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

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

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

Transposition of the yeast retroviruslike element Ty3 is dependent on the cell cycle.

Host cell cycle genes provide important functions to retroviruses and retroviruslike elements. To define some of these functions, the cell cycle dependence of transposition of the yeast retroviruslike element Ty3 was examined. Ty3 is unique among retroviruslike elements because of the specificity of its integration, which occurs upstream of genes transcribed by RNA polymerase III. A physical assay for Ty3 transposition which takes advantage of this position-specific integration was developed. The assay uses PCR to amplify a product of Ty3 integration into a target plasmid that carries a modified tRNA gene. By using the GAL1 upstream activating sequence to regulate expression of Ty3, transposition was detected within one generation of cell growth after Ty3 transcription was initiated. This physical assay was used to show that Ty3 did not transpose when yeast cells were arrested in G1 during treatment with the mating pheromone alpha-factor. The restriction of transposition was not due to changes in transcription of either Ty3 or tRNA genes or to aspects of the mating pheromone response unrelated to cell cycle arrest. The block of the Ty3 life cycle was reversed when cells were released from G1 arrest. Examination of Ty3 intermediates during G1 arrest indicated that Ty3 viruslike particles were present but that reverse transcription of the Ty3 genomic RNA into double-stranded DNA had not occurred. In G1, the Ty3 life cycle is blocked after particle assembly but before the completion of reverse transcription

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

Transfer RNA Genes Are Genomic Targets for De Novo Transposition of the Yeast Retrotransposon Ty3

Insertions of the yeast element Ty3 resulting from induced retrotransposition were characterized in order to identify the genomic targets of transposition. The DNA sequences of the junctions between Ty3 and flanking DNA were determined for two insertions of an unmarked element. Each insertion was at position -17 from the 5' end of a tRNA-coding sequence. Ninety-one independent insertions of a marked Ty3 element were studied by Southern blot analysis. Pairs of independent insertions into seven genomic loci accounted for 14 of these insertions. The DNA sequence flanking the insertion site was determined for at least one member of each pair of integrated elements. In each case, insertion was at position -16 or -17 relative to the 5' end of one of seven different tRNA genes. This proportion of genomic loci used twice for Ty3 integration is consistent with that predicted by a Poisson distribution for a number of genomic targets roughly equivalent to the estimated number of yeast tRNA genes. In addition, insertions upstream of the same tRNA gene in one case were at different positions, but in all cases were in the same orientation. Thus, genomic insertions of Ty3 in a particular orientation are apparently specified by the target, while the actual position of the insertion relative to the tRNA-coding sequence can vary slightly