[Show abstract][Hide abstract] ABSTRACT: Mammals express the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolyl-neuraminic acid (Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). Neu5Gc is synthesized from Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sia-lyllactose receptor (sialic acid—a2,6Gal) confirm that the type of terminal sialic acid con-tributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains.
[Show abstract][Hide abstract] ABSTRACT: Post-transcriptional regulation of gene expression through 5'UTR-encoded cis-acting elements is an important mechanism for the control of protein expression levels. Through controlling specific aspects of translation initiation, expression can be tightly regulated whilst remaining responsive to cellular requirements. With respect to Cystic Fibrosis, the overexpression of CFTR protein trafficking mutants, such as delta-F508, is of great biological and clinical interest. By understanding the post-transcriptional mechanisms that regulate CFTR expression, new procedures can be developed to enhance CFTR expression in homozygous delta-F508 cystic fibrosis patients. We have identified the key elements of a complex negative regulatory mechanism that is encoded within the human CFTR 5'UTR and show how these elements act in combination to restrict CFTR gene expression to a consistently low level in a transcript-specific manner. This study shows, for the first time, that endogenous human CFTR expression is post-transcriptionally regulated through a 5'UTR-mediated mechanism. We show that the very low levels of endogenous CFTR expression, compared with other low expression genes, are maintained through the co-operative inhibitory effects of an upstream open reading frame and a thermodynamically stable RNA secondary structure.
Human Molecular Genetics 09/2014; 24(4). DOI:10.1093/hmg/ddu501 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, suggesting that MYB may be a therapeutic target in these diseases. However, realization of this potential requires (i) a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis; and (ii) an approach for developing an effective therapeutic. We previously showed that the interaction of Myb with the coactivator CBP/p300 is essential for its transforming activity. Here we show, using cells from Booreana mice which carry a mutant allele of Myb, that this interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type mice, Booreana cells transduced with AML1-ETO9a or MLL-AF9 retroviruses fail to generate leukemia upon transplantation into irradiated recipients. Finally, we have begun to explore the molecular mechanisms underlying these observations by gene expression profiling. This identified several genes previously implicated in myeloid leukemogenesis and HSC function as being regulated in a Myb-p300-dependent manner. These data highlight the importance of the Myb-p300 interaction in myeloid leukemogenesis and suggest disruption of this interaction as a potential therapeutic strategy for AML.
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis (CF) is characterized as a single-gene disorder with a simple, autosomal recessive mode of inheritance. However, translation of cystic fibrosis transmembrane conductance regulator (CFTR) genotype into CF phenotype is influenced by nucleotide sequence variations at multiple genetic loci, and individuals heterozygous for CFTR mutations are predisposed to a range of CFTR-related conditions, such as disseminated bronchiectasis. CF disease severity and CFTR-related conditions are more akin to complex, multifactorial traits, which are increasingly being associated with mutations that perturb gene expression. We have identified a patient with disseminated bronchiectasis, who is heterozygous for a single-nucleotide substitution in the CFTR 5' untranslated region (UTR) (c.-34C>T). The c.-34C>T mutation creates an upstream AUG codon and upstream open reading frame that overlaps, and is out of frame with, the CFTR protein coding sequence. Using luciferase reporter constructs, we have shown that the c.-34C>T mutation decreases gene expression by 85-99%, by reducing translation efficiency and mRNA stability. This is the first CFTR regulatory mutation shown to act at a posttranscriptional level that reduces the synthesis of normal CFTR (Class V), and reaffirms the importance of regulatory mutations as a genetic basis of multifactorial phenotypes.
Human Mutation 10/2011; 32(10):E2266-82. DOI:10.1002/humu.21545 · 5.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The three extant genera of lungfish (Order: Dipnoi), together with the coelacanth Latimeria spp., represent the closest living relatives to the tetrapods and as such signify an important stage in vertebrate evolution. We have previously found that Neoceratodus forsteri possesses a duplex retina comprised of rods and three morphologically distinct types of cones. Cones are distributed non-homogeneously across the retina with increased density in the temporal region, indicating a forward facing visual axis subserving increased spatial resolution. The molecular evolution of the photoreceptor visual pigments was examined using RT-PCR on retinal cDNA from juvenile lungfish. All five vertebrate opsin families (LWS, SWS1, SWS2, RH1 and RH2) were found to be expressed. Comparative phylogenetic analysis of the RH1 opsin gene (using the neighbour-joining method) in N. forsteri and other vertebrates, including the African lungfish Protopterus spp., places the Dipnoi and coelacanth together as a sister group to the tetrapods with a bootstrap value of 99%. Examination of the amino acid sequence of the N. forsteri RH1 opsin gene reveals alanine at site 292 and tyrosine at site 261, both of which are important spectral tuning sites in other vertebrates. Substitutions at these sites have previously been shown to significantly shift the spectral sensitivity of the pigment to longer wavelengths, which would be an advantage in the long wavelength-transmitting riverine environment of the lungfish. In the closely-related coelacanth the RH1 opsin gene possesses serine at site 292 and phenylalanine at site 261, which shift the maximal wavelength of the visual pigment into shorter wavelengths, an advantage in a short wavelength-rich marine environment. The mutation to serine within the coelacanth lineage is not present in amphibia and must have occurred after the divergence from Dipnoans. Molecular genetic studies of visual pigments in these extant “fossils” continue to further our understanding of the evolution of colour vision and tuning sensitivity in vertebrates. This work has been supported by the Australian Research Council.
[Show abstract][Hide abstract] ABSTRACT: The correlation between ontogenetic changes in the spectral absorption characteristics of retinal photoreceptors and expression of visual pigment opsins was investigated in the black bream, Acanthopagrus butcheri. To establish whether the spectral qualities of environmental light affected the complement of visual pigments during ontogeny, comparisons were made between fishes reared in: (1) broad spectrum aquarium conditions; (2) short wavelength-reduced conditions similar to the natural environment; or (3) the natural environment (wild-caught). Microspectrophotometry was used to determine the wavelengths of spectral sensitivity of the photoreceptors at four developmental stages: larval, post-settlement, juvenile and adult. The molecular sequences of the rod (Rh1) and six cone (SWS1, SWS2A and B, Rh2Aalpha and beta, and LWS) opsins were obtained and their expression levels in larval and adult stages examined using quantitative RT-PCR. The changes in spectral sensitivity of the cones were related to the differing levels of opsin expression during ontogeny. During the larval stage the predominantly expressed opsin classes were SWS1, SWS2B and Rh2Aalpha, contrasting with SWS2A, Rh2Abeta and LWS in the adult. An increased proportion of long wavelength-sensitive double cones was found in fishes reared in the short wavelength-reduced conditions and in wild-caught animals, indicating that the expression of cone opsin genes is also regulated by environmental light.
[Show abstract][Hide abstract] ABSTRACT: Lampreys are one of the two surviving groups of jawless vertebrates, whose ancestors arose more than 540 million years ago. Some species, such as Geotria australis, are anadromous, commencing life as ammocoetes in rivers, migrating downstream to the sea, and migrating back into rivers to spawn. Five photoreceptor types and five retinal cone opsin genes (LWS, SWS1, SWS2, RhA, and RhB) have previously been identified in G. australis. This implies that the ancestral vertebrates possessed photopic or cone-based vision with the potential for pentachromacy. Changes in the morphology of photoreceptors and their spectral sensitivity are encountered during differing aquatic phases of the lamprey lifecycle. To understand the molecular basis for these changes, we characterized the visual pigments and measured the relative levels of opsin expression over two lifecycle phases that are accompanied by contrasting ambient light environments. By expressing recombinant opsins in vitro, we show that SWS1, SWS2, RhA, and RhB visual pigments possess lambda(max) values of 359, 439, 497, and 492 nm respectively. For the LWS visual pigment, we predict a lambda(max) value of 560 nm based on key spectral tuning sites in other vertebrate LWS opsins. Quantitative reverse transcriptase-polymerase chain reaction reveals that the retinal opsin genes of G. australis are differentially regulated such that the visual system switches from a broad sensitivity across a wide spectral range to a much narrower sensitivity centered around 490-500 nm on transition from marine to riverine conditions. These quantitative changes in visual pigment expression throughout the lifecycle may directly result from changes in the lighting conditions of the surrounding milieu.
The FASEB Journal 10/2007; 21(11):2713-24. DOI:10.1096/fj.06-8057com · 5.04 Impact Factor