Evidence for a retroviral insertion in TRPM1 as the cause of congenital stationary night blindness and leopard complex spotting in the horse

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ²=1022.00, p<<0.0005), and CSNB, testing 43 horses (χ2=43, p<<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.Rebecca R. Bellone … David L. Adelson, Sim Lin Lim … et al

The population biology and evolutionary significance of Ty elements in Saccharomyces cerevisiae

The basic structure and properties of Ty elements are considered with special reference to their role as agents of evolutionary change. Ty elements may generate genetic variation for fitness by their action as mutagens, as well as by providing regions of portable homology for recombination. The mutational spectra generated by Ty 1 transposition events may, due to their target specificity and gene regulatory capabilities, possess a higher frequency of adaptively favorable mutations than spectra resulting from other types of mutational processes. Laboratory strains contain between 25–35 elements, and in both these and industrial strains the insertions appear quite stable. In contrast, a wide variation in Ty number is seen in wild isolates, with a lower average number/genome. Factors which may determine Ty copy number in populations include transposition rates (dependent on Ty copy number and mating type), and stabilization of Ty elements in the genome as well as selection for and against Ty insertions in the genome. Although the average effect of Ty transpositions are deleterious, populations initiated with a single clone containing a single Ty element steadily accumulated Ty elements over 1,000 generations. Direct evidence that Ty transposition events can be selectively favored is provided by experiments in which populations containing large amounts of variability for Ty1 copy number were maintained for ∼100 generations in a homogeneous environment. At their termination, the frequency of clones containing 0 Ty elements had decreased to ∼0.0, and the populations had became dominated by a small number of clones containing >0 Ty elements. No such reduction in variability was observed in populations maintained in a structured environment, though changes in Ty number were observed. The implications of genetic (mating type and ploidy) changes and environmental fluctuations for the long-term persistence of Ty elements within the S. cerevisiae species group are discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42799/1/10709_2004_Article_BF00133718.pd

Ty3 integrase mutants defective in reverse transcription or 3'-end processing of extrachromosomal Ty3 DNA

Ty3, a retroviruslike element in Saccharomyces cerevisiae, encodes an integrase (IN) which is essential for position-specific transposition. The Ty3 integrase contains the highly conserved His-Xaa(3-7)-His-Xaa(23-32)-Cys-Xaa(2)-Cys and Asp, Asp-Xaa(35)-Glu [D,D(35)E] motifs found in retroviral integrases. Mutations were introduced into the coding region for the Ty3 integrase to determine the effects in vivo of changes in conserved residues of the putative catalytic triad D,D(35)E and the nonconserved carboxyl-terminal region. Ty3 viruslike particles were found to be associated with significant amounts of linear DNA of the approximate size expected for a full-length reverse transcription product and with plus-strand strong-stop DNA. The full-length, preintegrative DNA has at each 3' end 2 bp that are removed prior to or during integration. Such 3'-end processing has not been observed for other retroviruslike elements. A mutation at either D-225 or E-261 of the Ty3 integrase blocked transposition and prevented processing of the 3' ends of Ty3 DNA in vivo, suggesting that the D,D(35)E region is part of the catalytic domain of Ty3 IN. Carboxyl-terminal deletions of integrase caused a dramatic reduction in the amount of Ty3 DNA in vivo and a decrease in reverse transcriptase activity in vitro but did not affect the apparent size or amount of the 55-kDa reverse transcriptase in viruslike particles. The 115-kDa viruslike particle protein, previously shown to react with antibodies to Ty3 integrase, was shown to be a reverse transcriptase-IN fusion protein. These results are consistent with a role for the integrase domain either in proper folding of reverse transcriptase or as part of a heterodimeric reverse transcriptase molecule.</jats:p

Characterization of a transpositionally active Ty3 element and identification of the Ty3 integrase protein

Ty3 is a Saccharomyces cerevisiae retrotransposon associated with tRNA genes. Two Ty3 elements have been cloned and characterized. The complete nucleotide sequence for one element, Ty3-2, was reported previously (L. J. Hansen, D. L. Chalker, and S. B. Sandmeyer, Mol. Cell. Biol. 9:5245-5256, 1988). However, this element is incapable of autonomous transposition. The complete DNA sequence of a transpositionally competent Ty3 element, Ty3-1, is presented here. Its sequence translates into two overlapping open reading frames, TYA3-1 and TYB3-1, which encode proteins with homology to the proteins specified by the retroviral gag and pol genes, respectively. Comparison of the Ty3-1 nucleotide sequence to Ty3-2 suggests that the TYB3-2 open reading frame of Ty3-2 is truncated by the deletion of a single nucleotide, which causes a frameshift mutation. Restoration of the reading frame with insertion of a single adenine by site-directed mutagenesis converted Ty3-2 into a transpositionally active element, Ty3-2(+ A). Western blot analysis with antibodies made against synthetic peptides identified integrase (IN) proteins in viruslike particle preparations from cells expressing Ty3 elements. Cells expressing Ty3-1 and Ty3-2 (+A) produce antibody-reactive proteins with approximate molecular masses of 61 and 58 kilodaltons (kDa), while cells expressing Ty3-2 produce reactive proteins of approximately 52 and 49 kDa. Together, these data show that the 61- or 58-kDa protein, or both, provides the integrase function of Ty3.</jats:p

Adjacent pol II and pol III promoters: transcription of the yeast retrotransposon Ty3 and a target tRNA gene.

The Saccharomyces cerevisiae retrotransposon Ty3 integrates 16 to 19 basepairs upstream of tRNA genes in a region where sequences have been shown to affect the expression of tRNA genes in vivo and in vitro. Sigma, the isolated long terminal repeat of Ty3, is also found in this region. The purpose of these experiments was to elucidate the effects of Ty3 and sigma expression on that of an associated SUP2 tRNA(Tyr) gene in vivo. SUP2 pre-tRNA levels were moderately increased when SUP2 was associated with Ty3 or sigma in either orientation. These increases were independent of Ty3 or sigma promoter activity. The presence of Ty3 or sigma also increased the usage of a minor SUP2 transcription initiation site 2 basepairs upstream of the major initiation site and within the 5 basepair direct repeat flanking Ty3 and sigma. Transcription from an isolated sigma directed toward the tRNA gene was observed to extend through the tRNA gene. In contrast to the lack of an effect of sigma induction on pre-tRNA(Tyr) levels, levels of this sigma transcript were increased when the SUP2 promoter was inactivated by a single basepair mutation

Coordinate transcriptional regulation of type I procollagen genes by Rous sarcoma virus.

Chicken embryo fibroblasts infected with a strain of Rous sarcoma virus containing a temperature-sensitive mutation in the gene coding for pp60src, a protein kinase, undergo changes in collagen synthesis within 4 h after a temperature shift. Cells shifted from the restrictive to the permissive temperature for transformation show decreasing levels of collagen synthesis and increasing levels of kinase activity; the reverse occurs when infected cells are shifted from the permissive to the restrictive temperature. Levels of type I procollagen mRNAs coding for pro alpha 1 and pro alpha 2 chains, measured by hybridization to nick-translated cloned alpha 1 and alpha 2 cDNA, decreased simultaneously soon after a reduction in temperature and reached a new steady state at about 50 h after the shift. In order to test for regulation at the transcriptional level, nuclei were isolated from normal and Rous sarcoma virus-transformed chicken embryo fibroblasts and allowed to transcribe in the presence of [alpha-32P]UTP. Procollagen mRNA sequences in newly synthesized and in total RNA from transformed cell preparations were both about 5-fold lower than the levels in normal cell preparations. We conclude that the coordinate decrease in procollagen mRNAs observed in Rous sarcoma virus-transformed chicken embryo fibroblasts is caused primarily by a decrease in the transcription of the type I procollagen genes, a decrease which is directly or indirectly mediated by the pp60src protein kinase

Sites of RNA polymerase III transcription initiation and Ty3 integration at the U6 gene are positioned by the TATA box.

The function of a TATA element in RNA polymerase (EC 2.7.7.6) III transcription of a naturally TATA-containing U6 snRNA gene and a naturally TATA-less tRNA gene was probed by transcription and Ty3 transposition analyses. Deletion of the TATA box from a U6 minigene did not abolish transcription and Ty3 integration but changed the positions of initiation and insertion. Insertion of the U6 TATA box at three positions upstream of the TATA-less SUP2 tRNA(Tyr) gene resulted in novel transcription initiation and Ty3 integration patterns that depended upon position of the insertion. Nevertheless, the predominant tRNA gene initiation sites were not affected by insertion of the TATA sequence and remained at a fixed distance from the internal box A promoter element. Insertions of the TATA box upstream of a SUP2 box A mutant affected the level of transcription and restricted the use of upstream start sites, but they neither enhanced the use of TATA-dependent initiation sites nor restored expression to the level of the wild-type gene. We conclude that (i) the U6 TATA box is essential in vivo for correct initiation but not for transcription, (ii) a TATA box does not compensate for a weak box A sequence and so cannot perform equivalently, and (iii) the TATA-binding protein, and probably components of transcription factor IIIB, are present on the target at the time of Ty3 integration