Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish,Oryzias latipes

In the medaka fish (Oryzias latipes) many mutants for body color have been isolated. A typical example is the recessive oculocutaneous albino mutant i, which has amelanotic skin and red-colored eyes with no tyrosinase activity. To cast light on the molecular basis of the albino mechanism, we performed Southern blot analysis of genomic DNA from the mutant with an authentic tyrosinase gene probe; the results demonstrate that an extra 1.9 kb fragment is present inside the first exon. The insertion is responsible for the oculocutaneous albinism. About 80 copies of this fragment are present in the genomes of albino-i and wild-type fish; these repeated sequences are here designated Tol1 elements and the particular element found in the tyrosinase gene of albino-i is denoted Tol1-tyr. The nucleotide sequence of Tol1-tyr shows that the fragment (i) carries terminal inverted repeats of 14 bp, and (ii) is flanked by duplicated 8 by segments of the host chromosome. These are properties of DNA-mediated transposable elements. Comparison of the nucleotide sequence of Tol1-tyr with other sequences in DNA databases, with special attention to sequences of transposable elements known to date, did not reveal any similarity. Thus, Tol1 constitutes a hitherto unknown family of DNA transposable elements.     

Reversion mutation of ib oculocutaneous albinism to wild‐type pigmentation in medaka fish

We have previously identified three naturally occurring mutations in the medaka fish tyrosinase gene caused by transposable element insertions. Tyr‐i^b is one of these, containing the Tol2 element in the promoter region. Its homozygous carriers exhibit a weak oculocutaneous albino phenotype. We report here spontaneous reversion of the albino phenotype to the wild‐type pigmentation, associated with excision of the Tol2 element. The newly arising mutant gene is inherited in the Mendelian fashion. Thus, oculocutaneous albinism is not strictly irreversible, at least in this organism and the results also indicate that the insertion of the Tol2 element is the main, and possibly the only, cause of the i^b albinism. Importantly our data also suggest that medaka fish possess an active transposase.     

Color reversion of the albino medaka fish associated with spontaneous somatic excision of the Tol‐1 transposable element from the tyrosinase gene

The medaka fish albino mutant, i¹ is one of the Tomita collection of medaka pigmentation mutants which exhibits a complete albino phenotype, because of inactivation of the tyrosinase gene due to insertion of a transposable element, Tol‐1. Recently, mosaic black‐pigmented i¹ medaka fish have arisen in one of our laboratory breeding populations. Their pigmented cells have been observed in all of the tissues, including the eye and skin, in which melanin is detectable in the wild type. In this study, we analyzed the tyrosinase gene of revertants and showed Tol‐1 to have been precisely excised from the gene, suggesting a causal relationship. Mosaic patterns of pigmentation indicate spontaneous somatic excision of the element from the tyrosinase gene. To our knowledge, this is the first transposable element with somatic excision activity demonstrated phenotypically in vertebrates. The pattern of pigmentation in mosaic revertants indicates frequencies of melanin pigments to be consistent with the numbers of melanophores per unit area of body sites, such as the eyes, head and dorsal trunk.     

Oculocutaneous Albinism in the i6 Mutant of the Medaka Fish is Associated with a Deletion in the Tyrosinase Gene

Three mutant alleles (i¹, i⁴, and i⁵) of the tyrosinase gene in the i locus of the medaka fish Oryzias latipes have hitherto been described, all being associated with transposable element insertion. We have recently identified another allele causing a complete albino phenotype in homozygous carriers and named it i⁶. Sequence comparison between the tyrosinase gene for the i⁶ allele (Tyr-i⁶) and the wild-type gene previously obtained (Tyr-i ⁺) revealed three deletions of 8, 44, and 245 bp. The first two deletions reside in an intron and are differences in the number of tandem tetranucleotide repeats that are polymorphic even among wild-type genes, and, thus, not likely to be responsible for the i⁶ albino phenotype. The largest deletion spans over the last 180 bp of the second intron and the first 65 bp of the third exon. Because of this deletion, the Tyr-i⁶ gene lacks the branch point sequence and the acceptor site for the second intron, both being considered to be necessary for normal RNA splicing. Therefore, the 245-bp deletion is likely to be responsible for the albino phenotype. With a mutant gene of this type, unlike ones bearing transposable element insertions, the possibility of reversion mutations to the wild-type would be negligible. Therefore, fish having the i^e/i⁶ genotype should serve as superior recipients for the tyrosinase gene in rescue experiments.     

Reversion mutation of ib oculocutaneous albinism to wild-type pigmentation in medaka fish

We have previously identified three naturally occurring mutations in the medaka fish tyrosinase gene caused by transposable element insertions. Tyr-i(b) is one of these, containing the Tol2 element in the promoter region. Its homozygous carriers exhibit a weak oculocutaneous albino phenotype. We report here spontaneous reversion of the albino phenotype to the wild-type pigmentation, associated with excision of the Tol2 element. The newly arising mutant gene is inherited in the Mendelian fashion. Thus, oculocutaneous albinism is not strictly irreversible, at least in this organism and the results also indicate that the insertion of the Tol2 element is the main, and possibly the only, cause of the i(b) albinism. Importantly our data also suggest that medaka fish possess an active transposase.     

Albinism Due to Transposable Element Insertion in Fish

The i locus of the medaka fish, Oryzias latipes, is responsible for tyrosinase expression, and several mutant alleles have been identified. The genotype i¹/i¹ exhibits a complete albino phenotype, having pale orange-red skin and red eyes. This mutant lacks in vivo tyrosinase activity. The genotype i⁴/i⁴, on the other hand, shows a quasi-albino phenotype with skin as bright as that of i¹/i¹ but with red-wine-colored eyes. At the light microscope level, reduced pigmentation is observed both in the skin and eyes of this mutant. The tyrosinase genes for the i¹ and the i⁴ alleles were cloned and sequenced, and compared with that of the wild-type tyrosinase gene. The i¹ allele was found to contain a 1.9-kb transposable element in the 1st exon, and the i⁴ allele was found to contain a 4.7-kb transposable element in the 5th exon. Both i¹ and i⁴ are alleles that were found in a commercial breeding population. The insertion of a transposable element thus appears to constitute a natural cause of mutations that cause albinism in this organism.     

Genetic mapping of the dominant albino locus in rainbow trout (Oncorhynchus mykiss)

Albinism in animals is generally a recessive trait, but in Japan a dominant oculocutaneous albino (OCA) mutant strain has been isolated in rainbow trout (Oncorhyncus mykiss). After confirming that this trait is not due to a tyrosinase gene mutation that causes OCA1 (tyrosinase-negative OCA), we combined the amplified fragment length polymorphism (AFLP) technique with bulked segregant analysis (BSA) to map the gene involved in dominant oculocutaneous albinism. Four AFLP markers tightly linked to the dominant albino locus were identified. One of these markers was codominant and we have it converted into a GGAGT-repeat microsatellite marker, OmyD-AlbnTUF. Using this pentanucleotide-repeat DNA marker, the dominant albino locus has been mapped on linkage group G of a reference linkage map of rainbow trout. The markers identified here will facilitate cloning of the dominant albino gene in rainbow trout and contribute to a better understanding of tyrosinase-negative OCA in animals.     

Tol1转座子研究进展

Tol1和Tol2是在青鳉基因组中发现的具有自主活性的DNA转座子,而Tol1转座子的自主活性是新近才发现的,因此对它的报道较少。较之Tol2,Tol1可以携带更大片段的DNA进行转座,且Tol1的转座不受转座酶"过量表达抑制"的影响。研究已证实,Tol1转座子在秀丽线虫、斑马鱼、爪蟾和人等多种生物中具有转座活性。因此,在动物转基因和基因功能研究等方面有重要的应用前景。从Tol1转座子的结构特征、转座机制和作为基因转移载体的优点,以及应用研究等方面进行了简要的综述。     

Color reversion of the albino medaka fish associated with spontaneous somatic excision of the Tol-1 transposable element from the tyrosinase gene

The medaka fish albino mutant, i(1) is one of the Tomita collection of medaka pigmentation mutants which exhibits a complete albino phenotype, because of inactivation of the tyrosinase gene due to insertion of a transposable element, Tol-1. Recently, mosaic black-pigmented i(1) medaka fish have arisen in one of our laboratory breeding populations. Their pigmented cells have been observed in all of the tissues, including the eye and skin, in which melanin is detectable in the wild type. In this study, we analyzed the tyrosinase gene of revertants and showed Tol-1 to have been precisely excised from the gene, suggesting a causal relationship. Mosaic patterns of pigmentation indicate spontaneous somatic excision of the element from the tyrosinase gene. To our knowledge, this is the first transposable element with somatic excision activity demonstrated phenotypically in vertebrates. The pattern of pigmentation in mosaic revertants indicates frequencies of melanin pigments to be consistent with the numbers of melanophores per unit area of body sites, such as the eyes, head and dorsal trunk.     

Gene Transfer and Cloning of Flanking Chromosomal Regions Using the Medaka Fish Tol2 Transposable Element

For the ultimate purpose of developing genetic tools using the medaka fish Tol2 transposable element, we examined whether it can transfer a marker gene into the fish genome and also be applied for cloning of chromosomal regions adjacent to insertion points. An internal region of Tol2 was removed and replaced with the green fluorescent protein (GFP) gene and a bacterial plasmid replication origin. This modified Tol2 clone was microinjected into fertilized eggs together with messenger RNA for the Tol2 transposase. The GFP gene was found to be integrated into chromosomes and transmitted to subsequent generations. Restriction enzyme digestion of genomic DNA of a transformant fish, followed by ligation and introduction into bacteria, produced a plasmid containing the entire element and flanking chromosomal regions. Sequencing analysis of this clone demonstrated transposition of the element in the germline of the first generation. Thus, the basic requirements for a gene transfer vector and gene tagging system were fulfilled. Received July 30, 2001; accepted October 4, 2001     

Characterization of a Tc1-like transposable element in zebrafish (Danio rerio)

We have characterized Tdr1, a family of Tc1-like transposable elements found in the genome of zebrafish (Danio rerio). The copy number and distribution of the sequence in the zebrafish genome have been determined, and by these criteria Tdr1 can be classified as a moderately repetitive, interspersed element. Examination of the sequences and structures of several copies of Tdr1 revealed that a particular deletion derivative, 1250 by long, of the transposon has been amplified to become the dominant form of Tdr1. The deletion in these elements encompasses sequences encoding the N-terminal portion of the putative Tdr1 transposase. Sequences corresponding to the deleted region were also detected, and thus allowed prediction of the nucleotide sequence of a hypothetical full-length element. Well conserved segments of Tc1-like transposons were found in the flanking regions of known fish genes, suggesting that these elements have a long evolutionary history in piscine genomes. Tdr1 elements have long, 208 by inverted repeats, with a short DNA motif repeated four times at the termini of the inverted repeats. Although different from that of the prototype C. elegans transposon Tc1, this inverted repeat structure is shared by transposable elements from salmonid fish species and two Drosophila species. We propose that these transposons form a subgroup within the Tc1-like family. Comparison of Tc1-like transposons supports the hypothesis that the transposase genes and their flanking sequences have been shaped by independent evolutionary constraints. Although Tc1-like sequences are present in the genomes of several strains of zebrafish and in salmonid fishes, these sequences are not conserved in the genus Danio, thus raising the possibility that these elements can be exploited for gene tagging and genome mapping.     

Transposable elements in the fungal plant pathogenFusarium oxysporum

The genome of the fungal plant pathogenFusarium oxysporum contains at least six different families of transposable elements. Representatives of both DNA transposons and retrotransposons have been identified, either by cloning of dispersed repetitive sequences (Foret andpalm) or by trapping in the nitrate reductase gene (Fot1, Fot2 Impala andHop).Fot1 andImpala elements are related to theTc1 andmariner class of transposons. These transposable elements can affect gene structure and function in several ways: inactivation of the target gene through insertion, diversification of the nucleotide sequence by imprecise excisions, and probably chromosomal rearrangements as suggested by the extensive karyotype variation observed among field isolates. Comparisons of the distribution of these elements inFusarium populations have improved our understanding of population structure and epidemiology and provided support for horizontal genetic transfer. Also they could be developed as genetic tools for tagging genes, a cloning strategy that is particularly promising in imperfect fungi.     

Identification and structural characterization of further DNA elements in the potato and pepper genomes homologous to the transposable element-like insertion Tst1

Summary The molecular cloning and nucleotide sequence of elements from potato and pepper that are related to the recently identified Tst1 element are described. Sequence analysis reveals considerable conservation of sequences internal to both the Tst1 element and two of the related elements identified here. In six potato clones analysed, the II by inverted repeat first identified in the Tst1 element is conserved. Several of the elements are flanked by an 8 by direct repeat. DNA fragments which were amplified from several pepper genomes by polymerase chain reaction (PCR) amplification using the inverted repeat as sequence primers also display considerable conservation of sequences internal to the Tst1 element. These data further support the possibility that Tst1 is a non-autonomous transposable element and that Tst1 might be the first example of a transposable element which occurs in several genera of solanaceous plants.     

Novel non-autonomous transposable elements on W chromosome of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

The sex chromosomes of the silkworm Bombyx mori are designated ZW(XY) for females and ZZ (XX) for males. Numerous long terminal repeat (LTR) and non-LTR retrotransposons, retroposons and DNA transposons have accumulated as strata on the W chromosome. However, there are nucleotide sequences that do not show the characteristics of typical transposable elements on the W chromosome. To analyse these uncharacterized nucleotide sequences on the W chromosome, we used whole-genome shotgun (WGS) data and assembled data that was obtained using male genome DNA. Through these analyses, we found that almost all of these uncharacterized sequences were non-autonomous transposable elements that do not fit into the conventional classification. It is notable that some of these transposable elements contained the Bombyx short interspersed element (Bm1) sequences in the elements. We designated them as secondary-Bm1 transposable elements (SBTEs). Because putative ancestral SBTE nucleotide sequences without Bm1 do not occur in the WGS data, we suggest that the Bm1 sequences of SBTEs are not carried on each element merely as a package but are components of each element. Therefore, we confirmed that SBTEs should be classified as a new group of transposable elements.     

Remobilization of <Emphasis Type="Italic">Tol2</Emphasis> transposons in <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>

Background  

The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency.     

A transposon-like sequence with short terminal inverted repeats in the nuclear genome of Chlamydomonas reinhardtii

A 1.2 kb DNA sequence, flanked by a potential seven base target-site duplication, was found inserted into a TOC1 transposable element from Chlamydomonas reinhardtii. The insertion sequence, named TOC2, is a member of a family of repeated DNA sequences that is present in all the C. reinhardtii strains tested. It resembles class II transposable elements: it possesses short 14 bp imperfect terminal repeats that begin AGGAGGGT, and sub-terminal direct repeats located within 250 bp of the termini. No large open reading frames were found. The terminal bases and length of target-site duplication are important in classifying transposable elements. On this basis TOC2 does not fall readily into existing families of class II transposable elements found in plants.