Conserved elements in the 3′ untranslated regions of c-fos and actin mRNAs

In this study, the nucleotide sequences of the 3 untranslated regions (UTR) of the mouse and human c-fos genes, and the rat and human -actin genes were examined. It is shown (i) that the 3 UTR of c-fos is highly conserved between mouse and man, (ii) that multiple copies of a 12 bp element occur, in clusters, in the 3 UTR both of c-fos and of -actin. This conserved 12 bp element is analogous to the putative repressor binding site previously identified (Renan,Bioscience Reports,5 (1985), 739–753). These findings provide additional support for the proposal that regulatory signals are located in the 3 UTR's of certain genes.     

Nucleotide sequence of two 3′-termini of rRNA in the sea urchin Lytechinus variegatus

The 3′-terminus of 26 S rRNA from the sea urchin Lytechinus variegatus has been determined by oligonucleotide fingerprinting, S1 nuclease mapping and terminal nucleotide analysis. There are two species of 26 S rRNA of approximately equal abundance, one 19 nucleotides longer than the other.     

Conservation of the 3′-untranslated regions of calmodulin mRNAs in cetaceans

Three separate calmodulin (CaM) genes (I, II and III) encoding an identical CaM protein but differing in the 5- and 3-untranslated regions of each of the three mRNAs are present and highly conserved in all mammals (so far examined). Primers complementary to the 3- untranslated region (3UTR) of each of the three mRNAs occurring in human, rat and mouse were synthesized and used to amplify regions of the 3UTR from genomic DNA isolated from cetaceans, specifically from the bottled-nosed dolphin (Tursiops truncates), the pygmy sperm whale (Kogia breviceps) and the humpback whale (Megaptera novaeangliae). Using several primers and PCR conditions, the three CaM genes were identified in all three species by this method with one exception. The sequenced regions of the 3UTRs of the three genes of the cetaceans exhibited a high percentage identity when compared to the corresponding regions of these three CaM mRNAs isolated from humans (85-96%). These partial sequences of the 3UTR regions and the corresponding regions for humans, rats and mice that were available from the database were aligned and a phylogenetic tree was constructed. The three CaM genes from all species showed a close phylogenetic relationship based on these 3UTR sequences. Such high conservation of the 3UTRs suggests a specialized and significant function for this region in mammals.     

Evolution of the human sarcomeric-actin genes: Evidence for units of selection within the 3′ untranslated regions of the mRNAs

Summary The complete 3 untranslated region (3UTR) sequence of the human skeletal-actin gene has been compared with the corresponding regions of the rat and chicken skeletal-actin genes. This comparison reveals that the skeletal-actin 3UTR is composed of conserved and nonconserved segments. By using genomic Southern transfer blots and thermal stability (T_m) measurements, we found that the cardiac-actin gene 3UTR also consists of conserved and nonconserved segments. Comparison of human andXenopus laevis cardiac-actin mRNA sequences confirms the presence of a region of high similarity in the 3UTR. We conclude that subsegments of the 3UTRs of both skeletal- and cardiac-actin genes of birds and mammals are under considerable selective pressure. This suggests that these conserved sequences may have functional roles in actin-gene expression or regulation, and that these roles might be different for each actin isoform.     

Extensive sequence variation in the 3′ untranslated region of the KRAS gene in lung and ovarian cancer cases

While cancer is a serious health issue, there are very few genetic biomarkers that predict predisposition, prognosis, diagnosis, and treatment response. Recently, sequence variations that disrupt microRNA (miRNA)-mediated regulation of genes have been shown to be associated with many human diseases, including cancer. In an early example, a variant at one particular single nucleotide polymorphism (SNP) in a let-7 miRNA complementary site in the 3′ untranslated region (3′ UTR) of the KRAS gene was associated with risk and outcome of various cancers. The KRAS oncogene is an important regulator of cellular proliferation, and is frequently mutated in cancers. To discover additional sequence variants in the 3′ UTR of KRAS with the potential as genetic biomarkers, we resequenced the complete region of the 3′ UTR of KRAS in multiple non-small cell lung cancer and epithelial ovarian cancer cases either by Sanger sequencing or capture enrichment followed by high-throughput sequencing. Here we report a comprehensive list of sequence variations identified in cases, with some potentially dysregulating expression of KRAS by altering putative miRNA complementary sites. Notably, rs712, rs9266, and one novel variant may have a functional role in regulation of KRAS by disrupting complementary sites of various miRNAs, including let-7 and miR-181.     

Regulatory small RNAs from the 3′ regions of bacterial mRNAs

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Efficient cap-dependent translation of mammalian mRNAs with long and highly structured 5′-untranslated regions in vitro and in vivo

According to the generally accepted scanning model proposed by M. Kozak, the secondary structure of the 5′-untranslated region (5′-UTR) of eukaryotic mRNA can only inhibit the translation initiation by counteracting migration of the 40S ribosomal subunit along the mRNA polynucleotide chain. The existence of efficiently translatable mRNAs with long and highly structured 5′-UTRs is incompatible with the cap-dependent scanning mechanism. Such mRNAs are expected to use alternative ways of translation initiation to be efficiently translated, primarily the mechanism of internal ribosome entry mediated by internal ribosome entry sites (IRESs), special RNA structures that reside in the 5′-UTR. The paper shows that this viewpoint is incorrect and is probably based on experiments with mRNA translation in rabbit reticulocyte lysate. This cell-free system fails to adequately reflect the relative translation efficiencies observed for different mRNAs in vivo. Five structurally similar mRNAs with either short leaders of the β-globin and β-actin mRNAs or long and highly structured 5′-UTRs of the c-myc, LINE-1, and Apaf-1 mRNAs displayed comparable translation activities in transfected cells and an entire cytoplasmic extract of cultivated cells. Translation activity proved to strongly depend on the presence of a cap at the 5′ end.     

Evaluation of seed storage-protein gene 5′ untranslated regions in enhancing gene expression in transgenic rice seed

5′ untranslated regions (UTRs) are important sequence elements that modulate the expression of genes. Using the β-glucuronidase (GUS) reporter gene driven by the GluC promoter for the rice-seed storage-protein glutelin, we evaluated the potential of the 5′-UTRs of six seed storage-protein genes in enhancing the expression levels of the foreign gene in stable transgenic rice lines. All of the 5′-UTRs significantly enhanced the expression level of the GluC promoter without altering its expression pattern. The 5′-UTRs of Glb-1 and GluA-1 increased the expression of GUS by about 3.36- and 3.11-fold, respectively. The two 5′-UTRs downstream of the Glb-1, OsAct2 and CMV35S promoters also increased GUS expression level in stable transgenic rice lines or in transient expression protoplasts. Therefore, the enhancements were independent of the promoter sequence. Real-time quantitative RT-PCR analysis showed that the increase in protein production was not accompanied by alteration in mRNA levels, which suggests that the enhancements were due to increasing the translational efficiencies of the mRNA. The 5′-UTRs of Glb-1 and GluA-1, when combined with strong promoters, might be ideal candidates for high production of recombinant proteins in rice seeds.     

A sequence similar to bacterial transposable IS elements present in the 5′ untranslated region of the bovine butanediol dehydrogenase cDNA

A sequence similar to prokaryotic transposable elements was identified in the long 5′ untranslated region (5′UTR) of the butanediol dehydrogenase cDNA isolated from a bovine brain λgt11 library. Several observations suggested that this sequence could be related to bacterial IS elements: (a) 58% nucleotide sequence identity, (b) 56% amino acid sequence identity, and (c) the presence of inverted terminal repeats. However, nucleotide sequence analyses of the 5′UTR bovine cDNA showed the presence of chain-terminating nucleotide substitutions that would render it incapable of encoding a functional transposase. Finally, it was observed that different vertebrate genomes have sequences related to this putative transposable element. This revised version was published online in July 2006 with corrections to the Cover Date.     

Seasonal changes in the carotenoids of the sea urchin Strongylocentrotus dröbachiensis

• 1.1. The carotenoids of S. dröbachiensis ovaries and eggs were echinenone, β-carotene, zeaxanthin, isocryptoxanthin, and small amounts of a fucoxanthin isomer. The major carotenoids of the gut wall and gut contents were fucoxanthin, mainly isomerized, β-carotene and zeaxanthin. Echinenone was detectable but isocryptoxanthin was not.
• 2.2. Seasonal variation in ovarian carotenoids was studied. Echinenone constituted between 79 and 85% of the total, except in spawned out ovaries where it fell to 50%, reflecting preferential incorporation of echinenone into eggs.
• 3.3. Evidence is presented for the synthesis of echinenone from β-carotene via isocryptoxanthin in the ovary rather than in the gut wall.
• 4.4. The possible significance of the degree of oxidation of animal carotenoids is discussed briefly.

     

Evolution of the single copyαA-crystallin gene: differently sized mRNAs of mammals and birds show homology in their 3′ non-coding regions

A-Crystallin, a major structural polypeptide of the vertebrate eye lens, is evolutionarily highly conserved. We have analyzed the corresponding nucleic acid sequences in both genomic DNA digests as well as in lens cytoplasmic RNA preparations from a wide variety of vertebrates by blot hybridization with cloned rat A₂-crystallin cDNA probes. The probes are not able to hybridize under any conditions to RNA and DNA derived from fishes and amphibia, but do show substantial homology with the sequences of mammals, birds and reptiles. The A-crystallin gene, which has been isolated from a hamster gene library occurs only once in the haploid genome. Coding and 3-untranslated regions of A₂-crystallin mRNA are conserved among all mammals and birds examined. However, the regions comprising the conserved sequences are differently represented in the ultimate mRNA. The A₂-mRNA 3-non-coding regions of reptiles and birds are 300–550 bases longer than those of mammals. Some rodents produce next to the A₂-mRNA another messenger that encodes the A^lns-polypeptide possessing an insertion of 22/23 amino acid residues between positions 63 and 64 of the A₂-polypeptide chain. A₂ and A^lns-mRNA are generated from a single gene as major and minor species, respectively, in a proportion which is similar to the ratio of the polypeptides foundin vivo andin vitro. The size heterogeneity of the A₂-mRNA from most mammals examined is due to the variable size of the poly(A) tail.     

Genome organization and variation in the 3′-partial sequence of garlic latent virus in China

Ten different isolates of a carlavirus were detected by degenerate PCR from 12 garlic samples collected from 6 provinces in China, and the complete genome sequence of the Zhejiang isolate ZJ1 and 3′-terminal sequences of 9 other isolates were determined. The RNA genome of isolate ZJ1 consisted of 8363nts excluding the 3′-poly (A) tail, and the genome organization was similar to other carlaviruses with 6 open reading frames encoding a replicase, TGB1, TGB2, TGB3, CP and NABP respectively. Sequence comparisons showed that all 10 isolates were Garlic latent virus (GarLV). The variations in the TGB2, TGB3 and NABP were more significant than those in the CP. High homology was also detected between those isolates and Shallot latent virus (ShLV). Phylogenetic analysis suggested that GarLV isolates from garlic can be divided into 4 main groups and Chinese isolates belonged to each group. This is the first reported molecular analysis of members of the genus Carlavirus in China.