Sequence and translation initiation properties of the xenopus TGFbeta5, PDGF-A, and PDGF-alpha receptor 5' untranslated regions

The properties of the architecturally complex Xenopus laevis TGFbeta5, PDGF-A and PDGF-alpha receptor 5'UTRs were investigated. 5' extended cDNAs were obtained by 5'RACE, resulting in long 5'UTRs (478-710 nt) with multiple upstream AUGs (3-13), andthe potential to fold into stable structures. Injection studies suggested that the cloned PDGF-alphaR 5'UTR contains an intron. Splicing at potential 5' and 3' splice sites would result in a non-complex 5'UTR of 142 nt. The above mentioned 5'UTR characteristics are inhibitory for ribosomal scanning. Indeed, relative to the beta-globin 5'UTR, the complex 5'UTRs strongly repressed initiation of protein synthesis in pre-MBT Xenopus embryos. However, later in embryogenesis, the inhibition was partly relieved. The results show temporal translational control by these 5'UTRs. Transgenic embryos showed that the 5'UTRs allowed translation throughout the embryo; spatial control could not be observed. Interestingly, a fragment in the PDGF-A 5'UTR highly similar to an element in the human PDGF-A 5'UTR is complementary to Xenopus 18S ribosomal RNA. None of these Xenopus 5'UTRs contains an IRES, as determined by injecting bicistronic constructs.     

Introns of Entamoeba histolytica and Entamoeba dispar.

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar, indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to the corresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences G     

Lipid Class and Fatty Acid Composition of the Protozoan Parasite of Oysters, Perkinsus marinus Cultivated in Two Different Media

The meront stage of the oyster protozoan parasite, Perkinsus marinus, cultivated in two media with different fatty acid profiles was analyzed for its fatty acid and lipid class composition. The composition of fatty acids in the prezoosporangium stage of the parasite as well as that of the host oyster were investigated. Although the lipid class composition of meronts was dominated by phospholipids and triacylglycerol, there was no triaclgycerol detected in either culture medium. Despite the difference in fatty acid composition of the two media, the fatty acid composition of meronts in each medium was dominated by 14:0, 16:0, 18:0, 18:1(n-9), 20: (n-9), 18:2(n-6) and 20:4(n-6), a profile that differed from its host. The quantities of total lipids and fatty acids in meronts increased as the number of meronts increased and far exceeded the initial amounts in the media and in the initial cell inoculum. The meronts harvested 25 d post-inoculation, had about 3 to 6 times higher total lipids and 4 to 13 times higher fatty acids than the amounts contained in the media. The fatty acid profiles of both prezoosporangia and oysters resembled each other and consisted primarily of 16:0, 20:4(n-6), 20:5(n-3), 22:2delta7,15, and 22:6(n-3). These results indicate that during meront proliferation, the parasite synthesizes certain fatty acids and lipid classes. For development from meront to prezoosporangium, the parasite may rely on its host for lipid resources.     

Chloroplast ribosomal protein S7 of Chlamydomonas binds to chloroplast mRNA leader sequences and may be involved in translation initiation

Certain mutations isolated in the 5' untranslated region (5'UTR) of the chloroplast rps7 gene in Chlamydomonas reduce expression of reporter genes. Second site suppressors in this 5'UTR sequence restore reporter expression. 5'UTR sequences with the original mutations fail to bind a 20-kD protein, one of five proteins that bind to leaders of several chloroplast genes. However, 5'UTRs from suppressed mutants restore binding to this protein but do not bind a 47-kD protein present on the wild type and the original mutant 5'UTRs. The 20-kD protein was shown to be the S7 protein of the chloroplast ribosomal small subunit encoded by rps7, whereas the 47-kD protein was shown to be RB47, a poly(A) binding protein. Our data are consistent with the hypothesis that the S7 protein plays either a general or a specific regulatory role in translation initiation in the chloroplast.     

Introns of Entamoeba histolytica and Entamoeba dispar

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar,indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to thecorresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences GTTTGTT and TAG, respectively. Consistent with the close phylogenetic relationship of E. histolytica and E. dispar, the position and length of introns is conserved between the two species but the degree of sequence identity is reduced compared to orthologous coding regions.     

Functional and structural analysis of the internal ribosome entry site present in the mRNA of natural variants of the HIV-1

The 5'untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5'UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5'UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5'UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5'UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5'UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5'UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed.     

Genome-wide functional analysis of human 5' untranslated region introns

Background  

Approximately 35% of human genes contain introns within the 5' untranslated region (UTR). Introns in 5'UTRs differ from those in coding regions and 3'UTRs with respect to nucleotide composition, length distribution and density. Despite their presumed impact on gene regulation, the evolution and possible functions of 5'UTR introns remain largely unexplored.     

Effects of triclosan on growth, viability and fatty acid synthesis of the oyster protozoan parasite Perkinsus marinus

Perkinsus marinus, a protozoan parasite of the Eastern oyster Crassostrea virginica, has severely impacted oyster populations from the Mid-Atlantic region to the Gulf of Mexico coast of North America for more than 30 yr. Although a chemotherapeutic treatment to reduce or eliminate P. marinus from infected oysters would be useful for research and hatchery operations, an effective and practical drug treatment does not currently exist. In this study, the antimicrobial drug triclosan 5-chloro-2-(2,4 dichlorophenoxy) phenol, a specific inhibitor of Fab1 (enoyl-acyl-carrier-protein reductase), an enzyme in the Type II class of fatty acid synthetases, was tested for its effects on viability, proliferation and fatty acid synthesis of in vitro-cultured P. marinus meronts. Treatment of P. marinus meront cell cultures with concentrations of > or = 2 microM triclosan at 28 degrees C (a temperature favorable for parasite proliferation) for up to 6 d stopped proliferation of the parasite. Treatment at > or = 5 microM at 28 degrees C greatly reduced the viability and fatty acid synthesis of meront cells. Oyster hemocytes treated with > or = 20 microM triclosan exhibited no significant (p < 0.05) reduction in viability relative to controls for up to 24 h at 13 degrees C. P. marinus meronts exposed to > or = 2 microM triclosan for 24 h at 13 degrees C exhibited significantly (p < 0.05) lower viability relative to controls. Exposure of P. marinus meronts to triclosan concentrations of > or = 20 microM resulted in > 50% mortality of P. marinus cells after 24 h. These results suggest that triclosan may be effective in treating P. marinus-infected oysters.