We demonstrate that in Saccharomyces cerevisiae, the tandem array of ribosomal RNA genes [RDNl) is a target for integration of the Tyl retrotransposon that resuhs in silencing of Tyl transcription and transposition. Tyl elements transpose into random rDNA repeat units and are mitotically stable. In addition, we have found that mutation of several putative modifiers of RDNl chromatin structure abolishes silencing of Tyl elements in the rDNA array. Disruption of SIR2, which elevates recombination in RDNl, or TOPI, which increases psoralen accessibility in rDNA, or HTAl-HTBl, which reduces histone H2A-H2B levels and causes localized chromatin perturbations, abolishes transcriptional silencing of Tyl elements in RDNl. Furthermore, deletion of the gene for the ubiquitin conjugating enzyme Ubc2p, which ubiquitinates histones in vitro, derepresses not only Tyl transcription but also mitotic recombination in RDNl. On the basis of these results, we propose that a specialized chromatin structure exists in RDNl that silences transcription of the Tyl retrotransposon.

The molecular identity of a gene which encodes the pore-forming subunit (alpha1G) of a member of the family of low-voltage-activated, T-type, voltage-dependent calcium channels has been described recently. Although northern mRNA analyses have shown alpha1G to be expressed predominantly in the brain, the detailed cellular distribution of this protein in the central nervous system (CNS) has not yet been reported. The current study describes the preparation of a subunit specific alpha1G riboprobe and antiserum which have been used in parallel in situ mRNA hybridization and immunohistochemical studies to localize alpha1G in the mature rat brain. Both alpha1G mRNA and protein were widely distributed throughout the brain, but variations were observed in the relative level of expression in discrete nuclei. Immunoreactivity for alpha1G was typically localized in both the soma and dendrites of many neurons. Whilst alpha1G protein and mRNA expression were often observed in cells known to exhibit T-type current activity, some was also noted in regions, e.g. cerebellar granule cells, in which T-type activity has not been described. These observations may reflect differences between the subcellular distribution of channels that can be identified by immunohistochemical methods compared with electrophysiological techniques.

Ty1 retrotransposons in Saccharomyces cerevisiae are maintained in a state of transpositional dormancy. We isolated a mutation, rtt100-1, that increases the transposition of genomic Ty1 elements 18-to 56-fold but has little effect on the transposition of related Ty2 elements. rtt100-1 was shown to be a null allele of the FUS3 gene, which encodes a haploid-specific mitogen-activated protein kinase. In fus3 mutants, the levels of Ty1 RNA, protein synthesis, and proteolytic processing were not altered relative to those in FUS3 strains but steady-state levels of TyA, integrase, and reverse transcriptase proteins and Ty1 cDNA were all increased. These findings suggest that Fus3 suppresses Ty1 transposition by destabilizing viruslike particle-associated proteins. The Fus3 kinase is activated through the mating-pheromone response pathway by phosphorylation at basal levels in naive cells and at enhanced levels in pheromone-treated cells. We demonstrate that suppression of Ty1 transposition in naive cells requires basal levels of Fus3 activation. Substitution of conserved amino acids required for activation of Fus3 derepressed Ty1 transposition. Moreover, epistasis analyses revealed that components of the pheromone response pathway that act upstream of Fus3, including Ste4, Ste5, Ste7, and Ste11, are required for the posttranslational suppression of Ty1 transposition by Fus3. The regulation of Ty1 transposition by Fus3 provides a haploid-specific mechanism through which environmental signals can modulate the levels of retrotransposition.Retroviruses and endogenous retrovirus-like elements are ubiquitous in the eucaryotic kingdom and have been involved in the formation of a significant portion of the typical eucaryotic genome (45). Hence, eucaryotes have evolved many types of regulatory mechanisms to control the replication and mobility of retroelements. However, only a few host genes that control retroviral replication in vertebrates have been identified. A recent example is the mouse Fv1 gene, whose product is derived from the gag domain of an endogenous retroelement and is postulated to inhibit murine leukemia virus replication by interacting with the viral capsid protein (4).Aside from the infectivity of the retroviral particle, the steps of retrotransposition are analogous to retroviral replication. A well-characterized model system to study host regulation of retrovirus-like elements is the Ty1 retrotransposon in the yeast Saccharomyces cerevisiae (8,50). Ty1 elements have two long terminal repeats (LTRs) surrounding a central region consisting of two overlapping open reading frames: TyA, which encodes a structural capsid protein, and TyB, which encodes protease (PR), integrase (IN), and reverse transcriptase (RT) activities. Replication of Ty1 occurs in the following sequence of events: a chromosomal Ty1 element is transcribed from LTR to LTR by RNA polymerase II into a terminally redundant RNA that is polyadenylated and transported to the cytoplasm. TyA and TyA-TyB fusion protein are synthesized from the full-leng...

Ty1 retrotransposons in the yeast Saccharomyces cerevisiae are maintained in a genetically competent but transpositionally dormant state. When located in the ribosomal DNA (rDNA) locus, Ty1 elements are transcriptionally silenced by the specialized heterochromatin that inhibits rDNA repeat recombination. In addition, transposition of all Ty1 elements is repressed at multiple posttranscriptional levels. Here, we demonstrate that Sgs1, a RecQ helicase required for genome stability, inhibits the mobility of Ty1 elements by a posttranslational mechanism. Using an assay for the mobility of Ty1 cDNA via integration or homologous recombination, we found that the mobility of both euchromatic and rDNA-Ty1 elements was increased 32-to 79-fold in sgs1⌬ mutants. Increased Ty1 mobility was not due to derepression of silent rDNA-Ty1 elements, since deletion of SGS1 reduced the mitotic stability of rDNA-Ty1 elements but did not stimulate their transcription. Furthermore, deletion of SGS1 did not significantly increase the levels of total Ty1 RNA, protein, or cDNA and did not alter the level or specificity of Ty1 integration. Instead, Ty1 cDNA molecules recombined at a high frequency in sgs1⌬ mutants, resulting in transposition of heterogeneous Ty1 multimers. Formation of Ty1 multimers required the homologous recombination protein Rad52 but did not involve recombination between Ty1 cDNA and genomic Ty1 elements. Therefore, Ty1 multimers that transpose at a high frequency in sgs1⌬ mutants are formed by intermolecular recombination between extrachromosomal Ty1 cDNA molecules before or during integration. Our data provide the first evidence that the host cell promotes retrotransposition of monomeric Ty1 elements by repressing cDNA recombination.DNA helicases catalyze the unwinding of duplex DNA into individual DNA strands (42). A plethora of DNA helicases within cells is involved in DNA replication, repair, recombination, and transcription. Members of the RecQ family of DNA helicases are involved in the maintenance of genome stability in all organisms characterized, from bacteria to humans (7). Mutations in the SGS1 gene, which encodes the only RecQ homologue in Saccharomyces cerevisiae, result in elevated levels of mitotic homologous and illegitimate recombination, increased rates of chromosomal nondisjunction, and accelerated aging (21,56,61,62,65). Similarly, mutations in human genes encoding the RecQ homologues RecQL4 (35, 49), WRN (67), and BLM (18) give rise to rare hereditary disorders that are characterized by genome instability and a pronounced predisposition to cancer. Notably, expression of either WRN or BLM in yeast complements the hyperrecombination phenotypes of an sgs1 mutant (65). These findings suggest that the mechanisms by which Sgs1 preserves genetic stability in yeast will serve as a paradigm for the role of RecQ homologues in human disease.The SGS1 gene was originally isolated in a screen for genetic interaction with DNA topoisomerase III (21). Both topoisomerase III and Sgs1 repress recombination of DNA repe...

Most Ty1 retrotransposons in the genome of Saccharomyces cerevisiae are transpositionally competent but rarely transpose. We screened yeast mutagenized by insertion of the mTn3-lacZ/LEU2 transposon for mutations that result in elevated Ty1 cDNA-mediated mobility, which occurs by cDNA integration or recombination. Here, we describe the characterization of mTn3 insertions in 21 RTT (regulation of Ty1 transposition) genes that result in 5- to 111-fold increases in Ty1 mobility. These 21 RTT genes are EST2, RRM3, NUT2, RAD57, RRD2, RAD50, SGS1, TEL1, SAE2, MED1, MRE11, SCH9, KAP122, and 8 previously uncharacterized genes. Disruption of RTT genes did not significantly increase Ty1 RNA levels but did enhance Ty1 cDNA levels, suggesting that most RTT gene products act at a step after mRNA accumulation but before cDNA integration. The rtt mutations had widely varying effects on integration of Ty1 at preferred target sites. Mutations in RTT101 and NUT2 dramatically stimulated Ty1 integration upstream of tRNA genes. In contrast, a mutation in RRM3 increased Ty1 mobility >100-fold without increasing integration upstream of tRNA genes. The regulation of Ty1 transposition by components of fundamental pathways required for genome maintenance suggests that Ty1 and yeast have coevolved to link transpositional dormancy to the integrity of the genome.

No abstract

A variant mouse plasmacytoma (MPC)‐associated translocation chromosome has arisen by pericentric inversion and exchange of the distal segments of a Robertsonian 6;15 fusion chromosome in the CAK TEPC 1198 mouse plasmacytoma, as described earlier. In situ hybridization was performed on the normal and the inverted Rb chromosomes, using myc and kappa probes. On the normal Rb chromosome, myc was in the 15 D2/3 region, whereas kappa hybridized in the 6 C2 area, as expected. On the inverted Rb chromosome, myc remains on the centrometric side of the translocation breakpoint on the chromosome 15‐derived portion, whereas kappa has moved to the chromosome 6‐derived segment that joined the same breakpoint on the telomeric side. Taken together with our recent demonstration that the murine c‐myc locus is oriented ‘head up’ on chromosome 15, and with the results of Cory and co‐workers concerning the relationship between the kappa gene and the associated pvt‐1 region in the CAK TEPC 1198 tumor, the following conclusions can be drawn: (i) in the variant translocation of the CAK TEPC 1198 MPC, the breakage occurs 3′ of the c‐myc gene, as in the human Burkitt lymphoma‐associated variant translocations; (ii) the pvt‐1 gene on chromosome 15 is distal to the myc gene; (iii) the kappa light chain locus is oriented ‘head up’ on mouse chromosome 6 and faces pvt‐1 and, beyond it, c‐myc, in a head‐to‐tail configuration.

We have developed a powerful new tool for the physical analysis of genomes called Ty1-mediated chromosomal fragmentation and have used the method to map 24 retrotransposon insertions into two different mousederived yeast artificial chromosomes (YACs). Expression of a plasmid-encoded GAL1:Ty1 fusion element marked with the retrotransposition indicator gene, ade2AI, resulted in a high fraction of cells that sustained a single Ty1 insertion marked with ADE2. Strains in which Ty1ADE2 inserted into aYAC were identified by cosegregation of the ADE2 gene with the URA3-marked YAC. Ty1ADE2 elements also carried a site for the endonuclease I-DmoI, which we demonstrate is not present anywhere in the yeast genome. Consequently, I-DmoI cleaved a single chromosome or YAC at the unique site of Ty1ADE2 insertion, allowing rapid mapping of integration events. Our analyses showed that the frequency of Ty1ADE2 integration into YACs is equivalent to or higher than that expected based on random insertion. Remarkably, the 50-kb transcription unit of the mouse Steel locus was shown to be a highly significant hotspot for Ty1 integration. The accessibility of mammalian transcription units to Ty1 insertion stands in contrast to that of yeast transcription units.