Translational Frame Research Articles

The simian foamy virus type 1 transcriptional transactivator (Tas) binds and activates an enhancer element in the gag gene.

Simian and human foamy viruses (SFV and HFV) encode a transcriptional transactivator, Tas, which governs the levels of viral transcripts initiated by both the promoter in the long terminal repeat (LTR) and the internal promoter (IP) located within the env gene of these viruses. Tas-responsive target elements,(TRE) LTR in the LTR and (TRE) IP in the env gene, are located 5' of the TATA box in both viral promoters and function as orientation- and position-independent enhancers. We have identified a strong Tas-responsive element, designated TRE (GP), near the 3' end of the gag gene and preceding the pol gene of SFV-1. In transient-expression assays with plasmids containing reporter genes, a 59-bp DNA fragment containing TRE (GP) (nucleotides 2224 to 2282) functioned as an enhancer element, dependent on Tas, in several cell types and in the context of a heterologous basal promoter. DNase footprinting revealed that the fusion protein glutathione S-transferase-Tas, purified from genetically engineered bacteria, interacts with about 40 hp (nucleotides 2237 to 2279) in the TRE (GP). A low degree of sequence homology was noted between TRE (GP) and TRE (IP). In virus-infected cells, novel transcripts with 5' ends immediately upstream from the reverse transcriptase translation frame (nucleotides 2611 to 5778) were identified. Upstream of the start site for these transcripts is a TATA box (nucleotides 2575 to 2579), which was required for transcription in transient-expression assays. Although a spliced mRNA initiated in the viral LTR is implicated in the synthesis of the HFV Pol polyprotein which encodes protease, reverse transcriptase, and integrase, it is possible that SFV-1 contains a promoter within the pol gene for initiating a reverse transcriptase transcript. Taken together, these studies define a novel Tas-responsive enhancer element, which binds the viral transactivator, and a potential promoter within the pol gene.

Jasmonate signalling can be uncoupled from abscisic acid signalling in barley: identification of jasmonate-regulated transcripts which are not induced by abscisic acid.

Jasmonate and abscisic acid induce several identical mRNAs and proteins in barley. In order to study whether both hormones act through the same signalling pathway, we identified four transcripts induced by jasmonic acid methylester (JM) in leaf segments of barley (Hordeum vulgare L. cv. Salome). These newly identified transcripts were not induced by abscisic acid within the tested times of 2-72 h. This finding supports the conclusion that jasmonate signalling in barley is independent of abscisic acid, in contrast to the wound-induction signal cascade of proteinase-inhibitor II in tomato and potato. Of the four isolated cDNAs, the putative translation frame of one was homologous to caffeic acid methyltransferase, another was homologous to chalcone synthase, and the C-terminus of the third showed homology to two proteins from rice (a salt-induced protein and a root-specific protein); the last cDNA was not homologous to any sequences in the databases. The new cDNAs will be valuable tools for studying jasmonate signal transduction in barley.

A molecular defect in loricrin, the major component of the cornified cell envelope, underlies Vohwinkel's syndrome

Terminal keratinocyte differentiation involves coordinated expression of several functionally interdependent genes, many of which have been mapped to the epidermal differentiation complex (EDC) on chromosome 1q21. We have identified linkage of Vohwinkel's syndrome in an extended pedigree to markers flanking the EDC region with a maximum multipoint lod score of 14.3. Sequencing of the loricrin gene revealed an insertion that shifts the translation frame of the C-terminal Gly- and Gln/Lys-rich domains, and is likely to impair cornification. Our findings provide the first evidence for a defect in an EDC gene in human disease, and disclose novel insights into perturbations of cornified cell envelope formation.

The Neurofibromatosis Type I Messenger RNA Undergoes Base-Modification RNA Editing

A functional mooring sequence, known to be required for apolipoprotein B (apoB) mRNA editing, exists in the mRNA encoding the neurofibromatosis type I (NF1) tumor suppressor. Editing of NF1 mRNA modifies cytidine in an arginine codon (CGA) at nucleotide 2914 to a uridine (UGA), creating an in frame translation stop codon. NF1 editing occurs in normal tissue but was several-fold higher in tumors. In vitro editing and transfection assays demonstrated that apoB and NF1 RNA editing will take place in both neural tumor and hepatoma cells. Unlike apoB, NF1 editing did not demonstrate dependence on rate-limiting quantities of APOBEC-1 (the apoB editing catalytic subunit) suggesting that different trans-acting factors may be involved in the two editing processes.

An iterative algorithm for correcting sequencing errors in DNA coding regions.

Insertion and deletion (indel) sequencing errors in DNA coding regions disrupt DNA-to-protein translation frames, and hence make most frame-sensitive coding recognition approaches fail. This paper extends the authors' previous work on indel detection and "correction" algorithms, and presents a more effective algorithm for localizing indels that appear in DNA coding regions and "correcting" the located indels by inserting or deleting DNA bases. The algorithm localizes indels by discovering changes of the preferred translation frames within presumed coding regions, and then "corrects" them to restore a consistent translation frame within each coding region. An iterative strategy is exploited to repeatedly localize and "correct" indels until no more indels can be found. Test results have shown that this improved algorithm can detect and "correct" more indels while not worsening the rate of introduction of false indels when compared to the authors' previous work.

A novel antithrombin gene mutation: slippage and mispairing as a mechanism of genetic disease

There is a high degree of genetic heterogeneity underlying antithrombin deficiency indicating that a number of genetic mechanisms are responsible for the disorder. We report the identification of a five nucleotide (CAGAA) deletion in exon 2 of the antithrombin gene that results in a shift in the frame of translation of the mRNA and introduces a premature STOP signal in codon 70. The deleted nucleotides represent one repeat of a duplicated sequence within codons 36-39. The deletion may have arisen by slippage and mispairing of the repeated sequences at the replication fork during DNA synthesis.

Evidence suggesting cis action by the TnaC leader peptide in regulating transcription attenuation in the tryptophanase operon of Escherichia coli

Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and transcription attenuation. Elevated levels of tryptophan induce transcription antitermination at one or more Rho factor-dependent termination sites in the leader region of the operon. Induction requires translation of a 24-residue coding region, tnaC, located in the 319-nucleotide transcribed leader region preceding tnaA, the structural gene for tryptophanase. In the present paper, we show that two bacterial species that lack tryptophanase activity, Enterobacter aerogenes and Salmonella typhimurium, allow tryptophanase induction and tna operon regulation when they carry a plasmid containing the E. coli tna operon. The role of tnaC in induction was examined by introducing mutations in a 24-nucleotide segment of tnaC of E. coli surrounding and including the crucial Trp codon 12. Some mutations resulted in a noninducible phenotype; these mostly introduced nonconservative amino acid substitutions in TnaC. Other mutations had little or no effect; these generally were in third positions of codons or introduced conservative amino acid replacements. A tryptophan-inserting, UGA-reading glutamine suppressor tRNA was observed to restore partial regulation when Trp codon 12 of tnaC was changed to UGA. Stop codons introduced downstream of Trp codon 12 in all three reading frames established that induction requires translation in the natural tnaC reading frame. Our findings suggest that the TnaC leader peptide acts in cis to prevent Rho-dependent termination.

Nucleosomes reconstituted in vitro on mouse mammary tumor virus B region DNA occupy multiple translational and rotational frames.

The mouse mammary tumor virus acquires a highly reproducible chromatin structure when integrated into cellular DNA. Previous studies have suggested that the LTR is arranged as a series of six phased nucleosomes, that occupy specific positions on the LTR. On the basis of nucleosome reconstitution studies using DNA from the B region of the LTR, it has been argued that this sequence directs a uniquely positioned nucleosome. Here we demonstrate in vitro that reconstituted B region nucleosomes adopt at least five distinct translational positions in two rotational frames on a 206 bp fragment of DNA. We have resolved an initial reconstitute into its component species using nondenaturing gel electrophoresis, and precisely mapped the positions of each species using a hydroxyl radical footprinting assay. To confirm the nucleosome positions determined with the hydroxyl radical assay, nucleosome boundaries were mapped using exonuclease III. Comparison of the results from the hydroxyl radical footprinting and exonuclease III assays revealed a symmetrical pattern of overdigestion by exonuclease III which made unequivocal determination of nucleosome boundaries dubious. We conclude that the general use of exonuclease III to map the positions of nucleosomes may lead to incorrect assignment of position, and that assignment of position through the determination of the nucleosome pseudo-dyad from hydroxyl radical footprinting data represents a superior method of analysis.

Nucleosome positioning on the MMTV LTR results from the frequency-biased occupancy of multiple frames.

The translational positions of nucleosomes in the promoter region of the mouse mammary tumor virus (MMTV) were defined at high resolution. Nucleosome boundaries were determined in primer extension assays using full-length single-stranded mononucleosomal DNA prepared from cells treated with formaldehyde, a reversible protein-DNA cross-linking agent. Multiple boundaries were observed in both the nucleosome A (Nuc-A) and Nuc-B region of the promoter, indicating multiple nucleosome translational frames. The different nucleosome frames in both the Nuc-A and Nuc-B regions were occupied unequally. The most frequently occupied frames were found clustered within 50-60 bases of each other, resulting in a distribution centered in the positions defined previously at low resolution for Nuc-A and Nuc-B. The most abundant 5' ends of the frames in the B region were found between -235 and -187, and the 3' ends between -86 and -36, whereas in the A region the most abundant 5' ends were between -22 and +42, and the 3' ends between +121 and +186. Although frames in the Nuc-B region of the LTR extend at a low frequency in the 5' direction toward the Nuc-C region, there is a sharp discontinuity in the 3' direction toward Nuc-A, suggesting the presence of a boundary constraint in the A-B linker. The positions and relative occupancies of nucleosome frames, in either the B or the A region, did not change when the promoter was activated with dexamethasone.

Translation of the open reading frame encoded by comS, a gene of the srf operon, is necessary for the development of genetic competence, but not surfactin biosynthesis, in Bacillus subtilis.

A small open reading frame, comS of the srf operon, is the site of mutations that impair competence development in Bacillus subtilis. comS open reading frame translation was required for competence, as was confirmed by the suppression of a comS amber mutation [comS(Am)] by the nonsense suppressor sup-3. comS(Am), when introduced into the srf operon, eliminated late competence gene expression but had no significant effect on surfactin production.

A Similar DNA-binding Motif in NFAT Family Proteins and the Rel Homology Region

The cyclosporin-sensitive factor NFATp cooperates with Fos and Jun family proteins to regulate transcription of the interleukin 2 gene in activated T cells. We have defined a 187-amino-acid fragment of NFATp, located centrally within the protein sequence, as the minimal region required for DNA binding and for complex formation with Fos and Jun. The sequence of this region of NFATp shows a low degree of similarity to the Rel homology region. One specific short sequence in NFATp (RAHYETEG), located near the NH2 terminus of the DNA-binding domain, resembles a highly conserved sequence (RFRYxCEG) that is located near the NH2 terminus of the Rel homology region and that has been implicated in DNA binding by Rel family proteins. Mutational analysis demonstrates that the residues in this sequence that are identical in NFATp and Rel family proteins contribute to DNA binding by NFATp. Further, mutation of the threonine residue in this sequence to cysteine, as in Rel proteins, confers on NFATp a sensitivity to sulfhydryl modification similar to that of Rel family proteins. The results suggest that NFATp and Rel family proteins bind to DNA using similar structural motifs.

Bovine Viral Diarrhea Serologic Diagnostic Reagents Prepared from Bacterially Expressed Recombinant Proteins

Bovine viral diarrhea virus (BVDV) is a positive-stranded RNA virus and is classified in the Pestivirus genus of the family Flaviviridae. The BVDV genome has been cloned and sequenced, and a map of protein-encoding genes has also been established. According to this map, the BVDV genome encodes at least 4 primary gene products: p20, gp116, p125, and p133. There is evidence that the glycoprotein precursor gp116 gives rise to gp48, gp25, and gp53 through proteolytic processes, and p125 gives rise to p80 by proteolysis when cells are infected with cytopathic BVDV. The p125 is produced only in cells infected with noncytopathic BVDV. When an antibody against BVDV is analyzed by using radioimmunoprecipitation or western blots, gp48, gp25, gp53, and p80 (or p125) are detected. 1,2,8,9 Thus, these proteins are good candidates for specific enzyme-linked immunosorbent assays (ELISAs). Currently, virus neutralization (VN) is the standard serologic test for BVDV, and virus-neutralizing antibody binds primarily to gp53. 5,11 Other diagnostic tests, such as wholevirus ELISA, fluorescent antibody, and immunoperoxidase staining, also are used and likely detect additional viral proteins. All of these tests require specialized tissue culture equipment. Consequently, these procedures are often impractical for screening a large number of samples. Recombinant ELISA is now widely accepted for serodiagnosis of many important human and veterinary diseases. This test is a simple, rapid, highly sensitive, and inexpensive tool for screening for an antibody. In this report, we describe the production of large amounts of gp48, gp25, and p80 recombinant proteins, use of these recombinant proteins in the ELISA, and comparison of the ELISA with the standard VN test for the serodiagnosis of bovine viral diarrhea (BVD) in cattle. To express gp48, gp25, and p80, each gene fragment was cloned into the expression vector pGex2T or pGex3X. In each construct, the correct translational frame required for expression of glutathione-S-transferase (GST) fusion protein was created. The gp48 and p80 restriction fragments were isolated from pBV4-gp62 and pBV4-p80 clones. The construction and production of these recombinant proteins have been previously described. A 359-base pair (bp) fragment of the gp48 region (nucleotides 1295-1653) and a 918-bp

A plant plasma membrane proton-ATPase gene is regulated by development and environment and shows signs of a translational regulation.

A proton-pumping ATPase is present in the plasma membrane of plant cells where it sustains transport-related functions. This enzyme is encoded by a family of genes that shows signs of both transcriptional and post-transcriptional regulation. The regulation of pma1, one of the Nicotiana plumbaginifolia H+-ATPase genes, was characterized with the help of the beta-glucuronidase (gusA) receptor gene in transgenic plants. pma1 is active in the root epidermis, the stem cortex, and guard cells. This activity depends on developmental and growth conditions. For instance, pma1 activity in guard cells was strongly enhanced when the plant material (young seedlings or mature leaves) was incubated in liquid growth medium. pma1 is also expressed in several tissues of the reproductive organs where active transport is thought to occur but where scarcely any ATPase activity has been identified, namely in the tapetum, the pollen, the transmitting tissue, and the ovules. Several pma genes have a long 5'untranslated region (leader sequence) containing an upstream open reading frame (URF). Analysis of translational and transcriptional fusions with gusA in transgenic plants suggests that the pma1 leader sequence might activate translation of the main open reading frame, even though the URF is translated by a large majority of the scanning ribosomes. As confirmation, transient expression experiments showed that the pma1 leader causes a fourfold post-transcriptional increase of main open reading frame expression. Deletion of the URF by site-directed mutagenesis stimulated the main open reading frame translation 2.7-fold in an in vitro translational assay. These results are consistent with a regulatory mechanism involving translation reinitiation. Altogether, they suggest a fine, multilevel regulation of H+-ATPase activity in the plant.

Nucleosomal location of the STE6 TATA box and Mat alpha 2p-mediated repression.

It has been proposed that yeast MATa cell-specific genes are repressed in MAT alpha cells by the Mat alpha 2p repressor-directed placement of a nucleosome in a position that incorporates the TATA box of the MATa-specific gene close to the nucleosomal pseudodyad. In this study, we address this proposal directly with a series of plasmids designed to place the MATa-specific STE6 TATA box at different locations in a nucleosome and in the internucleosomal linker. These plasmids contain different lengths of synthetic random DNA between the Mat alpha 2p operator and the TATA box of the STE6 promoter, which is located upstream of a lacZ reporter gene in a multicopy plasmid. We show that in MAT alpha cells, a nucleosome is retained in an identical translational frame relative to the Mat alpha 2p operator in all the constructs investigated, irrespective of the sequence of the DNA wrapped onto the histone octamer. This result shows that the nucleosomal organization of the STE6 promoter in MAT alpha cells is not conferred by the sequence of the promoter itself. No expression of the lacZ reporter gene was detectable in MAT alpha cells in any of the constructs, even with the TATA box located in a short internucleosomal linker. These data indicate that repression of MATa-specific genes in MAT alpha cells does not require the precise translational placement of the TATA box close to the nucleosomal pseudodyad; the gene remains repressed when the TATA box is located within the investigated 250-bp region in the organized chromatin domain abutting the Mat alpha 2p operator in MAT alpha cells and may remain repressed with the TATA box located anywhere within this organized repression domain.

Nucleosomal Location of the STE6 TATA Box and Matα2p-Mediated Repression

It has been proposed that yeast MATa cell-specific genes are repressed in MAT alpha cells by the Mat alpha 2p repressor-directed placement of a nucleosome in a position that incorporates the TATA box of the MATa-specific gene close to the nucleosomal pseudodyad. In this study, we address this proposal directly with a series of plasmids designed to place the MATa-specific STE6 TATA box at different locations in a nucleosome and in the internucleosomal linker. These plasmids contain different lengths of synthetic random DNA between the Mat alpha 2p operator and the TATA box of the STE6 promoter, which is located upstream of a lacZ reporter gene in a multicopy plasmid. We show that in MAT alpha cells, a nucleosome is retained in an identical translational frame relative to the Mat alpha 2p operator in all the constructs investigated, irrespective of the sequence of the DNA wrapped onto the histone octamer. This result shows that the nucleosomal organization of the STE6 promoter in MAT alpha cells is not conferred by the sequence of the promoter itself. No expression of the lacZ reporter gene was detectable in MAT alpha cells in any of the constructs, even with the TATA box located in a short internucleosomal linker. These data indicate that repression of MATa-specific genes in MAT alpha cells does not require the precise translational placement of the TATA box close to the nucleosomal pseudodyad; the gene remains repressed when the TATA box is located within the investigated 250-bp region in the organized chromatin domain abutting the Mat alpha 2p operator in MAT alpha cells and may remain repressed with the TATA box located anywhere within this organized repression domain.

Alteration of splice site selection by an exon mutation in the human glycophorin A gene.

We report the identification in human glycophorin A gene of a novel exon mutation that affects splice site selection. DNA mapping showed that the mutation has abolished a unique MspI site marking the exon III-intron 3 splice junction (ACCG/GT). Genomic sequencing confirmed the occurrence of a single G-->A transition at the terminal nucleotide position of exon III. Analysis of the mRNA composition demonstrated a partial inactivation of the altered 5' splice site as well as skipping of exons involving the alternative use of other constitutive splice sites. The full-length transcript with the cognate A change encodes a variant glycophorin with an arginine replacing a glycine at position 59 and defining the ERIK epitope, whereas the exon III-deleted transcript specifies a shorter glycophorin carrying the Sta antigen. Also identified were the misspliced mRNA species with exon I-IV and exon I-V connections generated by selection of 5' splice sites far distant from the mutated site. Although having a correct translation frame, the predicted polypeptides of such exon-skipping products were not assembled on the erythrocyte membrane probably due to the severe truncation of the signal sequence required for targeting and translocation. These observations reveal a new mechanism for antigenic diversity of human glycophorins.

New improved lacZ gene fusion vectors

New plasmid vectors suitable for creating fusions with the lacZ gene have been developed. These vectors represent an improvement over currently available vectors and possess the following features: (1) an undetectable background β-galactosidase (βGal) activity in the absence of fusion, (2) an extended multiple cloning site (MCS), and (3) the ability to conveniently subclone in any one of three translational frames. Medium- and high-copy-number versions of these vectors have been developed.

Silk moth chorion pseudogenes: Hallmarks of genomic evolution by sequence duplication and gene conversion

The part of the genetic locus of the domesticated silk moth, Bombyx mori, in which high cysteine (Hc) chorion genes of late developmental specificity reside contains regions encompassing gene-like sequences which exhibit properties distinct from those of functional Hc genes. One of these regions has been characterized and shown to contain a chorion pseudogene, psi HcB.15, which shares pronounced similarities with a transcribed chorion pseudogene, psi HcB.12/13, which was characterized previously. Both pseudogenes are homologous to HcB chorion genes but bear multiple single nucleotide substitutions and short segmental mutations (insertions and deletions) which introduce translational frame shifts and termination codons in the coding regions. Structural characteristics unique to the two pseudogenes suggest that psi HcB.15 was generated first from a functional HcB gene and gave rise subsequently to psi HcB 12/13 as a result of a sequence duplication event. The two pseudogenes can be distinguished from each other by the presence of distinct regions of similarity to the consensus sequence of functional HcB genes which appear to have arisen from gene-conversion-mediated correctional events. These findings lend support to the hypothesis that chorion pseudogene sequences represent reservoirs of genetic information that participates in the evolution of the chorion locus rather than relics of inactivated genes passively awaiting extinction.

Characterization of translational frame exception patients in Duchenne/Becker muscular dystrophy.

The clinical progression of Duchenne muscular dystrophy (DMD) patients with deletions can be predicted in 93% of cases by whether the deletion maintains or disrupts the translational reading frame (frameshift hypothesis). We have identified and studied a number of patients who have deletions that do not conform to the translational frame hypothesis. The most common exception to the frameshift hypothesis is the deletion of exons 3 to 7 which disrupts the translational reading frame. We identified a Becker muscular dystrophy (BMD) patient, an intermediate, and a DMD patient with this deletion. In all three cases, dystrophin was detected and localized to the membrane. One DMD patient with an inframe deletion of exons 4-18 produced no dystrophin. One patient with a mild intermediate phenotype and a deletion of exon 45, which shifts the reading frame, produced no dystrophin. Two patients with large inframe deletions had discordant phenotypes (exons 3-41, DMD; exons 13-48, BMD), but both produced dystrophin that localized to the sarcolemma. The DMD patient, 113, indicates that dystrophin with an intact carboxy terminus can be produced in Duchenne patients at levels equivalent to some Beckers. The dystrophin analysis from these patients, together with patients reported in the literature, indicate that more than one domain can localize dystrophin to the sarcolemma. Lastly, the data shows that although most patients show correlation of clinical severity to molecular data, there are rare patients which do not conform.

Code domains in tandem repetitive DNA sequence structures.

Traditionally, many people doing research in molecular biology attribute coding properties to a given DNA sequence if this sequence contains an open reading frame for translation into a sequence of amino acids. This protein coding capability of DNA was detected about 30 years ago. The underlying genetic code is highly conserved and present in every biological species studied so far. Today, it is obvious that DNA has a much larger coding potential for other important tasks. Apart from coding for specific RNA molecules such as rRNA, snRNA and tRNA molecules, specific structural and sequence patterns of the DNA chain itself express distinct codes for the regulation and expression of its genetic activity. A chromatin code has been defined for phasing of the histone-octamer protein complex in the nucleosome. A translation frame code has been shown to exist that determines correct triplet counting at the ribosome during protein synthesis. A loop code seems to organize the single stranded interaction of the nascent RNA chain with proteins during the splicing process, and a splicing code phases successive 5' and 3' splicing sites. Most of these DNA codes are not exclusively based on the primary DNA sequence itself, but also seem to include specific features of the corresponding higher order structures. Based on the view that these various DNA codes are genetically instructive for specific molecular interactions or processes, important in the nucleus during interphase and during cell division, the coding capability of tandem repetitive DNA sequences has recently been reconsidered.