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PubMed2000

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 delta, but not beta and gamma, factors to the rpL32 gene promoter as measured by gel mobility shift assays. This increased binding reflects a change in the activity of the delta 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 delta factor to the delta 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 delta factor.

Animals↗
PubMed1994

Regulation of splicing at an intermediate step in the formation of the spliceosome.

In vivo experiments have demonstrated that the ribosomal protein L32 of Saccharomyces cerevisiae brings about the inhibition of splicing of the transcript of its own gene through an RNA structure comprised largely of the first exon. We now show that L32, itself, binds specifically to this RNA. Splicing of the RPL32 transcript in vitro is blocked by the presence of L32. Furthermore, addition of the 75-nucleotide RNA representing the 5' end of the RPL32 transcript stimulates specifically the splicing of the RPL32 substrate, presumably by competing for L32 present in the extract. Use of RNAs carrying mutations shown to abolish the regulation of splicing, either as substrates or as competitors, confirmed that the in vitro reaction is a faithful representation of the situation in vivo. We conclude that the regulation of splicing occurs through the specific binding of L32 to an RNA structure within the first 75 nucleotides of the RPL32 transcript. The RPL32 substrate, bound to L32, forms a complex with U1 snRNP, the first step in spliceosome assembly. The presence of L32 prevents the ATP-dependent association of the U2 snRNP necessary to form a complete spliceosome.

Base Sequence↗
PubMed2006

[Transcription factor ZF5 regulates expression of mammalian gene containing GCC-triplet repeats in 5'-regulatory region in human hepatoma HepG2 cells].

Some nuclear proteins of human HeLa and HepG2 cells are capable of binding to GCC-triplet repeats--(GCC)n > 3 in 5'-regulatory regions of a number of mammalian genes--G-C-elements. According to our previous data, nucleotide sequence (GCC)4 in promoter of mouse ribosomal protein L32 gene (rpL32) between 17 and 6 bp upstream of transcription start site interacts to nuclear proteins from HepG2 cells, and may be considered as a GCC-element. We suggest that one of those proteins, with molecular weight about 52 kDa, which may interact with rpL32 GCC-element, is a known conservative mammalian transcription factor ZF5. DNA-binding domain of ZF5 contains a few Kruppel-like Zn-fingers (Cys2His2-type) interacting with the GC-rich nucleotide sequences in 5'-regulatory regions of a number of mammalian genes. Our results (obtained by EMSA) showed that recombinant GST-ZF5 fused protein containing ZF5 DNA-binding domain specifically binds a few GS-rich sequences: (GCC)g-9riplet repeats, 5'-GCGCGC-3' (known ZF5 consensus binding site) and (more preferable) the fragment (-24...+1 bp) of rpL32 promoter. The high affinity of ZF5 DNA-domain binding with the latter may be explained by the presence in this fragment of two overlapped subsequences, each being capable of binding to ZF5: (GCC)4 and 5'-GCGCGC- 3'. Zf5 cDNA was cloned from HepG2 cells by RT-PCR method, and then used for construction of the gene expression vector. It has been shown that Zf5 cDNA expression vector specifically down-regulates (in luciferase assays) the activity of rpL32 promoter (-155...+159) including the above mentioned GC-rich subsequences by cotransfection of HepG2 cells. Therefore, our results enable us to consider GCC-elements as a novel class of ZF5 targets in 5'-regulatory regions of mammalian genes.

Cell Line, Tumor↗
PubMed1984

The gene family encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing gene and an unmutated processed gene.

The family of approximately 16 genes encoding the mouse ribosomal protein L32 has been characterized by an analysis of a representative set of genomic clones. Surprisingly, this family contains only a single expressed intron-containing gene. This gene, termed rpL32 , has been completely sequenced and found to possess certain novel features including the presence in two of its introns of a sequence with high homology to the 5' end of U1 snRNA and a 5' terminal region exceptionally rich in pyrimidines. Most of the other members of the L32 family appear to be processed genes, some of which are identical or very similar to the rpL32 gene, except for the lack of introns. One unmutated gene was found to be integrated 28 nucleotides downstream of a canonical TATA box. However, despite this feature, the gene does not seem to be expressed, as judged by its extent of methylation compared to the expressed rpL32 gene.

Amino Acid Sequence↗
PubMed1996

An RNA structure involved in feedback regulation of splicing and of translation is critical for biological fitness.

While studies of the regulation of gene expression have generally concerned qualitative changes in the selection or the level of expression of a gene, much of the regulation that occurs within a cell involves the continuous subtle optimization of the levels of proteins used in macromolecular complexes. An example is the biosynthesis of the ribosome, in which equimolar amounts of nearly 80 ribosomal proteins must be supplied by the cytoplasm to the nucleolus. We have found that the transcript of one of the ribosomal protein genes of Saccharomyces cerevisiae, RPL32, participates in such fine tuning. Sequences from exon I of the RPL32 transcript interact with nucleotides from the intron to form a structure that binds L32 to regulate splicing. In the spliced transcript, the same sequences interact with nucleotides from exon II to form a structure that binds L32 to regulate translation, thus providing two levels of autoregulation. We now show, by using a sensitive cocultivation assay, that these RNA structures and their interaction with L32 play a role in the fitness of the cell. The change of a single nucleotide within the 5' leader of the RPL32 transcript, which abolishes the site for L32 binding, leads to detectably slower growth and to eventual loss of the mutant strain from the culture. Experiments designed to assess independently the regulation of splicing and the regulation of translation are presented. These observations demonstrate that, in evolutionary terms, subtle regulatory compensations can be critical. The change in structure of an RNA, due to alteration of just one noncoding nucleotide, can spell the difference between biological success and failure.

Base Sequence↗
PubMed1985

A processed pseudogene in an intron of the HLA-DP beta 1 chain gene is a member of the ribosomal protein L32 gene family.

A sequence in an intron of the human HLA-DP beta 1 gene was identified by its homology to the gene encoding ribosomal protein L32 (rpL32). It lacked introns indicating that it was derived from a processed rpL32 mRNA transcript. A human cDNA clone encoding rpL32 was isolated and compared to this human pseudogene and to several related mouse sequences, one of which is contained in an intron of the murine dihydrofolate reductase gene. Comparison of these sequences revealed that they were more related within species than between, suggesting that they became inserted in the genome after man and mouse diverged.

Animals↗
PubMed1997

Ribosomal protein L32 of Saccharomyces cerevisiae influences both the splicing of its own transcript and the processing of rRNA.

Ribosomal protein L32 of Saccharomyces cerevisiae binds to and regulates the splicing and the translation of the transcript of its own gene. Selecting for mutants deficient in the regulation of splicing, we have identified a mutant form of L32 that no longer binds to the transcript of RPL32 and therefore does not regulate its splicing. The mutation is the deletion of an isoleucine residue from a highly conserved hydrophobic domain near the middle of L32. The mutant protein supports growth, at a reduced rate, and is found at normal levels in mature ribosomes. However, in cells homozygous for the mutant gene, the rate of processing of the ribosomal RNA component of the 60S ribosomal subunit is severely reduced, leading to an insufficiency of 60S subunits. L32 must be considered a remarkable protein. Composed of only 104 amino acids, it appears to interact with three distinct RNA molecules to influence three different elements of RNA processing and function in three different locations of the cell: the processing of pre-rRNA in the nucleolus, the splicing of the RPL32 transcript in the nucleus, and the translation of the spliced RPL32 mRNA in the cytoplasm.

Amino Acid Sequence↗
PubMed2005

Selection of reference genes for gene expression studies in human neutrophils by real-time PCR.

BACKGROUND: Reference genes, which are often referred to housekeeping genes, are frequently used to normalize mRNA levels between different samples. However the expression level of these genes may vary among tissues or cells, and may change under certain circumstances. Thus the selection of reference gene(s) is critical for gene expression studies. For this purpose, 10 commonly used housekeeping genes were investigated in isolated human neutrophils. RESULTS: Initial screening of the expression pattern demonstrated that 3 of the 10 genes were expressed at very low levels in neutrophils and were excluded from further analysis. The range of expression stability of the other 7 genes was (from most stable to least stable): GNB2L1 (Guanine nucleotide binding protein, beta polypeptide 2-like 1), HPRT1 (Hypoxanthine phosphoribosyl transferase 1), RPL32 (ribosomal protein L32), ACTB (beta-actin), B2M (beta-2-microglobulin), GAPD (glyceraldehyde-3-phosphate dehydrogenase) and TBP (TATA-binding protein). Relative expression levels of the genes (from high to low) were: B2M, ACTB, GAPD, RPL32, GNB2L1, TBP, and HPRT1. CONCLUSION: Our data suggest that GNB2L1, HPRT1, RPL32, ACTB, and B2M may be suitable reference genes in gene expression studies of neutrophils.

Actins↗
PubMed1987

The yeast ribosomal protein L32 and its gene.

The yeast ribosomal protein gene RPL32 of Saccharomyces cerevisiae is of particular interest for two reasons: 1) it is adjacent to another ribosomal protein gene, RP29, whose divergent transcription may be driven from the same control sequences, and 2) it appears that the splicing of its transcript is regulated by the product of the gene, ribosomal protein in L32. RPL32 has been analyzed in detail. It is essential for cell growth. Its sequence predicts L32 to be a protein of 105 amino acids, somewhat basic near the NH2 terminus, rather acidic near the COOH terminus, and homologous to ribosomal protein L30 of mammals. The reading frame has been confirmed by partial NH2-terminal analysis of L32. The nucleotide sequence also predicts an intron of 230 nucleotides, which begins with the unusual sequence GTCAGT and ends 40 nucleotides downstream of the consensus sequence TAC-TAAC. The intron has been confirmed by determination of the sequence of a cDNA clone. Transcription initiates 58 nucleotides upstream of the AUG initiation codon, and the polyadenylation site occurs 100 nucleotides downstream of the termination codon. Regulation of the transcription of ribosomal protein genes has been linked to two related consensus sequences. Analysis of the intergenic region between RP29 and RPL32 reveals three copies of these sequences. A deletion removing all three sequences reduces synthesis of a L32-LacZ fusion protein by more than 90%. Some residual activity, however, remains.

Amino Acid Sequence↗
PubMed1995

Characterization of the pre-mRNA binding site for yeast ribosomal protein L32: the importance of a purine-rich internal loop.

The structure of the RNA binding target for Saccharomyces cerevisiae ribosomal protein L32 was examined using chemical and enzymatic probes as well as thermodynamic methods. In vivo, the production of yeast RPL32 is regulated by a feedback mechanism whereby RPL32 binds to the 5' end of its transcript and inhibits splicing. The binding site of ribosomal protein L32 on the L32 RNA transcript can be reduced to fewer than 30 nucleotides which compromise a stem-internal loop-stem structural motif. The internal loop is closed by a potential G-U pair, is asymmetric and contains mostly purines. The existence of the two helical regions was confirmed by chemical and enzymatic probing. The reactivity of the loop region suggests a structure intermediate between that of single and double-stranded RNA. Base stacking continues into the loop, but two loop bases are extremely reactive to chemical agents. The interaction between the model RNA and the protein is specific and has a dissociation constant of approximately 10 nM. Several of the loop bases are critical for protein binding, as demonstrated by mutational data and chemical protection and modification interference studies. The internal loop destabilizes the RNA, and allows the RNA to melt in an all-or-none fashion.

Aniline Compounds↗
PubMed1991

Structural basis for the regulation of splicing of a yeast messenger RNA.

In S. cerevisiae, ribosomal protein L32 regulates the splicing of the transcript of its own gene, RPL32. We have identified an RNA structure within the transcript that is responsible for this regulation. Initial deletions limited essential sequences to the 5' exon and the first few nucleotides of the intron. To take advantage of phylogenetic comparison of RNA structures, RPL32 was cloned from the closely related species, Kluyveromyces lactis. The splicing of its transcript is similarly regulated. Sequences conserved between the S. cerevisiae and K. lactis transcripts suggested a structure involving base pairing of a region encompassing the 5' splice site with another near the 5' end of the transcript. Analysis of numerous site-directed mutations supports this structure. We infer that stabilization of this structure by L32 inhibits splicing by precluding the interaction of U1 RNA with the 5' splice site.

Base Sequence↗
PubMed1989

An element downstream of the cap site is required for transcription of the gene encoding mouse ribosomal protein L32.

To identify the elements that regulate transcription of the mouse gene encoding ribosomal protein L32 (rpL32), we transfected monkey kidney (COS or CV-1) cells with mutants bearing progressive 5' deletions or an internal deletion in exon I and measured their transient expression by S1 nuclease protection analysis. When the mutant genes were tested in the vector pi SVHSplac, which contains a short segment of the oriregion of simian virus 40, maximum expression was observed with as little as 36 base pairs of 5' flanking sequence, and the mutant bearing the exon I deletion was expressed very efficiently. However, when the genes were tested in a simple prokaryotic (pUC) vector, the expression was increased 3- to 4-fold by sequences between -36 and -159, and the exon I segment was absolutely required for expression. Gel mobility-shift and methylation interference analyses revealed that a nuclear factor specifically binds to a GGCTGCCATC sequence within this exon I segment. These results, taken together with other recent findings, indicate that the elements involved in transcriptional regulation of the rpL32 gene are distributed over a 200-base-pair region that spans the cap site. The contributions of some of these elements are apparently masked in the presence of simian virus 40 ori-region elements.

Animals↗
PubMed1989

Equipotent mouse ribosomal protein promoters have a similar architecture that includes internal sequence elements.

The promoters of the mouse ribosomal protein genes rpL30, rpL32, and rpS16 are of equal strength, as indicated by in vivo measurements of polymerase loading and by their relative efficiency in driving the expression of a linked reporter gene. The equipotency of these promoters appears to derive from a remarkably similar architecture in which five or more elements are distributed over a 200-bp region that spans a polypyrimidine-embedded cap site. Three trans-acting factors are shared by the rpL30 and rpL32 promoters, one of which, delta, recognizes a common CNGCCATCT motif in the first (untranslated) exons. Site-specific mutagenesis demonstrated that delta-factor binding is critical for rpL30 promoter function. The repeated occurrence of this novel promoter architecture among ribosomal protein genes with very different coding specificities is most readily explained by convergent evolution.

Animals↗
PubMed1992

Visualization of a mammalian transcription initiation complex.

Various proteins required for the initiation of eukaryotic gene transcription by RNA polymerase II have been identified and characterized, but little is known about their organization into a functional unit. Here, we describe the appearance of the murine ribosomal protein (rp) L32 gene transcription initiation complex as determined by transmission electron microscopy. Using a fractionated nuclear extract enriched for transcription factors necessary for rpL32 gene transcription in vitro and a DNA fragment containing the rpL32 gene promoter, the transcription initiation complex was imaged by standard transmission electron microscopy. Quantitative image analysis demonstrated that the complex is a multilobed structure whose two-dimensional projections are approximately 24 x 34 nm in size. Looping of the DNA seen in these images suggests that the proteins residing at the promoter region associate with proteins several hundred base pairs distant to the RNA start site, with bending of the DNA allowing these interactions to occur.

Animals↗
PubMed2006

Stability of housekeeping genes in alveolar macrophages from COPD patients.

The stability of housekeeping genes is critical when performing gene expression studies. To date, there have been no studies that look at the stability of commonly used housekeeping genes in alveolar macrophages. Expression levels may be affected by culture, stimulation or disease severity. The present study investigated the expression level of 10 housekeeping genes and analysed the stability of their expression in alveolar macrophages from chronic obstructive pulmonary disease patients (n = 22) who were classified according to disease severity. Guanine nucleotide-binding protein, beta polypeptide 2-like 1 (GNB2L1), hypoxanthine phosphoribosyl transferase 1 (HPRT1) and ribosomal protein L32 (RPL32) were the most stably expressed in alveolar macrophages, irrespective of disease severity. There was no difference in the expression levels of 10 housekeeping genes between mild and moderate/severe patients. GNB2L1, HPRT1 and RPL32 were also stably expressed in alveolar macrophages cultured with no stimulation, or with interleukin-1beta, lipopolysaccharide or tumour necrosis factor-alpha stimulation. In conclusion, as fluctuations in the expression of some housekeeping genes were observed, including glyceraldehyde-3-phosphate dehydrogenase, it is recommended that guanine nucleotide binding protein, beta polypeptide 2-like 1 be used as a reference gene for alveolar macrophages in similar study designs, or that the stability of housekeeping genes be validated in alveolar macrophages prior to expression studies.

Aged↗
PubMed2026

Plastome evolution and phylogenomic relationships in Ajuga (Lamiaceae, Ajugoideae).

BACKGROUND: Ajuga is currently known to include approximately 69 species, with a combined distribution extending throughout Eurasia, Africa, and Australia. Its popularity and significance are largely based on an extensive history of medicinal and horticultural use. It is divided into two sections based on morphological characters, and this sectional classification is also reflected in pronounced geographic patterns. Although previous studies have largely focused on Ajuga sect. Ajuga in East Asia, A. sect. Chamaepithys, which ranges from the Mediterranean to Central Asia, remains insufficiently sampled, thereby limiting a comprehensive understanding of infrageneric sectional relationships within the genus. Here, we generated complete plastid genomes for 12 species representing both sections of the genus and used these data to characterize plastome structure and infer evolutionary relationships. RESULTS: In this study, 21 Ajuga plastomes were analyzed, including 12 newly sequenced plastomes and 9 previously published plastomes representing 19 species. Comparative analyses showed that all plastomes exhibited a highly conserved quadripartite structure, with genome sizes ranging from 149,963 to 150,740 bp and GC contents varying from 38.2% to 38.3%. Each plastome contained 133 genes, including 88 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. The boundaries between the inverted repeat (IR) and single-copy (SC) regions were also highly conserved across species. In addition, 796 simple sequence repeats (SSRs), 874 long repeat sequences (LRSs), and 12 highly variable regions (ccsA-ndhD, ndhF-rpl32, petA-psbJ, rpl32-trnL-UAG, rps2-rpoC2, trnH-GUG-psbA, trnK-UUU-rps16, trnP-UGG-psaJ, trnT-UGU-trnL-UAA, ycf15-trnL-CAA, ndhF, and ycf1) were identified among the 21 plastomes. Phylogenetic analyses based on four datasets and conducted using Maximum Likelihood and Bayesian Inference recovered two major clades corresponding to the traditionally recognized sectional classification, with one distributed from the Mediterranean to Central Asia and the other in East Asia. CONCLUSION: This study represents the most comprehensive plastome-based sampling of Ajuga to date, including representative species from the Mediterranean, Central Asia, and East Asia. Our results have significantly enhanced our understanding of its infrageneric relationships. The plastome resources generated in this study provide a valuable foundation for future research on species delimitation, phylogeny, and the evolutionary history of Ajuga.

Phylogeny↗
PubMed2026

Insights into phylogenetic relationships of Veronica species (Plantaginaceae) based on comparative chloroplast genomics.

INTRODUCTION: Veronica L. is one of the most species-rich genera in Plantaginaceae and several species have medicinal, horticultural, or ecological value. METHODS: In this study, the complete chloroplast genomes of three Veronica species were assembled and annotated using Illumina sequencing data. RESULTS: The plastomes exhibited a typical quadripartite structures, with total lengths of 150,202 bp for Veronica biloba L., 151,159 bp for Veronica ciliata Fisch. and 151,098 bp for Veronica vandellioides Maxim. Each genome contained 130-132 unique genes, including 86-87 protein-coding genes, 36-37 tRNA genes, and 8 rRNA genes. Comparative analyses of 24 Veronica plastomes indicated that the IR/SC junctions were largely conserved, although slight boundary shifts occurred around rps19, ndhF, and ycf1. Forward, palindromic, complement, and reverse repeats were detected, and A/T mononucleotide repeats were the dominant SSR type. Nucleotide diversity analysis identified rpl32-trnL, trnK-rps16, rpl32, ycf1, ndhF, accD, matK, and rpoB as highly variable regions. Phylogenetic analyses recovered Veronica as a well-supported monophyletic lineage and clarified the plastid positions of the three newly sequenced species. Divergence time estimation suggested that the estimation suggested of Veronica was around 14.9 Ma, with V. biloba, V. ciliata and V. vandellioides diverging approximately 3.9 Ma, 0.6 Ma, and 6.9 Ma, respectively. DISCUSSION: Because the analyses were based on plastid genomes, the inferred topology should be interpreted as chloroplast phylogenetic evidence rather than a complete species-history reconstruction. These results provide plastome resources and molecular evidence for taxonomy, species identification, and future evolutionary studies of Veronica.

Plantaginaceae↗
PubMed1992

Multivariate statistical analysis of electron micrographs of a mammalian transcription initiation complex.

We describe the appearance of the murine ribosomal protein (rp) L32 gene transcription initiation complex as determined by electron image analysis. Using a fractionated nuclear extract enriched for transcription factors necessary for rpL32 gene transcription in vitro and a DNA fragment containing the rpL32 gene promoter, the transcription initiation complex was prepared and viewed by standard transmission electron microscopy. Image analysis demonstrated that the complex was a multilobed structure.

Cells, Cultured↗