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PubMed1992

Histidine ammonia-lyase from Streptomyces griseus.

Histidine ammonia-lyase (histidase; HutH) has been purified to homogeneity from Streptomyces griseus and the N-terminal amino acid (aa) sequence used to clone the histidase-encoding structural gene, hutH. The purified enzyme shows typical saturation kinetics and is inhibited competitively by D-histidine and histidinol phosphate. High concentrations of K.cyanide inactivate HutH unless the enzyme is protected by the substrate or histidinol phosphate. On the basis of the nucleotide sequence, the hutH structural gene would encode a protein of 53 kDa with an N terminus identical to that determined for the purified enzyme. Immediately upstream from hutH is a region that strongly resembles a class of Streptomyces promoters active during vegetative growth; however, there is no obvious ribosome-binding site adjacent to the hutH translation start codon. The deduced aa sequence of an upstream partial open reading frame shows no similarity with other proteins, including HutP of Bacillus subtilis and HutU of Pseudomonas putida. Promoter-probe analysis indicates that promoter activity maps within the DNA surrounding the hutH start codon. Pairwise comparisons of the primary structures of bacterial and mammalian histidases, together with the unique kinetic properties and gene organization, suggest that streptomycete histidase may represent a distinct family of histidases.

Amino Acid Sequence↗

PubMed1995

Gene pvuIIW: a possible modulator of PvuII endonuclease subunit association.

The PvuII restriction-modification system has been found to contain three genes which code for a DNA methyltransferase (MTase), a restriction endonuclease (ENase) and a small protein required for expression of the ENase-encoding gene. In addition, there is a small open reading frame (ORF) within and opposite to the MTase-encoding gene. The region containing this ORF is transcribed, and the ORF has an excellent Shine-Dalgarno sequence with an ATA start codon. A closely related ORF is present in the SmaI system. The 28-amino-acid (aa) predicted peptide from the PvuII ORF resembles a region of the PvuII ENase at the dimer interface. We have cloned this ORF, giving it an ATG start codon and putting it under the control of an inducible promoter: induction leads to a slight but significant decrease in restriction of bacteriophage lambda. We also have obtained the 28-aa synthetic peptide, and are exploring the possibility that it modulates ENase subunit association. While this peptide has no detectable effect on dimeric PvuII ENase, it inhibits renaturation of urea-denatured ENase in a concentration-dependent manner. The ORF may represent an additional safeguard during establishment of the PvuII restriction-modification system in a new host cell, helping to delay the appearance of active ENase dimers, while the MTase accumulates and protects the host chromosome.

Amino Acid Sequence↗

PubMed1995

Structural organization and chromosomal assignment of the human prostacyclin receptor gene.

Prostacyclin receptor is a member of the prostanoid receptor family in the G protein-coupled receptor superfamily with seven transmembrane domains. We report here the isolation and structural organization of the human prostacyclin receptor gene. Southern blot analysis demonstrated a single copy of the human prostacyclin receptor gene in the human genome. The human prostacyclin receptor gene spanned approximately 7.0 kb and was composed of three exons separated by two introns. The first intron occurred in the 5'-untranslated region, 13 bp upstream to the ATG start codon. The second intron was located at the end of the sixth transmembrane domain, thereby separating it from the downstream coding region and the 3'-untranslated region. By primer extension analysis, the transcription initiation sites were mapped 870-872 bp upstream to the ATG start codon. The 1.2-kb human prostacyclin receptor 5'-flanking region lacked conventional TATA and CCAAT boxes, but it contained several cis-acting regulatory elements including an inverted CCAAT box (Y box) and two copies of SP-1 binding sites. Using human-rodent somatic hybrid cell DNA, the human prostacyclin receptor gene was assigned to human chromosome 19. The present study helps establish the genetic basis for prostacyclin receptor research and provides further insight into the molecular mechanisms underlying the prostanoid receptor family.

Amino Acid Sequence↗

PubMed1997

Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6.

A primer extension (toeprinting) assay was used to monitor selection by ribosomes of the first versus the second AUG codon as a function of introducing mutations on the 3' side (positions +4, +5 and +6) of the first AUG codon. Six different flanking codons starting with G (GCG, GCU, GCC, GCA, GAU and GGA) strongly augmented selection of AUG#1 when compared with matched mRNAs that had A or C instead of G in position +4. Augmentation by G in position +4 failed only when it was combined with U in position +5, as in the sequence augGUA. In contrast with the usual enhancing effect of introducing G in position +4, most mutations in position +5 had no discernible effect, as shown with the series augANA (where N = C, A, G or U) and the series augCNA. AUG codon recognition was also unaffected by mutations in position +6, as shown by testing four mRNAs that had augCCN as the start site. Thus the primary sequence context that augments the recognition of AUG start codons does not appear generally to extend beyond G in position +4. When the toeprinting assay was used with mRNAs that initiate translation at CUG instead of AUG, cugGAU was not recognized better than cugGGU, contradicting the hypothesis that initiation at non-AUG codons might be favored by A instead of G in position +5.

Base Sequence↗

PubMed1996

Identification of cis-acting DNA sequences involved in the transcription of the virulence regulatory gene spvR in Salmonella typhimurium.

The SpvR protein is a DNA-binding protein of the LysR family, required for the transcription of the spvABCD virulence operon of Salmonella typhimurium. An alternative sigma factor, sigma S (RpoS), in conjunction with SpvR, controls the transcription of the spvR gene. In this study, we used a combination of primer extension experiments and deletion/fusion analyses of the spvR gene to identify sequences involved in spvR transcription in S. typhimurium. When induced in the stationary phase of growth in rich medium or during carbon starvation, transcription of spvR in S. typhimurium is driven by a single promoter (spvRp1) and initiates 17 nucleotides upstream of the spvR start codon. The level of spvR transcription originating at spvRp1 was 20-fold higher in the wild-type strain than in the rpoS mutant. In both strains, however, transcription at spvRp1 requires the SpvR protein. 5' Deletions up to position -86, relative to the spvR start codon, did not inhibit inducibility by sigma S and/or SPVR. In contrast, 5' deletion up to -75 abolished the activation of spvRp1 by SpvR in both the wild-type strain and rpoS mutant. Within the 11-bp sequence lying between position -86 and position -75, a 10-bp consensus motif TNTNTGCANA, present in both the spvR and spvA promoter regions, was identified and may contain the DNA recognition site for SpvR. In addition, we detected initiation of transcription within the spvR coding region. This finding may have implications for comparative studies of regulation with spvR gene fusions.

Bacterial Proteins↗

PubMed1995

The bovine leukemia virus encapsidation signal is discontinuous and extends into the 5' end of the gag gene.

In order to define bovine leukemia virus (BLV) sequences required for efficient vector replication, a series of mutations were made in a BLV vector. Testing the replication efficiency of the vectors with a helper virus and helper plasmids allowed for separation of the mutant vectors into three groups. The replication efficiency of the first group was reduced by a factor of 7; these mutants contained deletions in the 5' end of the gag gene. The second group of mutants had replication reduced by a factor of 50 and had deletions including the 5' untranslated leader region. The third group of mutants replicated at levels comparable to those of the parental vector and contained deletions of the 3' end of the gag gene, the pol gene, and the env gene. Analysis of cytoplasmic and virion RNA levels indicated that vector RNA expression was not affected but that the vector RNA encapsidation was less efficient for group 1 and group 2 mutants. Additional mutations revealed two regions important for RNA encapsidation. The first region is a 132-nucleotide-base sequence within the gag gene (nucleotides 1015 to 1147 of the proviral DNA) and facilitates efficient RNA encapsidation in the presence of the second region. The second region includes a 147-nucleotide-base sequence downstream of the primer binding site (nucleotide 551) and near the gag gene start codon (nucleotide 698; gag begins at nucleotide 628) and is essential for RNA encapsidation. We conclude that the encapsidation signal is discontinuous; a primary signal, essential for RNA encapsidation, is largely in the untranslated leader region between the primer binding site and near the gag start codon. A secondary signal, which facilitates efficient RNA encapsidation, is in a 132-nucleotide-base region within the 5' end of the gag gene.

Animals↗

PubMed1998

Parameters influencing translational efficiency in aphthovirus IRES-based bicistronic expression vectors.

Initiation of translation in picornavirus RNAs occurs internally, mediated by an internal ribosome entry site (IRES) element. This property has been exploited to coexpress proteins from a single bicistronic transcription unit in eukaryotic cells. The region that separates the IRES element from the authentic initiator codon of the second gene plays an important role in the translation efficiency of this cistron. In the present report, we have analyzed the effect of sequence modifications in this region on the translation efficiency directed by the foot-and-mouth disease (FMDV) IRES in bicistronic expression vectors. Insertion of various sequences, which contained additional start codons and/or the capacity to form hairpins immediately downstream of the 3' border of the IRES, strongly reduced the translation efficiency of the second gene in bicistronic RNAs. Interestingly, an increase of distance per se did not have a deleterious effect on translation efficiency. The bicistronic vector studied here tolerated 95 nucleotides between the 3' border of the IRES and the authentic start codon, provided that out-of-frame AUG codons or hairpins were not present in this RNA segment. These results indicate that FMDV-derived bicistronic constructs are extremely well suited for use in eukaryotic expression vectors.

Animals↗

PubMed2000

Cloning and characterization of the proximal promoter region of the mouse glutamate-L-cysteine ligase regulatory subunit gene.

We describe upregulation of the mRNA for the mouse glutamate-cysteine ligase regulatory subunit gene (Glcl-r) in Hepa-1 cells treated with beta-napthoflavone (BNF) and tert-butylhydroquinone (tBHQ). A 2-kb fragment of the proximal promoter region of the gene was cloned and sequenced, and sequence analysis reveals a high degree of homology when compared to the human glutamate-cysteine ligase regulatory subunit gene promoter. Primer extension analysis indicates a major transcription start site 218 bp upstream of the translation start codon in a CpG-rich region, suggesting that transcription is Sp1 mediated. Reporter constructs containing nested deletion fragments of the Glcl-r promoter demonstrate that regulatory elements sufficient for basal and tBHQ-inducible expression lie between -273 and -787 bp relative to the translation start codon and that the distal promoter may contain negative regulatory elements.

Animals↗

PubMed2001

A novel polymorphism in the promoter of the RAGE gene is associated with non-small cell lung cancer.

The receptor for advanced glycosylation endproducts (RAGE) is abundant at both the transcriptional and translational level in normal lung but is not expressed in non-small cell lung cancer (NSCLC). In order to determine whether sequence variations might be responsible for the inactivation of RAGE in NSCLC, we investigated the RAGE gene in primary NSCLCs and in the corresponding normal tissues of nine patients. Although sequence analysis revealed no somatic, tumor-associated mutations, six novel sequence variants were identified: T-->A in the promoter region 388 bp upstream of the start codon: T-->A in exon 1 (Ala2Ala), C-->G in exon 3 (Val89Val), C-->T in intron 6, G-->C and C-->G in exon 10 (Arg365Ser and Arg369Gly). In addition, we detected a 63 bp deletion in the promoter region (358-421 bp upstream of the start codon) in one NSCLC patient. The T-->A transversion in the promoter region was detected in three of nine patients. Further analysis of this polymorphic locus in 54 NSCLC patients and 59 non-cancer controls revealed a significant difference in the genotype distribution between NSCLC patients and controls. Interestingly, the AA genotype was more common in NSCLC patients (20.8%) than in controls (3.5%). The cumulative occurrence of the AA variant in NSCLC suggests that this genotype is a putative risk factor for NSCLC development.

Aged↗

PubMed1994

Sequence analysis of a unique temperature phage: mycoplasma virus L2.

Mycoplasma virus L2 is a quasi-spherical enveloped virion containing circular double-stranded DNA. L2 infection of Acholeplasma laidlawii host cells leads to a noncytocidal productive infection cycle followed by establishment of lysogeny in all (or most) infected cells, with viral DNA integrated into the host cell genome. The L2 genome has been sequenced and analyzed. L2 DNA is 11,965-bp long and contains 15 open reading frames (ORFs). One of these, ORF13*, has its start codon within and in the same reading frame as ORF13. The ORFs are clustered in four groups separated by noncoding intergenic regions, suggesting that gene expression involves transcription of genes in a cluster into polycistronic mRNA and translation of these genes via translational coupling or reinitiation. Fifteen L2 start codon sites have been defined and resemble those of eubacteria. The N-terminal sequences of two ORFs appear to be signal peptides, and the gene product of one of these may be an L2 virion integral membrane protein. The ORF 5 product has been tentatively identified as an integrase, based on its sequence similarity to site-specific recombinases. The putative attP integration site has been mapped to an intergenic region, 280-bp downstream from ORF 5. Two putative DNA replication ori sites have been mapped. Each is in an intergenic region and contains a DnaA-box bounded by A + T-rich 6-mer repeats.

Acholeplasma laidlawii↗

PubMed2005

Cloning and functional characterization of the rat alpha2B-adrenergic receptor gene promoter region: Evidence for binding sites for erythropoiesis-related transcription factors GATA1 and NF-E2.

In the rat, the alpha2B-adrenergic receptor (alpha2B-AR) is encoded by the rat non-glycosylated (RNG) gene and is primarily expressed in the kidney, brain and liver of adult animals. High levels of alpha2B-AR are also found during fetal life in the placenta, liver and blood, where it is borne by cells of the erythropoietic lineage. As a first step to define the mechanisms responsible for the spatio-temporal pattern of alpha2B-AR expression, a genomic fragment containing 2.8 kb of the 5'-flanking region, the ORF and approximately 20 kb of the 3'-flanking region of the RNG gene was isolated. RNase protection assays performed on RNA from placenta or kidney using a series of riboprobes permitted to locate the transcription start site 372 bases upstream from the start codon. Transient transfection of various cells, including rat proximal tubule in primary culture, with constructs containing luciferase as a reporter gene demonstrated that: (i) the 5'-flanking region exhibited a strong and sense-dependent transcriptional activity and (ii) the 332 bp fragment (-732/-401 relative to the start codon), which lacks a TATA box but contains Sp1 sites, is sufficient to drive expression. Analysis of chromatin susceptibility to DNaseI digestion identified two hypersensitive sites (HS1 and HS2) located 1.7 and 1.0 kb, respectively, upstream from ATG and containing recognition sequences for erythroid transcription factors. EMSA showed specific binding of GATA1 and NF-E2 to these elements. Taken together, the results suggest that the chromatin environment in the vicinity of these boxes plays a critical role for alpha2B-AR expression during fetal life.

Animals↗

PubMed1978

Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli.

The pheA structural gene of the phenylalanine operon of Escherichia coli is preceded by a transcribed leader region of about 170 nucleotide pairs. In vitro transcription of plasmids and restriction fragments containing the phe promoter and leader region yields a major RNA transcript about 140 nucleotides in length. This transcript, pheA leader RNA, has the following features: (i) a potential ribosome binding site and AUG translation start codon about 20 nucleotides from its 5' end; (ii) 14 additional in phase amino acid codons and a UGA stop codon after the AUG; 7 of these 14 are Phe codons; (iii) a 3'-OH terminus about 140 nucleotides from the 5' end (transcription termination occurs in an A.T-rich region which is subsequent to a G.C-rich region; just beyond the site of transcription termination there is a sequence corresponding to a ribosome binding site and the AUG translation start codon of the pheA structural gene); (iv) a sequence which would permit extensive intrastrand stable hydrogen bonding. In addition to G.C-rich stem structures, highly analogous to those proposed for the leader RNAs of the tryptophan operons of E. coli and Salmonella typhimurium [Lee, F. & Yanofsky, C. (1977) Proc. Natl. Acad. Sci. USA 74, 4365-4369], there is also extensive base-pairing possible between the phe codon region and a more distal region of the leader transcript. The roles of synthesis of the Phe-rich leader peptide and secondary structure of the leader transcript in the regulation of transcription termination at the attenuator of the phe operon are discussed.

Base Sequence↗

PubMed2006

Identification of human hepatic stimulator substance gene promoter and demonstration of dual regulation of AP1/AP4 cis-acting element in different cell lines.

Human hepatic stimulator substance (hHSS) is a newly identified growth-promoting factor in the liver. HSS is capable of stimulating hepatic regeneration in partial hepatectomized rats, thus, promoting growth of hepatic tumor cells. To understand and elucidate the transcriptional regulation of hHSS gene, the 4890bp of 5'-flanking region of the gene have been isolated and sequenced. The transcriptional start site, located at 248nt upstream from the ATG starting codon, was identified by 5'-rapid amplification cDNA end (5'-RACE). The classical promoter sequences, such as TATA box or GAATT were not identified in the promoter region, instead a GC-rich segment was formed (>70%) by expanding to a longer than 400bp, and immediately upstream from the ATG start codon. The transient transfection assays, using promoter deletion constructs, showed that hHSS promoter was efficiently capable in driving the reporter expression not only in HepG2 cells, but also in Cos7 cells. A region spanning nucleotides in the range of -447 to -358bp revealed a negative regulation on promoter activity in HepG2 cells, but with positive regulation in Cos7 and Hela cells. The promoter activity was obviously influenced by AP1/AP4 (-375/-369nt) mutation in these three cell lines. EMSAs showed that the site was recognized by AP1 in HepG2 cell, and only by an AP4 protein in Cos7 cells. The c-Jun bound to the promoter was further verified by supershift in HepG2 cells and human liver tissue. Chromatin immuno-precipitation (ChIP) demonstrated that there was a direct association of c-Jun with hHSS promoter in HepG2 cells. The c-Jun strongly suppressed hHSS promoter activity in transient expression analyses in HepG2 cells. Mutations in the AP1 binding sites rescued suppression caused by c-Jun, suggesting this was a direct regulation of the hHSS promoter. In contrast, there was no significant effect in c-Jun over-expressed Cos7 and Hela cells. The tissue-specific function of c-Jun in hHSS promoter activity may in part help explain the differences in biology function of hHSS between liver and non-liver cells.

Animals↗

PubMed1998

Efficient in vitro translational termination in Escherichia coli is constrained by the orientations of the release factor, stop signal and peptidyl-tRNA within the termination complex.

There have been contrasting reports of whether the positioning of a translational stop signal immediately after a start codon in a single oligonucleotide can act as a model template to support efficient in vitro termination. This paradox stimulated this study of what determines the constraints on the positioning of the components in the termination complex. The mini mRNA, AUGUGAA, was unable to support efficient in vitro termination in contrast to separate AUG/UGA(A) codons, unless the ribosomal interaction of the stop signal with the decoding factor, release factor 2, was stimulated with ethanol or with nucleotide-free release factor 3, or by using (L11-)-ribosomes which have a higher affinity for release factor 2, or unless the fMet-tRNA was first bound to 30S subunits independently of the mini mRNA. An additional triplet stop codon could restore activity of the mini mRNA, indicating that its recognition was not sterically restrained by the stop signal already within it. This suggests that in an initiation complex an adjoining start/stop signal is not positioned to support efficient decoding by release factor unless it is separated from the start codon. Site-directed crosslinking from mRNAs to components of the termination complex has shown that mRNA elements like the Shine-Dalgarno sequence and the codon preceding the stop signal can affect the crosslinking to release factor, and presumably the orientation of the signal to the factor.

Codon, Terminator↗

PubMed2000

The expression of full length Gp91-phox protein is associated with reduced amphotropic retroviral production.

BACKGROUND AND OBJECTIVE: As a single gene defect in mature bone marrow cells, chronic granulomatous disease (X-CGD) represents a disorder which may be amenable to gene therapy by the transfer of the missing subunit into hemopoietic stem cells. In the majority of cases lack of Gp91-phox causes the disease. So far, studies involving transfer of Gp91-phox cDNA, including a phase I clinical trial, have yielded disappointing results. Most often, low titers of virus have been reported. In the present study we investigated the possible reasons for low titer amphotropic viral production. DESIGN AND METHODS: To investigate the effect of Gp91 cDNA on the efficiency of retroviral production from the packaging cell line, GP+envAm12, we constructed vectors containing either the native cDNA, truncated versions of the cDNA or a mutated form (LATG) in which the natural translational start codon was changed to a stop codon. Following derivation of clonal packaging cell lines, these were assessed for viral titer by RNA slot blot and analyzed by non-parametrical statistical analysis (Whitney-Mann U-test). RESULTS: An improvement in viral titer of just over two-fold was found in packaging cells containing the start-codon mutant of Gp91 and no evidence of truncated viral RNA was seen in these cells. Further analysis revealed the presence of rearranged forms of the provirus in Gp91-expressing cells, and the production of truncated, unpackaged viral RNA. Protein analysis revealed that LATG-transduced cells did not express full-length Gp91-phox, whereas those containing the wild-type cDNA did. However, a truncated protein was seen in ATG-transduced cells which was also present in wild type cells. No evidence for the presence of a negative transcriptional regulatory element was found from studies with the deletion mutants. INTERPRETATION AND CONCLUSIONS: A statistically significant effect of protein production on the production of virus from Gp91-expressing cells was found. Our data point to a need to restrict expression of the Gp91-phox protein and its derivatives in order to enhance retroviral production and suggest that improvements in current vectors for CGD gene therapy may need to include controlled, directed expression only in mature neutrophils.

3T3 Cells↗

PubMed1998

Improved envelope function selected by long-term cultivation of a translation-impaired HIV-1 mutant.

The untranslated leader region of the human immunodeficiency virus (HIV) RNA genome contains multiple regulatory elements that fold into stable hairpin structures. Because extensive secondary structure can block the scanning of ribosomes, an alternative mechanism for HIV translation seems feasible. To study the mechanism of HIV-1 mRNA translation, a start codon was introduced in the leader region that will usurp scanning ribosomes. This upstream AUG mutation (uAUG) inhibited HIV gene expression, indicating that HIV-1 mRNA translation occurs via the regular scanning mechanism. Revertant viruses with increased replication capacity were obtained upon prolonged culturing of the mutant virus. To our surprise, the introduced start codon had not been inactivated in these phenotypic revertants. Instead, these revertants contain additional mutations in the envelope (Env) protein that stimulated HIV-1 replication. These second-site Env mutations did not specifically overcome the gene expression defect of the uAUG mutant, as the replication capacity of other HIV-1 mutants with an unrelated defect could also be improved. The uAUG construct appears to be a unique tool in forced HIV-1 adaptation studies because the deleterious uAUG mutation is stably maintained in the progeny, yielding phenotypic revertants with second-site mutations elsewhere in the viral genome.

Base Sequence↗

PubMed1993

Cloning, characterization and overexpression of the phytase-encoding gene (phyA) of Aspergillus niger.

Phytase catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. A gene (phyA) of Aspergillus niger NRRL3135 coding for extracellular, glycosylated phytase was isolated using degenerate oligodeoxyribonucleotides deduced from phytase amino acid (aa) sequences. Nucleotide (nt) sequence analysis of the cloned region revealed the presence of an open reading frame coding for 467 aa and interrupted once by an intron of 102 bp in the 5' part of the gene. The start codon is followed by a sequence coding for a putative signal peptide. Expression of phyA is controlled at the level of mRNA accumulation in response to inorganic phosphate levels. After cell growth in low-phosphate medium, a transcript of about 1.8 kb was visualized. Transcription of phyA initiates at at least seven start points within a region located 45-25 nt upstream from the start codon. In transformants of A. niger, expression of multiple copies of phyA resulted in up to more than tenfold higher phytase levels than in the wild-type strain.

6-Phytase↗

PubMed2003

Excision of the first intron from the gonadotropin-releasing hormone (GnRH) transcript serves as a key regulatory step for GnRH biosynthesis.

The mammalian gonadotropin-releasing hormone (GnRH) gene consists of four short exons (denoted as 1, 2, 3, and 4) and three intervening introns (A, B, and C). Recently, we demonstrated that excision of the first intron (intron A) from the GnRH transcript is regulated in a tissue- and developmental stage-specific fashion and is severely attenuated in hypogonadal (hpg) mouse because of its lack of exonic splicing enhancers (ESE) 3 and 4. In the present study, we examined the influence of intron A on translational efficiency, thereby establishing a post-transcriptional control over GnRH biosynthesis. First, we verified that an intron A-retained GnRH transcript is a splicing variant but not a splicing intermediate. Intron A-retained transcripts can be transported to the cytoplasm in contrast to intron B-containing transcripts, which are restricted to the nucleus. This result implicates the intron A-retained GnRH transcript as a splicing variant; it has a long 5'-untranslated region, as the GnRH prohormone open reading frame (ORF) begins on exon 2. We investigated whether an intron A-retained GnRH transcript can properly initiate translation at the appropriate start codon and found that intron A completely blocks the translation initiation of its downstream reporter ORF both in vivo and in vitro. The inhibition of translation initiation appears to be due to the presence of a tandem repeat of ATG sequences within intron A. Constructs bearing mutations of ATGs to AAGs restored translation initiation at the downstream start codon; the extent of this restoration correlated with the number of mutated ATGs. Besides the failure in the translation initiation of GnRH-coding region in the intron A-containing variant, the present study also suggests that the interference between mature GnRH mRNA and intron A-retained splicing variant could occur to lower the efficiency of GnRH biosynthesis in the GT1-1-immortalized GnRH-producing cell line. Therefore, our results indicate that the precise and efficient excision of intron A and the joining of adjacent exons may be a critical regulatory step for the post-transcriptional regulation of GnRH biosynthesis.

Alternative Splicing↗