFP patterns, we focused on 16 lines expressing eGFP in particular subsets of PR's. In particular, we describe three lines inserted near the spalt major, m-spondin and furrowed genes, whose respective expression patterns resemble those genes. These genes had not been reported to be expressed in the adult eye. These examples clearly show the ability of our screen to target genes expressed in the adult Drosophila eye.     

An enhancer trap line associated with a D-class cyclin gene in Arabidopsis

In yeast and animals, cyclins have been demonstrated to be important regulators of cell cycle progression. In recent years, a large number of A-, B-, and D-class cyclins have been isolated from a variety of plant species. One class of cyclins, the D-class cyclins, is important for progression through G1 phase of the cell cycle. In Arabidopsis, four D-class cyclins have been isolated and characterized (CYCLIN-D1;1, CYCLIN-D2;1, CYCLIN-D3;1, and CYCLIN-D4;1). In this report we describe the characterization of a fifth D-class cyclin gene, CYCLIN-D3;2 (CYCD3;2), from Arabidopsis. An enhancer trap line, line 5580, contains a T-DNA insertion in CYCD3;2. Enhancer trap line 5580 exhibits expression in young vegetative and floral primordia. In line 5580, T-DNA is inserted in the first exon of the CYCD3;2 gene; in homozygous 5580 plants CYCD3;2 RNA is not detectable. Even though CYCD3;2 gene function is eliminated, homozygous 5580 plants do not exhibit an obvious growth or developmental phenotype. Via in situ hybridization we demonstrate that CYCD3;2 RNA is expressed in developing vegetative and floral primordia. In addition, CYCD3;2 is also capable of rescuing a yeast strain that is deficient in G1 cyclin activity.     

The primary structure of chicken ribosomal protein L37a.

The amino acid sequence of chicken ribosomal protein L37a was deduced from the nucleotide sequence of a recombinant cDNA and its genomic DNA. Chicken ribosomal protein L37a has 92 amino acids and a molecular mass of 10,247 Da including the initiator methionine. The protein contains a typical Cys2Cys2 zinc finger motif, which may be involved in protein-RNA interaction.     

An enhancer trap in the ascidian Ciona intestinalis identifies enhancers of its Musashi orthologous gene

The enhancer trap technique, established in Drosophila melanogaster, is a very sophisticated tool. Despite its usefulness, however, there have been very few reports on enhancer traps in other animals. The ascidian Ciona intestinalis, a splendid experimental system for developmental biology, provides good material for developmental genetics. Recently, germline transgenesis of C. intestinalis has been achieved using the Tc1/mariner superfamily transposon Minos. During the course of that study, one Minos insertion line that showed a different GFP expression pattern from other lines was isolated. One fascinating possibility is that an enhancer trap event occurred in this line. Here we show that a Minos insertion in the Ci-Musashi gene was responsible for the altered GFP expression. Ci-Musashi showed a similar expression pattern to GFP. In addition, introns of Ci-Musashi have enhancer activity that can alter the expression pattern of nearby genes to resemble that of GFP in this line. These results clearly demonstrate that an enhancer trap event that entrapped enhancers of Ci-Musashi occurred in C. intestinalis.     

The Drosophila homologue of ribosomal protein L8.

We have cloned the gene encoding the Drosophila melanogaster homologue of ribosomal protein L8. It contains two introns: one in the 5' untranslated region and the second in the beginning of the ORF, and encodes a 256-residue protein which is highly conserved when compared with RpL8 proteins of other organisms. The gene is present as a single copy in the Drosophila genome and maps at position 62E6-7 on polytene chromosomes. It is expressed ubiquitously at all stages of development. It is located close to the gene encoding RpL12 and both are candidate targets of the Minute mutation, M(3)LS2, mapped in the region 62E-63A.     

An enhancer trap zebrafish line for lateral line development and regulation of six2b expression

Zebrafish lateral line system which is derived from neurogenic placodes has become a popular model for developmental biology since its formation involves cell migration, pattern formation, organogenesis, and hair cell regeneration. Transgenic lines play a crucial role in lateral line system study. Here, we identified an enhancer trap transgenic zebrafish line Et(gata2a:EGFP)189b (ET189b for short), which expressed enhanced green fluorescent protein (EGFP) in the pituitary, otic, and lateral line placodes and their derivatives. Especially, in neuromast, the accessory cells rather than hair cells were labeled by EGFP. Furthermore, we found the Tol2 transposon construct is integrated at the proximal upstream region of six2b gene locus. And EGFP expression of ET189b closely reflects the expression of endogenous six2b during development and after dkk1b over-expression. Taken together, our results indicated that ET189b is an ideal line for research on lateral line development and regulation of six2b expression.     

Binding of the human homolog of the Drosophila discs large tumor suppressor protein to the mitochondrial ribosomal protein MRP-S34

The human homolog of the Drosophila discs large tumor suppressor protein (hDLG) functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. Here we show that hDLG interacts through its PDZ domains with the carboxy-terminal S/TXV motif of the mitochondrial ribosomal protein S-34 (MRP-S34). Our results suggest that hDLG interacts with MRP-S34 prior to entry of MRP-S34 into the mitochondria and may be involved in the trafficking of MRP-S34.     

Identification and characterization of the gene for Drosophila L3 ribosomal protein

A cDNA clone that encodes a Drosophila homologue of ribosomal protein L3 was isolated from a Drosophila ovary gridded cDNA library. The Drosophila ribosomal protein L3 gene (RpL3) is highly conserved with ribosomal protein L3 genes in other organisms. It is a single copy gene and maps to position 86D5–10 on polytene chromosomes. A Minute gene in this region, M(3)86D, is a possible candidate to encode RPL3. RPL3 message is expressed ubiquitously. A partial RPL8 cDNA clone was also isolated and mapped to 62F.     

Isolation and molecular characterization of the ribosomal protein L6 homolog from Chlamydia trachomatis.

The cloning of a Chlamydia trachomatis eukaryotic cell-binding protein reported earlier from our laboratory (R. Kaul, K. L. Roy, and W. M. Wenman, J. Bacteriol. 169:5152-5156, 1987) represents an artifact generated by nonspecific recombination of chromosomal DNA fragments. However, the amino terminus of this plasmid-encoded fusion product demonstrated significant homology to Escherichia coli ribosomal protein L6. By using a 458-bp PstI-HindIII fragment of recombinant pCT161/18 (representing the 5' end of the cloned gene), we isolated and characterized a C. trachomatis homolog of the ribosomal protein L6 gene of E. coli. Sequence analysis of an 1,194-bp EcoRI-SacI fragment that encodes chlamydial L6 (designated CtaL6e) revealed a 552-bp open reading frame comprising 183 amino acids and encodes a protein with a molecular weight of 19,839. Interestingly, complete gene homology between C. trachomatis serovars L2 and J, each of which exists as a single copy per genome, was observed. Expression of a plasmid-encoded gene product is dependent on the lac promoter, since no product was obtained if the open reading frame was oriented in opposition to the lac promoter. Immunoblotting of purified ribosomes revealed functional, as well as antigenic, homology between the E. coli and C. trachomatis ribosomal L6 proteins.     

GAL4 enhancer trap targeting of the Drosophila sex determination gene fruitless

The fru4 allele of the sex determination gene fruitless is induced by insertion of a P[lacZ,ry+] enhancer trap element. This insert also acts to disrupt expression of the fru P1 promoter derived male-specific proteins, consequently impairing male courtship behavior. fru4 maps less than 2 kb upstream of the fru P3 promoter, whose function is essential for viability. We replaced this insert with a GAL4 element, P[GAL4,w+], recovering two lines with insertions in opposite orientations at the locus, one of which demonstrated fru-specific mutant phenotypes. Reporter expression of these lines recapitulated that of P3- and P4-derived proteins which, when correlated with a developmental and tissue specific survey of fru promoters' activities, uncovered a previously unsuspected complexity of fru regulation. These novel fru alleles provide the tools for manipulation of fru-expressing cells, allowing the consequent effects to be related back to specific fru functions and the regulatory units controlling these activities.     

Archaeal ribosomal protein L7 is a functional homolog of the eukaryotic 15.5kD/Snu13p snoRNP core protein

Recent investigations have identified homologs of eukaryotic box C/D small nucleolar RNAs (snoRNAs) in Archaea termed sRNAs. Archaeal homologs of the box C/D snoRNP core proteins fibrillarin and Nop56/58 have also been identified but a homolog for the eukaryotic 15.5kD snoRNP protein has not been described. Our sequence analysis of archaeal genomes reveals that the highly conserved ribosomal protein L7 exhibits extensive homology with the eukaryotic 15.5kD protein. Protein binding studies demonstrate that recombinant Methanoccocus jannaschii L7 protein binds the box C/D snoRNA core motif with the same specificity and affinity as the eukaryotic 15.5kD protein. Identical to the eukaryotic 15.5kD core protein, archaeal L7 requires a correctly folded box C/D core motif and intact boxes C and D. Mutational analysis demonstrates that critical features of the box C/D core motif essential for 15.5kD binding are also required for L7 interaction. These include stem I which juxtaposes boxes C and D, as well as the sheared G:A pairs and protruded pyrimidine nucleotide of the asymmetric bulge region. The demonstrated presence of L7Ae in the Haloarcula marismortui 50S ribosomal subunit, taken with our demonstration of the ability of L7 to bind to the box C/D snoRNA core motif, indicates that this protein serves a dual role in Archaea. L7 functioning as both an sRNP core protein and a ribosomal protein could potentially regulate and coordinate sRNP assembly with ribosome biogenesis.     

Identification of a mammalian mitochondrial homolog of ribosomal protein S7

Bovine mitochondrial small subunit ribosomal proteins were separated by two-dimensional electrophoresis. The region containing the most basic protein(s) was excised and the protein(s) present subjected to in-gel digestion with trypsin. Electrospray tandem mass spectrometry was used to provide sequence information on some of the peptide products. Searches of the human EST database using the sequence of the longest peptide analyzed indicated that this peptide was from the mammalian mitochondrial homolog of prokaryotic ribosomal protein S7 (MRP S7(human)). MRP S7(human) is a 28-kDa protein with a pI of 10. Significant homology to bacterial S7 is observed especially in the C-terminal half of the protein. Surprisingly, MRP S7(human) shows less homology to the corresponding mitochondrial proteins from plants and fungi than to bacterial S7.     

Zebrafish enhancer trap line showing maternal and neural expression of kctd15a

Potassium channel tetramerization domain containing proteins (KCTDs), which share a conserved BTB (Bric-a-brac, Tramtrack, Broad complex) domain at their N-terminus, are known to be involved in both developmental and neural processes. However, the developmental expression patterns and functional roles of most vertebrate KCTDs remain unknown. Using enhancer-trapping technology, we have identified a transgenic zebrafish line (ub49) where the vector insertion is in close proximity to kctd15a, and where transgenic marker (eGFP) expression closely reflects endogenous kctd15a expression. Both ub49 and kctd15a show strong maternal expression that suggests a functional role during epiboly and gastrulation. At later developmental stages, expression of eGFP in ub49 also shares the same spatiotemporal features as kctd15a in several neural tissues, including cranial placode precursors, retina, and different areas of the developing brain. In the retina, we observed eGFP labeling of the inner nuclear layer (INL), including a heterogenous population of amacrine cells, and both laminae of the inner plexiform layer (IPL). This expression pattern suggests that Kctd15a proteins have several context-dependent functional roles in both developmental and neural processes. The enhancer trap line, which is the first transgenic reporter of Kctd gene expression in vertebrates, also provides a novel tool to study kctd15a function in vivo.