Genes Rpl2 Research Articles

Dexamethasone stimulates ribosomal protein L32 gene transcription in rat myoblasts

Incubation of rat L6 myoblasts for 24 h with 10 −7 M dexamethasone, a glucocorticoid analogue, resulted in a 2.5-fold increase in the rate of ribosomal protein L32 (rpL32) gene transcription with a corresponding increase in the level of rpL32 mRNA. The increased rate of transcription was accompanied by a dramatic enhancement in binding of the δ, but not β and γ, factors to the rpL32 gene promoter as measured by gel mobility shift assays. This increased binding reflects a change in the activity of the δ factor since its level is unchanged by dexamethasone treatment. The presence of the glucocorticoid analogue RU38486 reversed the stimulating effect of dexamethasone on rpL32 gene transcription and binding of the δ factor to the δ element. These results suggest that the mechanism which enhances rpL32 gene transcription in dexamethasone-treated rat L6 myoblasts involves glucocorticoid-receptor mediated changes in the activity of the δ factor.

Genomic organization and chromosome location of the murine Rpl23 gene

The mouse gene coding for ribosomal protein L23 (Rpl23) has been fully sequenced, including 580 bp of the 5′ upstream region. The 5-kb gene comprises 5 exons and contains an unusually long (3,153 bp) third intron. The gene was mapped to the distal region of mouse chromosome 11, homologous to human chromosome 17q21→q22.

The ribosomal protein L34 gene from the mosquito, Aedes albopictus: exon–intron organization, copy number, and potential regulatory elements

We describe the structural analysis of genomic DNA encoding ribosomal protein (rp) L34 from the mosquito, Aedes albopictus. Comparison of genomic DNA sequences encompassing approximately 8 kb with the rpL34 cDNA sequence showed that the gene contains three exons and two introns, encoding a primary transcript with a deduced size of 6196 nucleotides from the transcription start site to the polyadenylation site. Exon 1, which is not translated, measures only 45 bp, and is separated from Exon 2 by a 359 bp intron. Exon 2 measures 78 bp, and contains the AUG translation initiation codon 14 nucleotides downstream of its 5′-end. Downstream of Exon 2 is a 5270 bp intron, followed by the remainder of the coding sequence in Exon 3, which measures 444 bp including the polyadenylation signal. We used a novel PCR-based procedure to obtain 1.7 kb of DNA upstream of the rpL34 gene. Like the previously described Ae. albopictus rpL8 gene and various mammalian rp genes, the DNA immediately upstream of the rpL34 gene lacks the TATA box, and the rpL34 transcription initiation site is embedded in a characteristic polypyrimidine tract. The 5′-flanking DNA contained a number of cis-acting elements that potentially interact with transcription factors characterized by basic domains, zinc-coordinating DNA binding domains, helix-turn-helix motifs, and beta scaffold factors with minor groove contacts. Particularly striking was the conservation of an AP-4 binding site within 100 nucleotides upstream of the transcription initiation site in both Aal-rpL34 and Aal-rpL8 genes. Comparison of Southern hybridization signals using probes from the 5′ and 3′-ends of the 5.3 kb second intron and the cDNA suggested that the Ae. albopictus rpL34 gene most likely occurs as a single expressed copy per haploid genome with restriction enzyme polymorphisms in the upstream flanking DNA and the likely presence of one or more pseudogenes.

Cloning cDNAs for Genes Preferentially Expressed during Fruit Growth in Cucumber

A cDNA library was constructed from poly(A)+RNA extracted from pollinated fruit of `PMR-142' cucumber (Cucumis sativus L.). Subtraction hybridization was made between the cDNAs and poly(A)+RNA from unpollinated fruit to isolate cDNA clones that corresponded to the genes preferentially expressed in the pollinated fruit. We isolated three cDNAs, which were 756, 826, and 998 nucleotides long and designated Csf1, Csf2, and Csf3, respectively. When fruit growth was triggered by pollination, auxin treatment and natural parthenocarpy, Csf2 was always expressed. Time course of expression of the Csf2 gene was nearly parallel to that of the fruit growth. Nucleotide sequences of the Csf cDNAs were fully determined. Homology of the deduced amino acid sequence for Csf1 showed 75% identity with a pea extensin. Only 37%, 33%, and 26% homology was found between Csf2 and bell pepper CaSn-2, tobacco FB7-4, and opium poppy gMLP15, respectively. The Csf3 sequence showed 68% identity with the large subunit of 60S ribosomal protein L3 of Arabidopsis thaliana.

The mouse H19 locus mediates a transition between imprinted and non-imprinted DNA replication patterns.

Genes subject to genomic imprinting generally occur in clusters of hundreds of kilobases. These domains exhibit several gamete of origin-dependent manifestations, including a pattern of asynchronous replication when studied by fluorescence in situ hybridization (FISH). We find a transition from asynchronous replication at the imprinted mouse H19 gene to synchronous replication at the downstream Rpl23 gene, the human homologue of which appears to be non-imprinted. Two-colour FISH demonstrates that this transition is due solely to a difference in replication timing between the upstream and downstream chromatin on the later-replicating (maternal) chromosome. This difference is lost in mice deleted for the H19 gene body and 9.9 kb of upstream DNA when this deletion is maternally inherited, with synchronous replication patterns extending over 110 kb upstream from the deleted area. No effect is seen when the deletion is paternally inherited. The presence of a boundary element in this region has been suggested by observations of position-independent expression of H19 -containing transgenes and the blocking of accessibility of downstream enhancers to the upstream Igf2 and Ins2 genes on the maternal chromosome. The FISH studies presented here demonstrate the insulation of replication patterns within the imprinted domain from downstream, non-imprinted chromatin, mediated by an element at the H19 locus which is subject to genomic imprinting.

S2F, a leaf-specific trans-acting factor, binds to a novel cis-acting element and differentially activates the RPL21 gene.

Tissue-specific factors control the differential expression of nuclear genes encoding plastid proteins. To identify some of these factors, the light-independent spinach RPL21 gene encoding the plastid ribosomal protein L21 was chosen as a model. The RPL21 promoter organization was defined by transient and stable transfections of RPL21 promoter deletion mutants fused to a reporter gene. The following results were obtained. (1) We identified a strong core promoter, spanning the transcription start site region, sufficient to drive high levels of gene expression. (2) We identified two non-overlapping positive and negative domains, located upstream from the core promoter region, that modulate core promoter activity independently of light. (3) We found that the positive domain contains a new cis-acting element, the S2 site, related to but different from the light-responsive GT-1 binding site. We show that the S2 site binds a leaf-specific nuclear factor (named S2F). The S2 site is conserved in the promoter region of many nuclear genes encoding plastid proteins. Experiments with transgenic tobacco plants confirmed that the S2 site is critical for positive domain activity in leaf tissues. The S2 site is thus identified as a new tissue-specific, light-independent regulatory element.

S2F, a Leaf-Specific trans-Acting Factor, Binds to a Novel cis-Acting Element and Differentially Activates the RPL21 Gene

S2F, a Leaf-Specific trans-Acting Factor, Binds to a Novel cis-Acting Element and Differentially Activates the RPL21 Gene

Polymorphism for the Presence of the rpL2 Intron in Chloroplast Genomes of Bauhinia (Leguminosae)

The intron of the chloroplast gene rpl2 is absent from relatively few lineages of angiosperms, and is thus potentially a useful phylogenetic character. A previous extensive survey of Leguminosae showed that the intron was missing from chloroplast genomes of one of the two species sampled from the large caesalpinioid genus Bauhinia. A survey of 78 species of this genus, representing all four subgenera and most of the currently recognized sections, shows that the intron is absent from numerous species of the genus, in a pattern that is correlated, albeit imperfectly, with taxonomic groupings. The intron is present in chloroplast genomes of two of three species sampled from the small subg. Elayuna, all but one species of subg. Phanera, and the monotypic subg. Barklya. In contrast, the intron appears to be absent from the gene in nearly all species of subg. Bauhinia. Three species in two sections of subg. Bauhinia were found to be polymorphic for the presence of the intron. Comparisons of the distribution of the intron with current taxonomic groupings and the topology of a recent morphological cladistic analysis suggest either that there may have been multiple independent losses of the intron in the genus or that a polymorphism has been retained across speciation events. The chloroplast gene rpl2, encoding the ribo- somal protein L2, is interrupted by an intron in

GA‐binding protein is involved in altered expression of ribosomal protein L32 gene

Differentiation of BC3H1 myoblasts to myocytes is accompanied by a 67% drop in the rate of rpL32 gene transcription. Addition of high concentrations of serum to resting myocyte populations stimulates cell growth and subsequent dedifferentiation to proliferating myoblasts with a return to the normal rate of rpL32 gene transcription. During these growth rate changes the binding activities of previously identified factors (β, γ, δ) which interact with the rpL32 gene promoter were examined by mobility shift assays. Binding of the β factor (an Ets related protein) to an oligonucleotide containing the β element was reduced significantly in myocycle nuclear extracts, but subsequent dedifferentiation increased binding within 30 min in either the presence or absence of the cycloheximide. Binding of the γ and δ factors to their respective elements changed only slightly during these processes. Dephosphorylation of either myoblast or myocyte extracts resulted in increased binding of the β factor suggesting that binding activity of the β factor is modulated by phosphorylation during the changes in BC3H1 myoblasts growth rate. In addition, mobility shift assays with recombinant GABP α and β proteins and their specific antibodies revealed that GABP proteins bind to the rpL32 gene promoter in a sequence dependent manner, and that similar proteins are present in BC3H1 myoblast/myocyte extracts. These results support the premise that the GABP heterodimer is the rpL32 β factor. Furthermore, during BC3H1 myoblast differentiation and dedifferentiation neither the levels of the GABP α and β proteins nor their respective mRNAs change. These results suggest that GABP is a constitutively expressed protein and is involved in regulating rpL32 gene by post-transcriptional modifications. J. Cell. Biochem. 65:287–307. © 1997 Wiley-Liss, Inc.

GA-binding protein is involved in altered expression of ribosomal protein L32 gene

Differentiation of BC3H1 myoblasts to myocytes is accompanied by a 67% drop in the rate of rpL32 gene transcription. Addition of high concentrations of serum to resting myocyte populations stimulates cell growth and subsequent dedifferentiation to proliferating myoblasts with a return to the normal rate of rpL32 gene transcription. During these growth rate changes the binding activities of previously identified factors (beta, gamma, delta) which interact with the rpL32 gene promoter were examined by mobility shift assays. Binding of the beta factor (an Ets related protein) to an oligonucleotide containing the beta element was reduced significantly in myocyte nuclear extracts, but subsequent dedifferentiation increased binding within 30 min in either the presence or absence of the cycloheximide. Binding of the gamma and delta factors to their respective elements changed only slightly during these processes. Dephosphorylation of either myoblast or myocyte extracts resulted in increased binding of the beta factor suggesting that binding activity of the beta factor is modulated by phosphorylation during the changes in BC3H1 myoblasts growth rate. In addition, mobility shift assays with recombinant GABP alpha and beta proteins and their specific antibodies revealed that GABP proteins bind to the rpL32 gene promoter in a sequence dependent manner, and that similar proteins are present in BC3H1 myoblast/myocyte extracts. These results support the premise that the GABP heterodimer is the rpL32 beta factor. Furthermore, during BC3H1 myoblast differentiation and dedifferentiation neither the levels of the GABP alpha and beta proteins nor their respective mRNAs change. These results suggest that GABP is a constitutively expressed protein and is involved in regulating rpL32 gene by post-transcriptional modifications.

The Chloroplast rpl2 Intron and ORF184 as Phylogenetic Markers in the Legume Tribe Desmodieae

A previous extensive survey showed that most but not all members of the papilionoid tribe Desmodieae lack the intron for the chloroplast gene rpl2 and partially or completely lack the open reading frame 0RF184. We extended the survey of Desmodieae to include all 26 genera recognized in the most recent comprehensive taxonomic treatment of the tribe, and use slot blot hybridization with gene- or intron-specific probes for all taxa, polymerase chain reaction amplification across the rpl2 intron region for a subset of taxa, and DNA sequencing across the rpl2 intron/exon boundary in representatives of selected genera (Desmodium, Lepesdeza, and Phylacium). Our results indicate that rpl2 intron is absent from both genera of the largest subtribe, Desmodiinae, and from Lespedezinae (Campylotropis, Kummerowia, and Lespedeza). It is indi- cated as present in both genera of Bryinae and in two Lespedezinae genera (Neocollettia and Phylacium). In contrast to the rpl2 intron, the pattern of 0RF184 loss in Desmodiinae and Lespedezinae was extremely complex and difficult to interpret from slot blots, but consistent either with partial deletion or extensive sequence divergence. Bryinae showed no such evidence of loss or partial deletion. In agreement with the distribution of the rpl2 intron, Bryinae have been considered to have affinities outside Desmodieae, while both Phylacium and Neocollettia are anomalous within Lespedezinae. Thus the rpl2 intron loss may represent a synapomorphy for a core Desmodieae (Desmodiinae and Lespedezinae).

A ribosomal protein L2 gene is transcribed, spliced, and edited at one site in rice mitochondria.

The mitochondrial ribosomal protein L2 gene (rpl2) is coded by two exons of 840 and 669 bp separated by an intron sequence of 1481 bp in the rice mitochondrial genome. The rpl2 gene is located three nucleotides upstream of the ribosomal protein S19 gene (rps19) and both genes are co-transcribed. cDNA sequence analysis identified splicing of the intron sequence from the rpl2 mRNA as well as RNA editing events. The deduced secondary structure of the rpl2 intron sequence shows the characteristic features of a group-II intron. A single RNA editing site is identified in rpl2 and six editing sites in rps19 transcripts. In addition, one editing site is observed in the 3 nucleotide intergenic region. Analysis of individual cDNA clones showed a different extent of RNA editing. The rice rpl2 intron is located at a different site and shows no significant nucleotide sequence similarity with the rpl2 intron of liverwort. However, 60% nucleotide sequence identity is observed between the rice rpl2 intron and the Oenothera nad5 intron in a 234 nucleotide region. The mitochondrial rpl2 sequence is absent from the pea mitochondrial genome and we consequently propose that the mitochondrial RPL2 protein is encoded by a nuclear gene in pea.

Changes in ribosomal protein rpl8 mRNA during the reproductive cycle of the mosquito, Aedes aegypti

Changes in the abundance of ribosomal protein rpL8 mRNA were compared with 18S rRNA and vitellogenin mRNA in fat body from adult female Aedes aegypti during the reproductive cycle. Levels of rpL8 mRNA began to increase within 2 h after adult eclosion, peaked at about 24 h post-eclosion, and remained high throughout the next 48 h. During this same period, rRNA abundance increased about 2-fold. After the bloodmeal, levels of rpL8 mRNA gradually decreased over the next 48 h, while rRNA levels increased about 4-fold within 16–24 h postbloodfeeding and eventually returned to previtellogenic levels. Vitellogenin mRNA was induced only after the bloodmeal, and disappeared by 48 h after feeding. After oviposition, rpL8 mRNA levels again increased to pre-bloodfeeding levels. These results indicate that rpL8 mRNA transcription in mosquito fat body is independent of rRNA transcription during the previtellogenic acquisition of competence and also during the post-bloodmeal ovarian cycle. Moreover, unlike the vitellogenin gene, the rpL8 gene is under post-transcriptional regulation in blood-fed females.

Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation.

A new vector, pFaadAII, for transformation of plastids of Nicotiana tabacum L. has been developed. It harbours a chimeric gene consisting of the aadA coding region from Escherichia coli, the 16S rDNA promoter from tobacco combined with a synthetic ribosome-binding site, a 500-bp fragment containing the 3' untranslated transcript region (UTR) of the Chlamydomonas rbcL gene and 3.75-kb (5') and 0.95-kb (3') tobacco plastome sequences allowing for targeting the foreign sequences to the intergenic region between the rpl32 and trnL genes of the tobacco plastome. The vector thus targets foreign sequences to the small single-copy region of the plastome, which has so far not been modified by transformation. Leaf protoplasts of Nicotiana tabacum L. were treated with polyethylene glycol (PEG) in the presence of the vector. The protocol for PEG treatment aiming at plastome transformation was optimized. Cell lines were cultured in the presence of spectinomycin and streptomycin using a novel and efficient protoplast culture and selection system. Regenerants were characterized by polymerase chain reaction (PCR) analysis, Southern hybridization and reciprocal crossing. The transformation procedure is described in detail and parameters influencing its efficiency are presented. Special effort is placed on analyzing suitable selection conditions. Only a proportion of the cell lines with a resistant phenotype could be confirmed by molecular analysis and/or reciprocal crossings to represent plastome transformants. Integration of the plastome specific aadA cassette into the nuclear genome accounted for a fraction of the resistant cell lines. Still, as many as 20-40 plastome transformants can be expected from the treatment of 10(6) protoplasts. Therefore, the improved protocol for PEG-mediated plastome transformation in combination with the new aadA-vector supplies a simple, reproducible and cost-efficient alternative to the biolistic procedure.

Phylogenetic affinities of the grasses to other monocots as revealed by molecular analysis of chloroplast DNA.

The distribution of structural alterations of the chloroplast genome found in grass chloroplast (cp) DNA in comparison with that of tobacco was systematically surveyed in the cpDNAs of monocots. Southern hybridization and/or PCR analyses for the detection of (1) three inversions in the large single-copy region, (2) loss of an intron in the rpoC1 gene, (3) an extra-sequence insertion in the rpoC2 gene, (4) the deletion of ORF2280, (5) rearrangements of the accD (ORF512) gene, and (6) non-reciprocal translocation of the rpl23 gene, were carried out on cpDNAs isolated from 58 species, 22 families, and 11 orders, which covered almost all families of monocots. These structural alterations of cpDNA mostly occurred at the family level. However, only part of the Restionaceae possessed the inversion that characterizes the lineage of grass differentiation. The order of mutational events made it possible to reconstruct grass phylogeny in monocots. Since no variations in structural alterations of the cpDNA were found among the Poaceae, grass plants were inferred to have originated from an ancestor harboring these structural alterations of the chloroplast genome. These phylogenetic relationships were supported by the sequence data of rbcL.

Multiple Independent Losses of Two Genes and One Intron from Legume Chloroplast Genomes

The losses of genes or introns from the chloroplast genome are of potential utility as phylogenetic characters. Previous broad surveys of angiosperms, along with sequencing studies in Pisum sativum, uncovered several instances of variation for such characters within the Legumi- nosae. Using a slot blot hybridization method that did not require high molecular weight DNA, probes for six genes and three introns typically found in angiosperm chloroplast genomes were used to screen 392 legume genera, representing all three subfamilies and 50 out of 51 tribes, and 48 genera from 26 other families of dicots. Few cases of chloroplast gene or intron loss from chloroplast genomes were observed among nonlegumes or within the legume subfamilies Mimo- soideae and Caesalpinioideae, but numerous apparent losses were observed in the large subfamily Papilionoideae. Certain cases of suspected losses were tested by polymerase chain reaction. The genes rpl2 and rbcL appear to be present in all taxa surveyed, and virtually all possess accD. In contrast, ORF184 and rpsl6 each appear to have been lost multiple, independent times within Leguminosae, while all members of the family appear to lack rp122. The intron of rpl2 has likely been lost at least four times within the family, and its absence is a synapomorphy for a core group of the papilionoid tribe Desmodieae. No convincing evidence of loss was observed among legumes for the introns of trnI and rpll6. These results indicate that gene or intron loss characters may often be homoplastic. Intron losses are a more reliable and valuable class of phylogenetic characters than are gene losses.

Sequence of a 17·1 kb DNA fragment from chromosome X of Saccharomyces cerevisiae includes the mitochondrial ribosomal protein L8

We have sequenced a continuous segment of 17,137 bp on chromosome X. Sequence analysis of this stretch revealed 14 open reading frames (ORFs) at least 100 amino acids long. One gene, encoding the mitochondrial 60S ribosomal protein L8, had already been sequenced. Four ORF products show weak homologies with known protein sequences. The nine remaining ORF products have no homologies with sequences in data banks.

Inefficient rpl2 splicing in barley mutants with ribosome-deficient plastids.

Analysis of transcript accumulation and splicing in plastids of four nuclear mutants of barley revealed that the ribosomal protein L2 (rpl2) gene transcripts containing a group II intron remained entirely unspliced, whereas the intron of the ribosomal protein L16 (rpl16) gene (linked with the rpl2 gene in the same operon) was removed in the mutant plastids. Also, the transcripts of other genes containing group II introns (ribosomal protein S16 gene, rps16; NADH dehydrogenase ND2 gene, ndhB; cytochrome f gene, petD; and intron-containing reading frame 170, irf170) and of the tRNA for leucine, trnL (UAA), possessing the only chloroplast group I intron, were found to be spliced. The mutants used in this investigation are considered to be nonallelic; this excludes the possibility that a single nuclear gene is responsible for the impaired splicing of rpl2 transcripts. The mutants, however, have a severe deficiency in chloroplast ribosomes in common; this deficiency is evident from the lack of the essential ribosomal protein L2 and from an extremely low steady state level of plastid rRNAs. From these results, we conclude that a functioning translational apparatus of the organelle is a prerequisite for splicing of the chloroplast rpl2 class II intron but not for splicing of at least five other group II intron-containing transcripts. This provides genetic evidence for a chloroplast DNA-encoded component (e.g., a maturase) involved in the splicing of rpl2 pre-mRNA.

Protein Substitution in Chloroplast Ribosome Evolution: A Eukaryotic Cytosolic Protein has Replaced its Organelle Homologue (L23) in Spinach

The chloroplast translational system differs from the eubacterial ones in containing several ribosomal proteins (RPs) that have no apparent homologues in eubacteria, and in having their RP genes distributed in two cellular genome compartments. The genes maintained in the organelle genome encode mainly ribosome assembly proteins. The discovery in spinach and related plants ( Caryophyllidae) of a disrupted chloroplast gene encoding the ribosome assembly protein L23 raised speculations about the transfer of the functional rpl23 gene to the nucleus or the evolutionary loss of L23 protein requirement. To solve this problem, we overexpressed in E. coli the intact rpl23 gene from corn ( Zea mays), purified the protein and raised antibodies. Based on immunoanalysis, we show that a prokaryotic-type L23 protein is absent in spinach. Concomittantly we have isolated a new protein from spinach chloroplast 50 S ribosomal subunits and determined its amino acid sequence. The data revealed an unexpectedly high sequence identity to the eukaryotic family of cytosolic L23 proteins (reported from yeast, trypanosome and rat), with conservation of a peptide motif responsible for the specific interaction of these proteins with domain III of 26 S and 23 S rRNA. We propose that the prokaryotic-type L23 protein in the chloroplast ribosomes of Caryophyllidae has been replaced by a homologue of the eukaryotic cytosolic L23 family. These results represent the first case of a protein (gene) substitution in chloroplast ribosome evolution, and open a new view on how the nuclear genome could progressively exert stronger over the chloroplast translational system. We also describe experiments on the incorporation of chloroplast L23 into E. coli ribosomes, its effect on cell growth, and an unexpected immuno cross-reaction between two chloroplast RP families.

Rps3 and rpl16 genes do not overlap in Oenothera mitochondria: GTG as a potential translation initiation codon in plant mitochondria?

Characterization of the Oenothera mitochondrial ribosomal gene cluster rps19-rps3-rpl16 shows the two genes rps3 and rpl16 to be separated by 9 nucleotides. The first codon of rpl16 is a GTG codon for valine and the only potential translational start. This GTG codon is conserved at the same position in maize, Petunia and Marchantia mitochondria, while sequences diverge upstream. These observations suggest that GTG at least at this position may act as translation initiation codon in plant mitochondria. Analysis of RNA editing suggests both genes to code for functional ribosomal proteins in Oenothera mitochondria. A duplication/recombination event at a decanucleotide in the intron of rps3 created a pseudogene missing part of the intron and the 3' exon.