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PubMed2000

Regulation of the furA and catC operon, encoding a ferric uptake regulator homologue and catalase-peroxidase, respectively, in Streptomyces coelicolor A3(2).

We isolated the catC gene, encoding catalase-peroxidase in Streptomyces coelicolor, using sequence homology with the katG gene from Escherichia coli. Upstream of the catC gene, an open reading frame (furA) encoding a homologue of ferric uptake regulator (Fur) was identified. S1 mapping analysis indicated that the furA gene was cotranscribed with the catC gene. The transcriptional start site of the furA-catC mRNA was mapped to the translation start codon ATG of the furA gene. The putative promoter contains consensus -10 and -35 elements similar to those recognized by sigma(HrdB), the major sigma factor of S. coelicolor. The transcripts were produced maximally at late-exponential phase and decreased at the stationary phase in liquid culture. The change in the amount of mRNA was consistent with that of CatC protein and enzyme activity. When the furA gene was introduced into S. lividans on a multicopy plasmid, the increased production of catC transcripts and protein product at late growth phase was inhibited, implying a role for FurA as the negative regulator of the furA-catC operon. FurA protein bound to its own promoter region between -59 and -39 nucleotides from the transcription start site. The binding affinity of FurA increased under reducing conditions and in the presence of metals such as Ni(2+), Mn(2+), Zn(2+), or Fe(2+). Addition of these metals to the growth medium decreased the production of CatC protein, consistent with the role of FurA as a metal-dependent repressor.

Amino Acid Sequence↗

PubMed2000

Nucleotide sequence of a 7-kb fragment of pACM1 encoding an IncM DNA primase and other putative proteins associated with conjugation.

A 7-kb fragment of pACM1 (fragment 90¿91) containing one or more kor (kill-override) loci was sequenced, and 28 open reading frames (ORFs; >/=50 codons) were identified. The nucleotide sequence has no significant homologs in the GenBank database except for a 1.3-kb region 98.6% identical to the iml (insensitivity to phage PhiM-mediated lysis) determinant fragment of IncM plasmid R446. Deduced amino acid sequences for several ORFs are homologous to those of known proteins, including the Sog DNA primases of IncI1 plasmids R64 and ColIb-P9 and the TraL, TraM, and TraN products of ColIb-P9. Two protein products of the putative primase ORF (ORF 1, 1100 amino acids) were detected by SDS-PAGE. The 158- and 107-kDa proteins were designated PriL and PriS, respectively. PriS is apparently produced by an in-frame reinitiation of the ORF 1 transcript at a second start codon located between a Sau96I site and a PstI site. The motif EGYATA, conserved among primases and associated with primase function, occurs in the first one-third of the deduced amino acid sequence of PriL and is not included in PriS. Partial suppression of the temperature-sensitive dnaG3 mutation in BW86 was demonstrated by recombinants that overexpressed both PriL and PriS, but not by constructs overexpressing only PriS. Therefore, primase function can be assigned to PriL. Fragment 90/91 represents a portion of the IncM tra region, which has not previously been examined in detail.

Amino Acid Sequence↗

PubMed2000

Rho-dependent transcription termination in the tna operon of Escherichia coli: roles of the boxA sequence and the rut site.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced transcription antitermination. Tryptophan induction prevents Rho-dependent transcription termination in the leader region of the operon. Induction requires translation of a 24-residue leader peptide-coding region, tnaC, containing a single, crucial Trp codon. Studies with a lacZ reporter construct lacking the tnaC-tnaA spacer region suggest that, in the presence of excess tryptophan, the TnaC leader peptide acts in cis on the ribosome translating tnaC to inhibit its release. The stalled ribosome is thought to block Rho's access to the transcript. In this paper we examine the roles of the boxA sequence and the rut site in Rho-dependent termination. Deleting six nucleotides (CGC CCT) of boxA or introducing specific point mutations in boxA results in high-level constitutive expression. Some constitutive changes introduced in boxA do not change the TnaC peptide sequence. We confirm that deletion of the rut site results in constitutive expression. We also demonstrate that, in each constitutive construct, replacement of the tnaC start codon by a UAG stop codon reduces expression significantly, suggesting that constitutive expression requires translation of the tnaC coding sequence. Addition of bicyclomycin, an inhibitor of Rho, to these UAG constructs increases expression, demonstrating that reduced expression is due to Rho action. Combining a boxA point mutation with rut site deletion results in constitutive expression comparable to that of a maximally induced operon. These results support the hypothesis that in the presence of tryptophan the ribosome translating tnaC blocks Rho's access to the boxA and rut sites, thereby preventing transcription termination.

Amino Acid Sequence↗

PubMed2000

Molecular cloning and transcriptional regulation of ompT, a ToxR-repressed gene in Vibrio cholerae.

In pathogenic Vibrio cholerae, at least 17 genes are co-ordinately regulated by ToxR. Most of these genes, including those that encode cholera toxin (CT), toxin co-regulated pilus (TCP), accessory colonization factor (ACF) and OmpU, are positively regulated. OmpT is the only identified protein under negative regulation of ToxR. To understand the molecular mechanism by which ToxR represses OmpT expression, we cloned ompT and characterized the ompT promoter and its interaction with ToxR. Sequence analysis revealed that ompT encodes a predicted 35.8 kDa outer membrane porin of V. cholerae. Primer extension analysis identified a transcriptional start site 104 bp upstream of the translational start codon. Both primer extension analysis and promoter fusion studies showed that ToxR represses OmpT expression at the transcriptional level. Promoter fusion studies also suggest that cyclic AMP receptor protein (CRP) is involved in ompT activation. Gel mobility shift assays combined with DNase I footprinting analysis demonstrated that ToxR mediates repression of ompT transcription by directly binding to an A/T-rich region between -95 and -30 of the ompT promoter. To further understand how the interaction of ToxR with different promoters results in its function as an activator or repressor, we have also mapped the regions on the ctxAB and toxT promoters to which ToxR binds. The regions protected by ToxR on each of these promoters are all A/T rich and large in size, although they are positioned differently relative to each transcriptional start site.

Amino Acid Sequence↗

PubMed1997

Additional organizational features of the murine folylpolyglutamate synthetase gene. Two remotely situated exons encoding an alternate 5' end and proximal open reading frame under the control of a second promoter.

Nucleotide sequence analysis of independently isolated clones from a mouse liver cDNA library identified two additional splice variants of folylpolyglutamate synthetase (FPGS) mRNA with novel sequence at the 5' end. These variants incorporate two new alternatives (exons A1a and A1b) of exon 1 in the murine FPGS gene which are also spliced to exon 2. Exon A1a encodes most of the 5'-untranslated region. Exon A1b encodes a downstream segment of the 5'-untranslated region, two ATG start codons, and a unique mitochondrial leader peptide as well as 15 additional amino acids of cytosolic FPGS not encoded by all previously identified (Roy, K., Mitsugi, K., and Sirotnak, F. M. (1996) J. Biol. Chem., 271, 23820-23827) splice variants. It was also found by direct sequencing of genomic fragments that although exon A1b is spliced to exon 2, these new alternatives (i.e. exons A1a and A1b) to exon 1 are found approximately 9.5 kilobases upstream from exons B1a, B1b, and B1c. Exons A1a and A1b are separated from each other by a 124-nucleotide intron. Sequencing of the region 5' to exon A1a revealed a nucleotide sequence that was promoter-like and different from the downstream promoter region in the content of putative cis-acting elements. Primer extension analysis identified a number of potential transcription start sites within the more 3' end of this region. FPGS RNA transcripts incorporating exons A1a and A1b were detected in both normal mouse tissues, particularly, liver and kidney, and also to a varying extent in tumors; FPGS RNA transcripts incorporating exons B1a, B1b, and B1c were detected mainly in tumors. Thus, transcription of the FPGS gene in this tissue-specific manner appears to reflect the different usage of alternates to exon 1 under the control of different promoters. An unusual splice variant identified infrequently in a mouse liver cDNA library was 2.67 kilobases in size and incorporated exons A1a and A1b and a segment of the downstream promoter region along with exons B1c and B1b and exons 2-15.

Alternative Splicing↗

PubMed1989

Nucleotide sequence of traQ and adjacent loci in the Escherichia coli K-12 F-plasmid transfer operon.

The F tra operon region that includes genes trbA, traQ, and trbB was analyzed. Determination of the DNA sequence showed that on the tra operon strand, the trbA gene begins 19 nucleotides (nt) distal to traF and encodes a 115-amino-acid, Mr-12,946 protein. The traQ gene begins 399 nt distal to trbA and encodes a 94-amino-acid, Mr-10,867 protein. The trbB gene, which encodes a 179-amino-acid, Mr-19,507 protein, was found to overlap slightly with traQ; its start codon begins 11 nt before the traQ stop codon. Protein analysis and subcellular fractionation of the products expressed by these genes indicated that the trbB product was processed and that the mature form of this protein accumulated in the periplasm. In contrast, the protein products of trbA and traQ appeared to be unprocessed, membrane-associated proteins. The DNA sequence also revealed the presence of a previously unsuspected locus, artA, in the region between trbA and traQ. The artA open reading frame was found to lie on the DNA strand complementary to that of the F tra operon and could encode a 104-amino-acid, 12,132-dalton polypeptide. Since this sequence would not be expressed as part of the tra operon, the activity of a potential artA promoter region was assessed in a galK fusion vector system. In vivo utilization of the artA promoter and translational start sites was also examined by testing expression of an artA-beta-galactosidase fusion protein. These results indicated that the artA gene is expressed from its own promoter.

Amino Acid Sequence↗

PubMed1997

Alternative translation of the proto-oncogene c-myc by an internal ribosome entry site.

The human proto-oncogene c-myc encodes two proteins, c-Myc1 and c-Myc2, from two initiation codons, CUG and AUG, respectively. It is also transcribed from four alternative promoters (P0, P1, P2, and P3), giving rise to different RNA 5'-leader sequences, the long sizes of which suggest that they must be inefficiently translated by the classical ribosome scanning mechanism. Here we have examined the influence of three c-myc mRNA 5'-leaders on the translation of chimeric myc-CAT mRNAs. We observed that in the reticulocyte rabbit lysate, these 5'-leaders lead to cap-independent translation initiation. To determine whether this kind of initiation resulted from the presence of an internal ribosome entry site (IRES), COS-7 cells were transfected with bicistronic vectors containing the different c-myc 5'-leaders in the intercistronic region. An IRES was identified, requiring elements located within the P2 leader, between nucleotides -363 and -94 upstream from the CUG start codon. This is the first demonstration of the existence of IRES-dependent translation for a proto-oncogene. This IRES could be a translation enhancer, allowing activation of c-myc expression under the control of trans-acting factors and in response to specific cell stimuli.

Animals↗

PubMed1999

Vitamin D receptor translation initiation codon polymorphism and markers of osteoporotic risk in older African-American women.

A polymorphism at the first of two potential translation initiation codons in the vitamin D receptor (VDR) gene defined by the FokI restriction endonuclease has been associated with reduced bone mineral density (BMD) among Caucasian, Asian, and Mexican-American women. We tested the hypothesis that the FokI polymorphism is related to markers of osteoporotic risk in 104 community-dwelling African-American women aged 65 years and older. Six percent of the African-American women had the ff genotype, 32% were heterozygous, and 63% had the FF genotype. FokI genotype frequencies did not differ from Hardy-Weinberg expectations. Hip and calcaneal BMD, calcaneal ultrasound attenuation and hip geometry from pelvic radiographs did not differ significantly by FokI genotypes or between women with and without the rare FokI allele. There was also no association between the FokI polymorphism and biochemical markers of bone turnover or fractional calcium absorption. We conclude that the VDR start codon polymorphism does not have a major influence on osteoporotic risk in older African-American women.

Aged↗

PubMed1991

GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae.

The GCD2 protein is a translational repressor of GCN4, the transcriptional activator of multiple amino acid biosynthetic genes in Saccharomyces cerevisiae. We present evidence that GCD2 has a general function in the initiation of protein synthesis in addition to its gene-specific role in translational control of GCN4 expression. Two temperature-sensitive lethal gcd2 mutations result in sensitivity to inhibitors of protein synthesis at the permissive temperature, and the gcd2-503 mutation leads to reduced incorporation of labeled leucine into total protein following a shift to the restrictive temperature of 36 degrees C. The gcd2-503 mutation also results in polysome runoff, accumulation of inactive 80S ribosomal couples, and accumulation of at least one of the subunits of the general translation initiation factor 2 (eIF-2 alpha) in 43S-48S particles following a shift to the restrictive temperature. The gcd2-502 mutation causes accumulation of 40S subunits in polysomes, known as halfmers, that are indicative of reduced 40S-60S subunit joining at the initiation codon. These phenotypes suggest that GCD2 functions in the translation initiation pathway at a step following the binding of eIF-2.GTP.Met-tRNA(iMet) to 40S ribosomal subunits. consistent with this hypothesis, we found that inhibiting 40S-60S subunit joining by deleting one copy (RPL16B) of the duplicated gene encoding the 60S ribosomal protein L16 qualitatively mimics the phenotype of gcd2 mutations in causing derepression of GCN4 expression under nonstarvation conditions. However, deletion of RPL16B also prevents efficient derepression of GCN4 under starvation conditions, indicating that lowering the concentration of 60S subunits and reducing GCD2 function affect translation initiation at GCN4 in different ways. This distinction is in accord with a recently proposed model for GCN4 translational control in which ribosomal reinitiation at short upstream open reading frames in the leader of GCN4 mRNA is suppressed under amino acid starvation conditions to allow for increased reinitiation at the GCN4 start codon.

Amino Acids↗

PubMed1998

The methyl group of the N6-methyl-N6-threonylcarbamoyladenosine in tRNA of Escherichia coli modestly improves the efficiency of the tRNA.

tRNA species that read codons starting with adenosine (A) contain N6-threonylcarbamoyladenosine (t6A) derivatives adjacent to and 3' of the anticodons from all organisms. In Escherichia coli there are 12 such tRNA species of which two (tRNA(Thr1)GGU and tRNA(Thr3)GGU) have the t6A derivative N6-methyl-N6-threonylcarbamoyladenosine (m6t6A37). We have isolated a mutant of E. coli that lacks the m6t6A37 in these two tRNA(Thr)GGU species. These tRNA species in the mutant are likely to have t6A37 instead of m6t6A37. We show that the methyl group of m6t6A37 originates from S-adenosyl-L-methionine and that the gene (tsaA) which most likely encodes tRNA(m6t6A37)methyltransferase is located at min 4.6 on the E. coli chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA(Thr)GGU to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m6t6A37-containing tRNA(Thr)GGU species. We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m6t6A37 in tRNA(Thr)GGU slightly reduces the efficiency of this tRNA to read cognate codon ACC.

Adenosine↗

PubMed2001

Leptin receptor 5'untranslated regions in the rat: relative abundance, genomic organization and relation to putative response elements.

Hypothalamic sensitivity to leptin has been suggested to be important for regulation of body fat mass. Mice heterozygous for a mutation in the leptin receptor (leptin-R) have an increased body fat mass suggesting that the abundance of leptin-R may be an important determinator of leptin sensitivity. Leptin-R cDNAs from several species contain alternative 5'untranslated regions (5'UTRs), suggesting that several distinct regulatory regions may exist. To investigate possible mechanisms by which leptin-R expression may be regulated, we searched for possible alternative 5'UTRs of the leptin-R in the rat and determined their location in relation to putative response elements. Four leptin-R 5'UTRs (exons 1A-1D), which diverged 23 bp upstream of the start codon, were identified by 5'Rapid Amplification of cDNA Ends (5'RACE) and sequencing. Exons 1B and 1C were present in 31 and 61%, respectively, of all leptin-R transcripts in the hypothalamus as determined by a ribonuclease protection assay. Analysis of the 5' flanking genomic sequences revealed an imperfect estrogen response element (ERE), two Spl-sites, three CCAAT-boxes and one octamer. Exons 1A and 1D corresponded to a putative second gene, encoding the OB-Receptor Gene Related Protein (OB-RGRP), which is transcribed from a promoter shared with the leptin-R. DNA sequencing revealed that the rat OB-RGRP had 98 and 97% homology with the mouse and human sequence, respectively. We report here that transcription of the rat leptin-R gene may generate transcripts with four alternative 5'UTRs. The presence of a putative ERE, close to the most frequently used transcriptional start sites of the leptin-R gene in the hypothalamus, provides a possible mechanism by which estrogen may exert its effects on food intake.

5' Untranslated Regions↗

PubMed2005

Alternative initiation and splicing in dicer gene expression in human breast cells.

INTRODUCTION: Dicer is a ribonuclease that mediates RNA interference both at the transcriptional and the post-transcriptional levels. Human dicer gene expression is regulated in different tissues. Dicer is responsible for the synthesis of microRNAs and short temporal (st)RNAs that regulate the expression of many genes. Thus, understanding the control of the expression of the dicer gene is essential for the appreciation of double-stranded (ds)RNA-mediated pathways of gene expression. Human dicer mRNA has many upstream open reading frames (uORFs) at the 5'-leader sequences (the nucleotide sequence between the 5'-end and the start codon of the major ORF), and we studied whether these elements at the 5'-leader sequences regulate the expression of the dicer gene. METHOD: We determined the 5'-leader sequences of the dicer mRNAs in human breast cells by 5'-RACE and S1-nuclease protection analysis. We have analyzed the functions of the 5'-leader variants by reporter gene expression in vitro and in vivo. RESULTS: We found that the dicer transcripts in human breast cells vary in the sequence of their 5'-leader sequences, and that alternative promoter selection along with alternative splicing of the 5'-terminal exons apparently generate these variations. The breast cell has at least two predominant forms of dicer mRNAs, one of which has an additional 110 nucleotides at the 5'-end. Sequence comparison revealed that the first 80 nucleotides of these mRNA isoforms are encoded by a new exon located approximately 16 kb upstream of the reported start site. There are 30 extra nucleotides added to the previously reported exon 1. The human breast cells studied predominantly express two 5'-leader variants of dicer mRNAs, one with the exons 2 and 3 (long form) and the other without them (short form). By reporter gene expression analysis we found that the exon 2 and 3 sequences at the 5'-leader sequences are greatly inhibitory for the translation of the mRNA into protein. CONCLUSION: Dicer gene expression in human breast cells is regulated by alternative promoter selection to alter the length and composition of the 5'-leader sequence of its mRNA. Furthermore, alternative splicing of its exon 2 and 3 sequences of their pre-mRNA creates a more translationally competent mRNA in these cells.

Alternative Splicing↗

PubMed1986

Pleiotropic effects induced by modification deficiency next to the anticodon of tRNA from Salmonella typhimurium LT2.

A strain of Salmonella typhimurium LT2 was isolated, which harbors a mutation acting as an antisuppressor toward an amber suppressor derivative, supF30, of tRNATyr1. The mutant is deficient in cis-2-methylthioribosylzeatin[N6-(4-hydroxyisopentenyl)-2-me thylthioadenosine, ms2io6A], which is a modification normally present next to the anticodon (position 37) in tRNA reading codons starting with uridine. The gene miaA, defective in the mutant, is located close to and counterclockwise of the purA gene at 96 min on the chromosomal map of S. typhimurium with the gene order mutL miaA purA. Growth rate of the mutant was reduced 20 to 50%, and the effect was more pronounced in media supporting fast growth. Translational chain elongation rate at 37 degrees C was reduced from 16 amino acids per s in the wild-type cell to 11 amino acids per s in the miaA1 mutant in the four different growth media tested. The cellular yield in limiting glucose, glycerol, or succinate medium was reduced for the miaAI mutant compared with wild-type cells, with 49, 41, and 57% reductions, respectively. The miaAI mutation renders the cell more sensitive or resistant toward several amino acid analogs, suggesting that the deficiency in ms2io6A influences the regulation of several amino acid biosynthetic operons. We suggest that tRNAPhe, lacking ms2io6A, translates a UUU codon in the early histidine leader sequence with lowered efficiency, leading to repression of the his operon.

Anticodon↗

PubMed2006

A mutation in the translation initiation codon of Gata-1 disrupts megakaryocyte maturation and causes thrombocytopenia.

We have generated mice from a N-ethyl-N-nitrosourea mutagenesis screen that carry a mutation in the translation initiation codon of Gata-1, termed Plt13, which is equivalent to mutations found in patients with acute megakaryoblastic leukemia and Down syndrome. The Gata-1 locus is present on the X chromosome in humans and in mice. Male mice hemizygous for the mutation (Gata-1Plt13/Y) failed to produce red blood cells and died during embryogenesis at a similar stage to Gata-1-null animals. Female mice that carry the Plt13 mutation are mosaic because of random inactivation of the X chromosome. Adult Gata-1Plt13/+ females were not anemic, but they were thrombocytopenic and accumulated abnormal megakaryocytes without a concomitant increase in megakaryocyte progenitor cells. Gata-1Plt13/+ mice contained large numbers of blast-like colony-forming cells, particularly in the fetal liver, but also in adult spleen and bone marrow, from which continuous mast cells lines were readily derived. Although the equivalent mutation to Gata-1Plt13 in humans results in production of GATA-1s, a short protein isoform initiated from a start codon downstream of the mutated initiation codon, Gata-1s was not detected in Gata-1Plt13/+ mice.

Alkylating Agents↗

PubMed2006

Sp1/3 and NF-1 mediate basal transcription of the human P2X1 gene in megakaryoblastic MEG-01 cells.

BACKGROUND: P2X1 receptors play an important role in platelet function as they can induce shape change, granule centralization and are also involved in thrombus formation. As platelets have no nuclei, the level of P2X1 expression depends on transcriptional regulation in megakaryocytes, the platelet precursor cell. Since nothing is known about the molecular mechanisms regulating megakaryocytic P2X1 expression, this study aimed to identify and functionally characterize the P2X1 core promoter utilized in the human megakaryoblastic cell line MEG-01. RESULTS: In order to identify cis-acting elements involved in the transcriptional regulation of P2X1 expression, the ability of 4.7 kb P2X1 upstream sequence to drive luciferase reporter gene expression was tested. Low promoter activity was detected in proliferating MEG-01 cells. This activity increased 20-fold after phorbol-12-myristate-13-acetate (PMA) induced differentiation. A transcription start site was detected 365 bp upstream of the start codon by primer extension. Deletion analysis of reporter constructs indicated a core promoter located within the region -68 to +149 bp that contained two Sp1 sites (named Sp1a and Sp1b) and an NF-1 site. Individual mutations of Sp1b or NF-1 binding sites severely reduced promoter activity whereas triple mutation of Sp1a, Sp1b and NF-1 sites completely abolished promoter activity in both untreated and PMA treated cells. Sp1/3 and NF-1 proteins were shown to bind their respective sites by EMSA and interaction of Sp1/3, NF-1 and TFIIB with the endogenous P2X1 core promoter in MEG-01 cells was demonstrated by chromatin immunoprecipitation. Alignment of P2X1 genes from human, chimp, rat, mouse and dog revealed consensus Sp1a, Sp1b and NF-1 binding sites in equivalent positions thereby demonstrating evolutionary conservation of these functionally important sites. CONCLUSION: This study has identified and characterized the P2X1 promoter utilized in MEG-01 cells and shown that binding of Sp1/3 and NF-1 to elements in the direct vicinity of the transcription start site is essential for basal transcription. Targeting the function of these transcription factors in megakaryocytes may therefore provide a basis for the future therapeutic manipulation of platelet P2X1 function.

Binding Sites↗

PubMed1984

The effect of point mutations affecting Escherichia coli tryptophan tRNA on anticodon-anticodon interactions and on UGA suppression.

tRNA species in Escherichia coli that translate codons starting with U contain 2-methyl-thio-N6-isopentenyl-adenosine in position 37, 3' adjacent to the anticodon. The role of this hypermodification in protein synthesis and trp operon attenuation has been investigated. Temperature-jump relaxation methods have been applied to study the interaction between E. coli tRNAPro, with anticodon VGG (V is uridine-5-oxyacetic acid) complementary to that of tRNATrp, and three species of E. coli tRNATrp: wild type tRNATrp (with ms2i6A37 and G24), UGA suppressor tRNATrp (with ms2i6A37 and A24 in the dihydrouridine stem but the same anticodon CCA), and the same suppressor molecule but ms2i6A-deficient as a result of the mutation miaA. Complex formation between tRNAPro and ms2i6A-containing tRNATrp shows thermodynamic parameters close to those found for several other pairs of tRNA with complementary anticodons. However, ms2i6A-deficient tRNATrp makes less stable complexes with tRNAPro, which dissociate eightfold faster. No effect on the complementary anticodon interaction of the mutation in the dihydrouridine stem can be detected. When the tRNA analogous to the opal codon, E. coli tRNASerIV (anticodon VGA) replaces tRNAPro in similar experiments, very weak complexes are observed with both normally hypermodified species of tRNATrp, the wild type and UGA suppressor; these show a lifetime about 50-fold shorter than with tRNAPro, but are again similar. No complex formation is detectable with the ms2i6A-deficient species. This may explain why the hypermodification is necessary for the efficient suppression of the UGA terminator of Q beta coat protein in vitro. The data on complexes with tRNAPro suggest that deficiency in ms2i6A may also reduce the efficiency of UGG reading. Thus, miaA may affect trp operon attenuation by slowing translation of the tandem UGG codons in the leader sequence. Temperature-jump differential spectra suggest that ms2i6 stabilizes the anticodon interaction by improved stacking of base 37.

Anticodon↗

PubMed1997

Structural analysis of the mouse prosaposin (SGP-1) gene reveals the presence of an exon that is alternatively spliced in transcribed mRNAs.

SGP-1/prosaposin can be secreted or targeted to the lysosomes where it is processed into smaller saposins A, B, C, and D required for the hydrolysis of glycosphingolipids. The deficiency of saposins B and C results in variant forms of metachromatic leukodystrophy and Gaucher's disease, respectively, which are characterized by lysosomal storage of undegraded glycosphingolipids. A required step to correct these genetic defects, or to understand the targeting mechanism of SGP-1 to the lysosomes, or to the extracellular space as well as its interaction with specific glycosphingolipids, is the analysis of the gene encoding this protein. Thus our investigation dealt with the molecular cloning of the mouse SGP-1 gene. Sequence analysis revealed that the mouse SGP-1 gene consists of 15 exons ranging from nine base pairs to 298 base pairs and 14 introns, which ranged from 89 base pairs to >8 kb in length. Our data show that saposin A is encoded by the exons 3, 4, and 5, saposin B by exons 6, 7, 8, and 9, saposin C by exons 10 and 11, and saposin D by exons 12, 13, and 14. The translation start codon is located within exon 1, and the translation stop codon is located within exon 15. The exon/intron boundaries were in accordance to the AG/GT consensus sequences. Our data also revealed that the SGP-1 gene has an exon consisting of the nine base pairs (CAG GAT CAG) encoding the three amino acids of saposin B, which may be alternatively spliced in the SGP-1 mRNA. The presence of the different forms of alternatively spliced mRNAs in various tissues was analyzed by RT-PCR. This approach demonstrated that prosaposin mRNAs of brain, heart, and muscle contain the nine base pairs of exon 8, whereas the transcripts from testis, lung, pancreas, spleen, and kidney do not contain this exon 8. Sequence comparison between the human and mouse prosaposin showed that exon 11 of mouse SGP-1 consists of 279 base pairs, whereas the human prosaposin gene consists of 187 base pairs. The extra 93 base pairs encode 31 amino acids corresponding to a proline-rich region located between saposin C and saposin D in the mouse prosaposin molecule. Finally, the availability of these genomic clones provides a starting point for further studies on the genetic role of specific sequences on the structure and function of SGP-1/prosaposin and its derived saposin proteins. In conclusion, we cloned and sequenced the mouse prosaposin (SGP-1) gene. The structural analysis of this gene revealed the presence of an exon that is alternatively spliced in transcribed mRNAs in a tissue-specific manner.

Alternative Splicing↗

PubMed2002

Gene knock-down in rainbow trout embryos using antisense morpholino phosphorodiamidate oligonucleotides.

Gene knock-down technology using antisense molecules has many applications for studying gene function, disrupting undesirable genetic traits, as well as providing effective therapy for a number of viral diseases. Encouraged by these applications, we developed a gene knock-down technique to interfere with gene expression using transgenic rainbow trout expressing the green fluorescent protein (GFP) gene as a model. One of the antisense morpholino phosphorodiamidate oligonucleotides (AMOs) used in this study (AtGFP-1) was 25 nucleotides in length and localized against codons 2 to 8 of GFP messenger RNA. Microinjection of AtGFP-1 into the blastodisc of fertilized eggs decreased the level of GFP gene expression in a dose-dependent manner, A comparison of the effects of various doses of AtGFP-1 suggested that 10 ng of AtGFP-1 was the optimal concentration in that it interfered with specific gene expression without being strongly toxic to trout embryos. Conversely, morpholino phosphorodiamidate oligonucleotides with the inverted AtGFP-1 sequence, which cannot bind to the target mRNA, did not inhibit GFP gene expression. AtGFP-1 did not affect the expression of nontargeted genes such as the skeletal muscle actin and foreign lacZ genes. These results also indicate that AtGFP-1 interfered with the expression of only the targeted gene. Western blot and reverse transcriptase polymerase chain reaction analyses revealed that the amount of GFP protein drastically decreased whereas the mRNA level was not affected by AtGFP-1, suggesting that AtGFP-1 blocked specific gene function at the translational level. Further, this gene inhibition persisted until the hatching stage. Another AMO, which was localized against the junction region between the 5? untranslated region and the starting codon of GFP mRNA (AtGFP-2), also caused inhibition effects. Thus AMOs can have potent and specific gene knock-down effects in trout embryos. This technology may be useful for examining the roles of selected genes and disrupting their expression during embryonic development of salmonid fish.

Journal Article↗