Interpreting cDNA sequences: Some insights from studies on translation
TL;DR: This review discusses some rules for assessing the completeness of a cDNA sequence and identifying the start site for translation and includes some advice for using in vitro translation systems for the expression of cDNAs.
Abstract: This review discusses some rules for assessing the completeness of a cDNA sequence and identifying the start site for translation. Features commonly invoked-such as an ATG codon in a favorable context for initiation, or the presence of an upstream in-frame terminator codon, or the prediction of a signal peptide-like sequence at the amino terminus-have some validity; but examples drawn from the literature illustrate limitations to each of these criteria. The best advice is to inspect a cDNA sequence not only for these positive features but also for the absence of certain negative indicators. Three specific warning signs are discussed and documented: (i) The presence of numerous ATG codons upstream from the presumptive start site for translation often indicates an aberration (sometimes a retained intron) at the 5' end of the cDNA. (ii) Even one strong, upstream, out-of-frame ATG codon poses a problem if the reading frame set by the upstream ATG overlaps the presumptive start of the major open reading frame. Many cDNAs that display this arrangement turn out to be incomplete; that is, the out-of-frame ATG codon is within, rather than upstream from, the protein coding domain. (iii) A very weak context at the putative start site for translation often means that the cDNA lacks the authentic initiator codon. In addition to presenting some criteria that may aid in recognizing incomplete cDNA sequences, the review includes some advice for using in vitro translation systems for the expression of cDNAs. Some unresolved questions about translational regulation are discussed by way of illustrating the importance of verifying mRNA structures before making deductions about translation.
Citations
More filters
TL;DR: The organ- and cell-specific expression of ACE2 and its unique cleavage of key vasoactive peptides suggest an essential role for ACE2 in the local renin-angiotensin system of the heart and kidney.
Abstract: ACE2, the first known human homologue of angiotensin-converting enzyme (ACE), was identified from 5' sequencing of a human heart failure ventricle cDNA library. ACE2 has an apparent signal peptide, a single metalloprotease active site, and a transmembrane domain. The metalloprotease catalytic domains of ACE2 and ACE are 42% identical, and comparison of the genomic structures indicates that the two genes arose through duplication. In contrast to the more ubiquitous ACE, ACE2 transcripts are found only in heart, kidney, and testis of 23 human tissues examined. Immunohistochemistry shows ACE2 protein predominantly in the endothelium of coronary and intrarenal vessels and in renal tubular epithelium. Active ACE2 enzyme is secreted from transfected cells by cleavage N-terminal to the transmembrane domain. Recombinant ACE2 hydrolyzes the carboxy terminal leucine from angiotensin I to generate angiotensin 1-9, which is converted to smaller angiotensin peptides by ACE in vitro and by cardiomyocytes in culture. ACE2 can also cleave des-Arg bradykinin and neurotensin but not bradykinin or 15 other vasoactive and hormonal peptides tested. ACE2 is not inhibited by lisinopril or captopril. The organ- and cell-specific expression of ACE2 and its unique cleavage of key vasoactive peptides suggest an essential role for ACE2 in the local renin-angiotensin system of the heart and kidney. The full text of this article is available at http://www. circresaha.org.
2,711 citations
TL;DR: The identification of MEN1 will enable improved understanding of the mechanism of endocrine tumorigenesis and should facilitate early diagnosis of multiple endocrine neoplasia-type 1.
Abstract: Multiple endocrine neoplasia-type 1 (MEN1) is an autosomal dominant familial cancer syndrome characterized by tumors in parathyroids, enteropancreatic endocrine tissues, and the anterior pituitary. DNA sequencing from a previously identified minimal interval on chromosome 11q13 identified several candidate genes, one of which contained 12 different frameshift, nonsense, missense, and in-frame deletion mutations in 14 probands from 15 families. The MEN1 gene contains 10 exons and encodes a ubiquitously expressed 2.8-kilobase transcript. The predicted 610-amino acid protein product, termed menin, exhibits no apparent similarities to any previously known proteins. The identification of MEN1 will enable improved understanding of the mechanism of endocrine tumorigenesis and should facilitate early diagnosis.
1,862 citations
Cambridge University Hospitals NHS Foundation Trust1, Wellcome Trust Sanger Institute2, Lund University3, Erasmus University Medical Center4, Radboud University Nijmegen5, European Bioinformatics Institute6, University of Oslo7, Oslo University Hospital8, Gachon University9, Netherlands Cancer Institute10, Université libre de Bruxelles11, University of Antwerp12, Harvard University13, University of Amsterdam14, University of Ulsan15, Hanyang University16, Memorial Sloan Kettering Cancer Center17, University of Texas MD Anderson Cancer Center18, French Institute of Health and Medical Research19, Ninewells Hospital20, ICM Partners21, University of Queensland22, University of Iceland23, Curie Institute24, University of Cambridge25, King's College London26, Institute of Cancer Research27, University of Bergen28, Singapore General Hospital29
TL;DR: This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operative, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.
Abstract: We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.
1,696 citations
TL;DR: The binding of Met-tRNA to ribosomes is mediated by a GTP-binding protein in both prokaryotes and eukaryotes, but the more complex structure of the eukARYotic factor (eIF-2) and its association with other proteins underlie some aspects of initiation unique to eUKaryotes.
1,459 citations
TL;DR: The cloning of the FMF gene promises to shed light on the regulation of acute inflammatory responses and is described as a member of a family of nuclear factors homologous to the Ro52 autoantigen.
1,424 citations
References
More filters
TL;DR: 5'-Noncoding sequences have been compiled from 699 vertebrate mRNAs and GCCA/GCCATGG emerges as the consensus sequence for initiation of translation in vertebrates.
Abstract: 5'-Noncoding sequences have been compiled from 699 vertebrate mRNAs. (GCC) GCCA/GCCATGG emerges as the consensus sequence for initiation of translation in vertebrates. The most highly conserved position in that motif is the purine in position -3 (three nucleotides upstream from the ATG codon); 97% of vertebrate mRNAs have a purine, most often A, in that position. The periodical occurrence of G (in positions -3, -6, -9) is discussed. Upstream ATG codons occur in fewer than 10% of vertebrate mRNAs-at-large; a notable exception are oncogene transcripts, two-thirds of which have ATG codons preceding the start of the major open reading frame. The leader sequences of most vertebrate mRNAs fall in the size range of 20 to 100 nucleotides. The significance of shorter and longer 5'-noncoding sequences is discussed.
5,077 citations
TL;DR: By analyzing the effects of single base substitutions around the ATG initiator codon in a cloned preproinsulin gene, ACCATGG is identified as the optimal sequence for initiation by eukaryotic ribosomes.
4,559 citations
TL;DR: The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation.
Abstract: The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained.
3,389 citations
Tel Aviv University1, National Institutes of Health2, Sheba Medical Center3, Yale University4, University of Texas Southwestern Medical Center5, University of California, Los Angeles6, Sapienza University of Rome7, Hacettepe University8, QIMR Berghofer Medical Research Institute9, Erasmus University Rotterdam10, University of Birmingham11, University of Sussex12
TL;DR: A gene, ATM, that is mutated in the autosomal recessive disorder ataxia telangiectasia was identified by positional cloning on chromosome 11q22-23 and encoded a putative protein that is similar to several yeast and mammalian phosphatidylinositol-3' kinases that are involved in mitogenic signal transduction, meiotic recombination, and cell cycle control.
Abstract: A gene, ATM, that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, cancer predisposition, radiation sensitivity, and cell cycle abnormalities. The disease is genetically heterogeneous, with four complementation groups that have been suspected to represent different genes. ATM, which has a transcript of 12 kilobases, was found to be mutated in AT patients from all complementation groups, indicating that it is probably the sole gene responsible for this disorder. A partial ATM complementary DNA clone of 5.9 kilobases encoded a putative protein that is similar to several yeast and mammalian phosphatidylinositol-3' kinases that are involved in mitogenic signal transduction, meiotic recombination, and cell cycle control. The discovery of ATM should enhance understanding of AT and related syndromes and may allow the identification of AT heterozygotes, who are at increased risk of cancer.
2,729 citations
TL;DR: The results show subtle differences between eukaryotic and prokaryotic sequences, but the general impression of signal sequences as being highly variable is reinforced.
2,053 citations