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Rescue from oculocutaneous albinism type 4 using medaka slc45a2 cDNA driven by its own promoter.

ABSTRACT: Patients and vertebrate mutants with oculocutaneous albinism type 4 (OCA4) have mutations in the solute carrier family 45 member 2 (slc45a2) gene. However, there is no empirical evidence for this gene-phenotype relationship. There is a unique OCA4 mutant in medaka (b) that exhibits albinism only in the skin, but the mechanism underlying this phenotype is also unknown. In this study, we rescued medaka OCA4 phenotypes, in both the eyes and the skin, by micro-injection of an slc45a2-containing genomic fragment or slc45a2 cDNA driven by its own 0.9-kb promoter. We also identified a spontaneous nucleotide change of 339 bp in the promoter as the b mutation. There are multiple transcription start sites in medaka slc45a2, as in its human ortholog, and only the shortest and eye-specific mRNA is transcribed with the b mutation. Interestingly, we further revealed a conserved pyrimidine (Py)-rich sequence of approximately 10 bp in the promoter by medaka-pufferfish comparative genomics and verified that it plays an indispensable role for expression of slc45a2 in the skin. Further studies of the 0.9-kb promoter identified in this study should provide insights into the cis/trans-regulatory mechanisms underlying the ocular and cutaneous expression of slc45a2.

SUBMITTER: Fukamachi S

PROVIDER: S-EPMC2248340 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Rescue from oculocutaneous albinism type 4 using medaka slc45a2 cDNA driven by its own promoter.

Fukamachi Shoji S Kinoshita Masato M Tsujimura Taro T Shimada Atsuko A Oda Shoji S Shima Akihiro A Meyer Axel A Kawamura Shoji S Mitani Hiroshi H

Genetics 20080201 2

Patients and vertebrate mutants with oculocutaneous albinism type 4 (OCA4) have mutations in the solute carrier family 45 member 2 (slc45a2) gene. However, there is no empirical evidence for this gene-phenotype relationship. There is a unique OCA4 mutant in medaka (b) that exhibits albinism only in the skin, but the mechanism underlying this phenotype is also unknown. In this study, we rescued medaka OCA4 phenotypes, in both the eyes and the skin, by micro-injection of an slc45a2-containing geno ...[more]

PMID: 18245373

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Project description:Oculocutaneous albinism (OCA) is a group of inherited disorders of melanin biosynthesis characterized by a generalized reduction in pigmentation of hair, skin and eyes. The prevalence of all forms of albinism varies considerably worldwide and has been estimated at approximately 1/17,000, suggesting that about 1 in 70 people carry a gene for OCA. The clinical spectrum of OCA ranges, with OCA1A being the most severe type with a complete lack of melanin production throughout life, while the milder forms OCA1B, OCA2, OCA3 and OCA4 show some pigment accumulation over time. Clinical manifestations include various degrees of congenital nystagmus, iris hypopigmentation and translucency, reduced pigmentation of the retinal pigment epithelium, foveal hypoplasia, reduced visual acuity usually (20/60 to 20/400) and refractive errors, color vision impairment and prominent photophobia. Misrouting of the optic nerves is a characteristic finding, resulting in strabismus and reduced stereoscopic vision. The degree of skin and hair hypopigmentation varies with the type of OCA. The incidence of skin cancer may be increased. All four types of OCA are inherited as autosomal recessive disorders. At least four genes are responsible for the different types of the disease (TYR, OCA2, TYRP1 and MATP). Diagnosis is based on clinical findings of hypopigmentation of the skin and hair, in addition to the characteristic ocular symptoms. Due to the clinical overlap between the OCA forms, molecular diagnosis is necessary to establish the gene defect and OCA subtype. Molecular genetic testing of TYR and OCA2 is available on a clinical basis, while, at present, analysis of TYRP1 and MATP is on research basis only. Differential diagnosis includes ocular albinism, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Griscelli syndrome, and Waardenburg syndrome type II. Carrier detection and prenatal diagnosis are possible when the disease causing mutations have been identified in the family. Glasses (possibly bifocals) and dark glasses or photocromic lenses may offer sufficient help for reduced visual activity and photophobia. Correction of strabismus and nystagmus is necessary and sunscreens are recommended. Regular skin checks for early detection of skin cancer should be offered. Persons with OCA have normal lifespan, development, intelligence and fertility.

Project description:The recent Zika virus (ZIKV) outbreak has been linked to severe pathogenesis. Here, we report the construction of a plasmid carrying a cytomegalovirus (CMV) promoter-expressed prototype 1947 Uganda MR766 ZIKV cDNA that can initiate infection following direct plasmid DNA transfection of mammalian cells. Incorporation of a synthetic intron in the nonstructural protein 1 (NS1) region of the ZIKV polyprotein reduced viral cDNA-associated toxicity in bacteria. High levels of infectious virus were produced following transfection of the plasmid bearing the wild-type MR766 ZIKV genome, but not one with a disruption to the viral nonstructural protein 5 (NS5) polymerase active site. Multicycle growth curve and plaque assay experiments indicated that the MR766 virus resulting from plasmid transfection exhibited growth characteristics that were more similar to its parental isolate than previously published 2010 Cambodia and 2015 Brazil cDNA-rescued ZIKV. This ZIKV infectious clone will be useful for investigating the genetic determinants of ZIKV infection and pathogenesis and should be amenable to construction of diverse infectious clones expressing reporter proteins and representing a range of ZIKV isolates. IMPORTANCE The study of ZIKV, which has become increasingly important with the recent association of this virus with microcephaly and Guillain-Barré syndrome, would benefit from an efficient strategy to genetically manipulate the virus. This work describes a model system to produce infectious virus in cell culture. We created a plasmid carrying the prototype 1947 Uganda MR766 ZIKV genome that both was stable in bacteria and could produce high levels of infectious virus in mammalian cells through direct delivery of this DNA. Furthermore, growth properties of this rescued virus closely resembled those of the viral isolate from which it was derived. This model system will provide a simple and effective means to study how ZIKV genetics impact viral replication and pathogenesis.

Dataset Information

Rescue from oculocutaneous albinism type 4 using medaka slc45a2 cDNA driven by its own promoter.

Publications

Rescue from oculocutaneous albinism type 4 using medaka slc45a2 cDNA driven by its own promoter.

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