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PubMed1983

In vitro translation of elastin mRNA and processing of the translated products and the signal sequence of elastin a.

Chick embryo aorta mRNAs were translated in a gel-filtered reticulocyte lysate. The translated products showed two elastin-related proteins (a and b; relative mass approximately 70 000). The translated elastin a protein was separated essentially free of the b protein by centrifugation and sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis. The a protein was then electroeluted from the SDS-polyacrylamide gel and a partial sequence was determined by automated Edman degradation. The NH2-terminal presequence of the elastin a protein is (formula; see text) The assigned sequence is identical to that reported for the b protein. Translation of chick embryo aorta mRNA in the presence of dog pancreas microsomal membranes segregated the a and b proteins into membrane vesicles and cotranslationally cleaved their respective presequences. The processed a and b elastin proteins were isolated together and NH2-terminal proline positions were determined. These are (formula; see text) These proline positions 4 and 8 are identical to those for NH2-terminal sequence for tropoelastin. This suggests that the signal peptidase removes the 24-residue signal peptide and thus directly generates the tropoelastin sequence, with no NH2-terminal prosequence as the intermediate.

Amino Acid Sequence↗

PubMed2004

Primary acute lymphoblastic leukemia cells use a novel promoter and 5'noncoding exon for the human reduced folate carrier that encodes a modified carrier translated from an upstream translational start.

The human reduced folate carrier (hRFC) is reported to be regulated by up to seven alternatively spliced noncoding exons (A1, A2, A, B, C, D, and E). Noncoding exon and promoter usage was analyzed in RNAs from 27 childhood acute lymphoblastic leukemia (ALL) specimens by real-time PCR and/or 5' rapid amplification of cDNA ends (5' RACE) assay. By real-time PCR, total hRFC transcripts in ALL spanned a 289-fold range. Over 90% of hRFC transcripts were transcribed with A1, A2, and B 5' untranslated regions (UTRs). Analysis of 5' RACE clones showed that the A1 + A2 5'UTRs contained A1 sequence alone or a fusion of A1 and A2, implying the existence of a single, alternatively spliced 1021-bp A1/A2 noncoding region. High frequency sequence polymorphisms (AGG deletion, C/T transition) identified in the A1/A2 region by 5'RACE were confirmed in normal DNAs. By reporter assays in HepG2 hepatoma and Jurkat leukemia cells, A1/A2 promoter activity was localized to a 134-bp minimal region. Translation from an upstream AUG in the A1/A2 noncoding region in-frame with the normal translation start resulted in synthesis of a larger ( approximately 7 kDa) hRFC protein with transport properties altered from those for wild-type hRFC. Although there was no effect on transcript or protein stabilities, in vitro translation from A1/A2 transcripts was decreased compared with those with the B 5'UTR. Our results document the importance of the hRFC A1/A2 upstream region in childhood ALL and an intricate transcriptional and posttranscriptional regulation of hRFC-A1/A2 mRNAs. Furthermore, they suggest that use of the A1/A2 5'UTR may confer a transport phenotype distinct from the other 5'UTRs due to altered translation efficiency and transport properties.

5' Untranslated Regions↗

PubMed2004

Lost in Translation: Obstacles to Translational Medicine.

When we launched the Journal of Translational Medicine a few months ago, we were interested primarily in exploring scientific consideration of this discipline. However, as editors of JTM, we have been contacted almost daily to discuss the problems faced by scientists and clinicians around the world who are challenging the traditional boundaries of science and medicine. Through these conversations, we have learned that translational medicine is in fact "lost in translation," inspiring much angst, many promises and some Federal appropriations. However, little has been done to substantively promote this important field. Authoritative reviews on the subject are available to the interested reader 1234567. In this article, we will address JTM's "constituency" to report what we've learned about the obstacles to translational medicine from the myriad of phone conversations and e-mail interactions.

Editorial↗

PubMed1999

Apontic binds the translational repressor Bruno and is implicated in regulation of oskar mRNA translation.

The product of the oskar gene directs posterior patterning in the Drosophila oocyte, where it must be deployed specifically at the posterior pole. Proper expression relies on the coordinated localization and translational control of the oskar mRNA. Translational repression prior to localization of the transcript is mediated, in part, by the Bruno protein, which binds to discrete sites in the 3' untranslated region of the oskar mRNA. To begin to understand how Bruno acts in translational repression, we performed a yeast two-hybrid screen to identify Bruno-interacting proteins. One interactor, described here, is the product of the apontic gene. Coimmunoprecipitation experiments lend biochemical support to the idea that Bruno and Apontic proteins physically interact in Drosophila. Genetic experiments using mutants defective in apontic and bruno reveal a functional interaction between these genes. Given this interaction, Apontic is likely to act together with Bruno in translational repression of oskar mRNA. Interestingly, Apontic, like Bruno, is an RNA-binding protein and specifically binds certain regions of the oskar mRNA 3' untranslated region.

3' Untranslated Regions↗

PubMed2002

JavaScript DNA translator: DNA-aligned protein translations.

There are many instances in molecular biology when it is necessary to identify ORFs in a DNA sequence. While programs exist for displaying protein translations in multiple ORFs in alignment with a DNA sequence, they are often expensive, exist as add-ons to software that must be purchased, or are only compatible with a particular operating system. JavaScript DNA Translator is a shareware application written in JavaScript, a scripting language interpreted by the Netscape Communicator and Internet Explorer Web browsers, which makes it compatible with several different operating systems. While the program uses a familiar Web page interface, it requires no connection to the Internet since calculations are performed on the user's own computer. The program analyzes one or multiple DNA sequences and generates translations in up to six reading frames aligned to a DNA sequence, in addition to displaying translations as separate sequences in FASTA format. ORFs within a reading frame can also be displayed as separate sequences. Flexible formatting options are provided, including the ability to hide ORFs below a minimum size specified by the user. The program is available free of charge at the BioTechniques Software Library (www.Biotechniques.com).

Amino Acid Sequence↗

PubMed2005

Breaking the translational barriers: the value of integrating biomedical informatics and translational research.

The conduct of translational health research has become a vital national enterprise. However, multiple barriers prevent the effective translation of basic science discoveries into clinical and community practice. New information technology (IT) applications could help address these barriers. Unfortunately, owing to a combination of organizational, technical, and social factors, neither physician-investigators and research staff nor their clinical and community counterparts have harnessed such applications. Recently, at the request of the Institute of Medicine's Clinical Research Roundtable, a qualitative study of these factors was conducted at several leading academic medical centers. We explore the current status of IT in the translational research domain, describe the qualitative results, and conclude with a proposed set of initiatives to further increase the integration of IT into translational research.

Biomedical Research↗

PubMed1990

Recombinant C5a stimulates transcription rather than translation of interleukin-1 (IL-1) and tumor necrosis factor: translational signal provided by lipopolysaccharide or IL-1 itself.

We investigated the effects of recombinant C5a (rC5a) on gene expression and synthesis of interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) in fresh human peripheral blood mononuclear cells (PBMC). Total (cell-associated and secreted) cytokine synthesis was measured. In the strict absence of endotoxin (lipopolysaccharide [LPS]), rC5a resulted in a small but statistically insignificant increase in immunoreactive IL-1 beta and TNF, as well as in IL-1 and IL-6 bioactivity. On the other hand, rC5a induced marked transcriptional activation of IL-1 beta and TNF in a dose-dependent fashion with an optimal concentration of 50 ng/mL. The rC5a-induced cytokine messenger RNA (mRNA) was not spontaneously translated into protein. At 50 ng/mL, rC5a induced the same levels of mRNA for IL-1 beta and TNF as 1 ng/mL of LPS, whereas LPS induced 12 times more IL-1 beta protein and 70 times more TNF protein than rC5a alone. The C5a-induced mRNA half-life was the same as that induced by LPS. Formyl-Meth-Leu-Phe (fMLP) did not induce cytokine transcription. Pretreatment with rC5a enhanced cytokine synthesis induced by other stimuli. After 2 hours of preincubation with rC5a, PBMC synthesized 3 to 10 times more IL-1 beta and TNF on subsequent stimulation by LPS or IL-1 itself. We conclude that rC5a provides primarily a transcriptional but not translational signal for IL-1 beta and TNF; the half-life of the untranslated mRNA is the same as that of translated message; rC5a-induced transcription upregulates PBMC for enhanced synthesis of these cytokines; and a translational signal can be provided by LPS or IL-1 itself.

Complement C5a↗

PubMed1989

Influence of the 5'-end region of aldehyde dehydrogenase mRNA on translational efficiency. Potential secondary structure inhibition of translation in vitro.

mRNA coding for rat liver mitochondrial aldehyde dehydrogenase was translated in rabbit reticulocytes 10 times more efficiently than was the corresponding bovine mRNA. Previous data showed that the mRNAs primarily differed in the noncoding 5'-end (K.-L. Guan and H. Weiner, manuscript submitted for publication to Arch. Biochem. Biophys.). Fusion mRNAs were constructed so that the 5'-untranslated region from one was attached to the coding sequence of the other. Furthermore, the mRNAs were mutated at position -3, a position implicated in translational regulation, so that both contained the same residues. Results indicated that the dramatic difference in translational rates between rat and bovine mRNAs was just partly due to the sequence around the initiation codon. Secondary structure prediction of mRNA indicated that stable secondary structure could exist in the 5'-end of bovine mRNA, whereas no such stable structure should exist in rat mRNA. Existence of the predicted secondary structure of bovine mRNA was substantiated by the observation that termination in DNA sequencing in the 5'-end of bovine cDNA occurred. It is concluded that stable secondary structure in the 5'-end region of bovine mitochondrial aldehyde dehydrogenase performs a major role in inhibiting translation. This inhibitory effect is related to the magnitude of free energy required to melt the secondary structure in order to reach the initiation AUG codon.

Aldehyde Dehydrogenase↗

PubMed1979

Post-translational modification of rat immunoglobulins synthesized in the Xenopus oocyte translation system.

The post-translational modification of rat immunoglobulin synthesised in Xenopus laevis oocytes was studied. The major products of translation of rat spleen poly-(A) containing mRNA were found to be assembled 7S immunoglobulin molecules indicating extensive modification of primary translation products. The possibility that these immunoglobulin molecules might include antibodies of defined specificity was investigated using spleen mRNA from rats hyperimmunized with ferritin and keyhole limpet haemocyanin. The presence of antibodies to immunizing antigen in oocyte translation products was determined by affinity chromatography on Sepharose-antigen columns and the synthesis of Sepharose-antigen binding antibodies was observed, equivalent to 2.5-3% of total immunoglobulins. The oocyte produced antibodies were of the same immunoglobulin class as the circulating antibodies from the immunized rats.

Animals↗

PubMed1997

Translational misreading: mutations in translation elongation factor 1alpha differentially affect programmed ribosomal frameshifting and drug sensitivity.

The translation elongation feactor 1alpha (EF-1alpha) catalyzes the critical step of delivering aminoacyl-tRNAs to the elongating ribosome. A series of Saccharomyces cerevisiae strains containing mutant alleles of the TEF2 gene encoding EF-1alpha have phenotypes consistent with effects on cellular processes related to translation. These include (1) conditional growth defects, (2) antibiotic sensitivity or resistance, (3) altered +1 or -1 ribosomal frameshifting efficiencies, and (4) altered maintenance of the killer phenotype. Although all the mutant alleles were isolated as dominant +1 frameshift suppressors, the effects of these mutations on the cell are quite different when present as the only form of EF-1alpha. Allele-specific effects are observed with regard to their ability to alter the efficiency of programmed +1 frameshifting as opposed to programmed -1 ribosomal frameshifting. The significantly altered efficiency of -1 frameshifting in strains containing the TEF2-4 and TEF2-9 mutant alleles further correlates with a reduced ability to maintain the killer phenotype and the M1 satellite virus of L-A, an in vivo assay of translational fidelity. In light of the proposed models regarding the different A- and P-site occupancy states required for +1 or -1 ribosomal frameshifting, these results aid analysis of interactions between EF-1alpha and the translational apparatus.

DNA-Binding Proteins↗

PubMed1987

Regulation of membrane IgM expression in secretory B cells: translational and post-translational events.

IgM secreting cells express little or no membrane IgM. This is not always due to absence of the relevant mRNA. To investigate the synthesis and processing of membrane (micron) and secreted (microseconds) polypeptides in secretory B cells, myeloma cells were transfected either with a plasmid containing an intact mu gene or with one only capable of directing micron (not microseconds) mRNA synthesis. Although myeloma transfectants could make abundant levels of micron mRNA, they did not express IgM on the cell surface. In the myeloma host, micron mRNA is translated some 5-fold less efficiently than microseconds mRNA. However, this translational control does not totally preclude micron synthesis, indicating post-translational regulatory events. No difference between micron and microseconds chains could be detected in their rate of assembly with light chains or in their stability, although both types of heavy chain were degraded more rapidly when synthesized in the absence of light chain, or when the hydrophobic nature of the leader sequence was destroyed by site-directed mutagenesis. However, whereas intracellular microseconds chains in IgM-secreting plasmacytoma were found to be concentrated in the Golgi, the micron chains were mainly located in the endoplasmic reticulum. Retention in the endoplasmic reticulum is also observed for both micron and microseconds when synthesized in the absence of light chain. We propose that it is the expansion of the endoplasmic reticulum that accompanies B cell to plasma cell differentiation which is in part responsible for the down-regulation of surface IgM expression. Such a mechanism may also affect the expression of other surface proteins.

Animals↗

PubMed1990

Translational or post-translational processes affect differentially the accumulation of isocitrate lyase and malate synthase proteins and enzyme activities in embryos and seedlings of Brassica napus.

We have analyzed the accumulation of the glyoxylate cycle enzymes isocitrate lyase and malate synthase in embryos and seedlings of Brassica napus L. The two enzyme activities and proteins begin to accumulate during late embryogeny, reach maximal levels in seedlings, and are not detected in young leaves of mature plants. We showed previously that mRNAs encoding the two enzymes exhibit similar qualitative patterns of accumulation during development and that the two mRNAs accumulate to different levels in both embryos and seedlings (L. Comai et al., 1989, Plant Cell 1, 293-300). In this report, we show that the relative accumulation of the proteins and activities do not correspond to these mRNA levels. In embryos and seedlings, the specific activities of isocitrate lyase and malate synthase are approximately constant. By contrast, the ratio of malate synthase protein to mRNA is 14-fold higher than that of isocitrate lyase. Differences in the translational efficiencies of the two mRNAs in vitro do not appear to account for the discrepancy between mRNA and protein levels. Our results suggest that translational and/or post-translational processes affect differentially the accumulation of the proteins.

Brassica↗

PubMed1993

Post-translational chemical modifications of proteins--III. Current developments in analytical procedures of identification and quantitation of post-translational chemically modified amino acid(s) and its derivatives.

1. The Chemical modifications of amino acids and their derivatives are mainly due to different post-translational enzymatic reactions. 2. The enzymatic reactions resulting in amino acids such as acetylation-, formylation, methylation-phosphorylation-, sulfation-, hydroxylation, ADP ribosylation-, carboxylation-, amidation-, adenylylation-, glycosylation-, ubiquitination-, prenylation and acylation are listed and analytical methods are reported and extensively reviewed. 3. The post-translationally modified cross-linking molecules after maturations such as desmosines, allo-desmosine, hydroxy-, lysylpyridinoline, 3-hydroxypyridinium derivatives, cyclopentenosine recently found in matured elastin, and in collagen, and pulcherosine a novel tyrosine-derived found in fertilization envelope of Sea Urchin embryo, di-tyrosine in resilin, gamma-glutamyl-lysine isopeptide cross-linking molecule etc. are listed and both physico-chemical and analytical methods are extensively reviewed and discussed. 4. Other consequences of post-translational modifications encountered in the analytical procedure such as N-terminal step-wise Edman degradation of glycosylated site(s), phosphorylated-site(s) and or sulfated-site(s) were also reported by us.

Amino Acid Sequence↗

PubMed1996

Messenger RNA translation in prokaryotes: GTPase centers associated with translational factors.

During the decoding of messenger RNA, each step of the translational cycle requires the intervention of protein factors and the hydrolysis of one or more GTP molecule(s). Of the prokaryotic translational factors, IF2, EF-Tu, SELB, EF-G and RF3 are GTP-binding proteins. In this review we summarize the latest findings on the structures and the roles of these GTPases in the translational process.

Amidohydrolases↗

PubMed1996

Translational initiation competence, 'leaky scanning' and translational reinitiation in areA mRNA of Aspergillus nidulans.

(1) AUG codons that either permit or prevent 'leaky scanning' of mRNA encoding AREA, the transcriptional activator mediating nitrogen metabolite repression in Aspergillus nidulans, have been identified. The consensus context for a strong initiation codon (i.e. one preventing 'leaky scanning') derived from this work is GXX AUG C/UCX. However, AUG codons which do not conform to this consensus are nevertheless able to initiate translation. (2) Translational reinitiation can occur within areA mRNA, although there is a limitation determined by the distance between the chain termination and reinitiation codons and/or the strength of the reinitiation codon. (3) The minimum size of the region of areA mRNA that is competent for initiation of translation has been determined to be 329 nucleotides (nt), and might be at least 975 nt if a 709 bp deletion does not alter it.

Amino Acid Sequence↗

PubMed1981

Mode of action of the heme-controlled translational inhibitor: relationship of eukaryotic initiation factor 2-stimulating protein to translation restoring factor.

We have purified the translation restoring factor (RF) and the eukaryotic initiation factor 2 (eIF-2) stimulating protein (ESP) to near homogeneity from the postribosomal supernatant and the ribosomal salt wash, respectively, of rabbit reticulocyte lysate. They were isolated in the form of eIF-2 complexes, apparently in a 1:1 ratio. Their virtually identical NaDodSO4/polyacrylamide gel electrophoretic patterns show, in addition to the eIF-2 alpha (38,000), beta (52,000), and gamma (54,000) bands, peptide bands at approximately 80, 65, 57, 40, and 32 kilodaltons. The apparent Mr of either complex is about 450,000, whereas that of free translation restoring factor (RF) is approximately 25,000. At 0.5 mM Mg2+, both ESP and RF stimulate ternary complex (eIF-2.GTP.Met-tRNAi) formation catalytically with unphosphorylated eIF-2. Phosphorylation of the eIF-2 alpha subunit by preincubation with eIF-2 alpha kinase and ATP, which virtually blocks eIF-2-ESP interaction, results in only partial blocking of the interaction with RF. This may explain the translation restoring activity of RF.

Animals↗

PubMed2002

Consequences of mevalonate depletion. Differential transcriptional, translational, and post-translational up-regulation of Ras, Rap1a, RhoA, AND RhoB.

Ras-related proteins are small GTPases that are post-translationally modified with mevalonate-derived isoprenoids. Although the effects of inhibition of isoprenylation on protein function have been examined, the consequences of depletion of isoprenoid pools on regulation of expression of isoprenylated proteins have yet to be investigated. In these studies we have shown that depletion of mevalonate results in increased total levels of Ras, Rap1a, RhoA, and RhoB in K562 cells. Cycloheximide and [(35)S]methionine pulse/pulse-chase experiments reveal that mevalonate depletion increases the de novo synthesis of Ras and RhoA and decreases the degradation of existing Ras and RhoA protein. Pretreatment with actinomycin D completely prevents the induced up-regulation of RhoB and only partially prevents the up-regulation of Ras, Rap1a, and RhoA. Although depletion of mevalonate does not alter steady state levels of Ras mRNA, there is an increase in RhoB mRNA. Our results are the first to demonstrate that mevalonate depletion induces up-regulation of Ras and Ras-related proteins by discrete mechanisms that include modulation of transcriptional, translational, and post-translational processes.

Antineoplastic Agents↗

PubMed2001

Translation and validation of the Spanish version of the RELATE questionnaire using a modified serial approach for cross-cultural translation.

This article describes the initial translation and validation of the Spanish version of the RELATionshhip Evaluation (RELATE) questionnaire with a sample of monolingual English speakers (n = 78), a sample of monolingual Spanish speakers (n = 18), and two samples of Spanish/English Bilinguals (n = 27 and n = 34). Cross-cultural and cross-language equivalence of the Spanish version of RELATE to the original English version were assessed using a Modified Serial Approach (MSA) for instrument translation. Face and content validity of the Spanish RELATE were established. Test-retest reliability indices obtained with the translated version among the monolingual and bilingual Spanish speaking groups were consistently equivalent to, and in some cases higher than, the baseline reliability obtained with the monolingual English speaking group. Applications of the Spanish version of RELATE and use of the MSA for researchers and practitioners are presented.

Adult↗