Genome evolution mediated by Ty elements in Saccharomyces.

How mobile genetic elements molded eukaryotic genomes is a key evolutionary question that gained wider popularity when mobile DNA sequences were shown to comprise about half of the human genome. Although Saccharomyces cerevisiae does not suffer such "genome obesity", five families of LTR-retrotransposons, Ty1, Ty2, Ty3, Ty4, and Ty5 elements, comprise about 3% of its genome. The availability of complete genome sequences from several Saccharomyces species, including members of the closely related sensu stricto group, present new opportunities for analyzing molecular mechanisms for chromosome evolution, speciation, and reproductive isolation. In this review I present key experiments from both the pre- and current genomic sequencing eras suggesting how Ty elements mediate genome evolution.

Chemical cleavage at aspartyl residues for protein identification.

An alternative method to enzymatic digestion for protein identification by mass spectrometry has been developed that is based on chemical cleavage by formic acid. This method was tested on gel-purified apomyoglobin and BSA, as well as unknown proteins that cofractionate with Tyl-virus-like particles from Saccharomyces cerevisiae. Cleavage at aspartyl residues was found to be efficient and specific, and this specificity of cleavage lent itself easily to database searches. Parallel digestions using trypsin were also performed. The formic acid cleavage method generated comparable or better results than tryptic digestion for protein identification.

Correct integration of model substrates by Ty1 integrase.

The retrovirus-like mobile genetic element of Saccharomyces cerevisiae, Ty1, transposes to new genomic locations via the element-encoded integrase (IN). Here we report that purified recombinant IN catalyzed correct integration of a linear DNA into a supercoiled target plasmid. Ty1 virus-like particles (VLPs) integrated donor DNA more efficiently than IN. VLP and IN-mediated insertions occurred at random sites in the target. Mg(2+) was preferred over Mn(2+) for correct integration, and neither cation enhanced nonspecific nuclease activity of IN. Products consistent with correct integration events were also obtained by Southern analysis. Recombinant IN and VLPs utilized many, but not all, linear donor fragments containing non-Ty1 ends, including a U3 mutation which has been shown to be defective for transposition in vivo. Together, our results suggest that IN is sufficient for Ty1 integration in vitro and IN interacts with exogenous donors less stringently than with endogenous elements.

The genomic RNA in Ty1 virus-like particles is dimeric.

The yeast retrotransposon Ty1 resembles retroviruses in a number of important respects but also shows several fundamental differences from them. We now report that, as in retroviruses, the genomic RNA in Ty1 virus-like particles is dimeric. The Ty1 dimers also resemble retroviral dimers in that they are stabilized during the proteolytic maturation of the particle. The stabilization of the dimer suggests that one of the cleavage products of TyA1 possesses nucleic acid chaperone activity.

Melatonin for treatment of sundowning in elderly persons with dementia - a preliminary study.

This pilot study investigated the impact of melatonin administration as a clinical intervention for improving sleep and alleviating sundowning in 11 elderly nursing home residents who suffer from dementia. Melatonin is a hormone produced and secreted by the pineal gland in response to darkness, which plays a major role in the induction and regulation of sleep. Melatonin production decreases with age. Age-related sleep disorders are frequently associated with disruption of circadian cycle rhythms, and sometimes with 'sundowning'. Sundowning refers to the manifestation of agitation and/or confusion in the evening hours. Agitation has been linked to sleep disorders. Analysis revealed a significant decrease in agitated behaviors in all three shifts, and a significant decrease in daytime sleepiness. There was a nonsignificant decrease in latency (time to fall asleep) during the evening shift and no significant changes were reported in night-time sleep ratings. The results of this study are important, because finding ways of decreasing sundowning in elderly persons may improve their well being, alleviate the burden of the caregivers, and even enable caregiving in a less restrictive environment.

Nucleotide excision repair/TFIIH helicases RAD3 and SSL2 inhibit short-sequence recombination and Ty1 retrotransposition by similar mechanisms.

Eukaryotic genomes contain potentially unstable sequences whose rearrangement threatens genome structure and function. Here we show that certain mutant alleles of the nucleotide excision repair (NER)/TFIIH helicase genes RAD3 and SSL2 (RAD25) confer synthetic lethality and destabilize the Saccharomyces cerevisiae genome by increasing both short-sequence recombination and Ty1 retrotransposition. The rad3-G595R and ssl2-rtt mutations do not markedly alter Ty1 RNA or protein levels or target site specificity. However, these mutations cause an increase in the physical stability of broken DNA molecules and unincorporated Ty1 cDNA, which leads to higher levels of short-sequence recombination and Ty1 retrotransposition. Our results link components of the core NER/TFIIH complex with genome stability, homologous recombination, and host defense against Ty1 retrotransposition via a mechanism that involves DNA degradation.

The Saccharomyces cerevisiae DNA recombination and repair functions of the RAD52 epistasis group inhibit Ty1 transposition.

RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or delta) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA.

New lines of host defense: inhibition of Ty1 retrotransposition by Fus3p and NER/TFIIH.

The genomes of all organisms examined contain transposons whose uncontrolled movement threatens genome function. Fortunately, host cells have evolved defense mechanisms to minimize the level of transposition. In this review we discuss recent work showing that proteins involved in signal transduction and RNA transcription/DNA repair inhibit Ty1 retrotransposition in the yeast Saccharomyces cerevisiae. On the basis of these examples, we hypothesize that the level of Ty1 retrotransposition may be modulated in response to environmental stress signals that affect cellular differentiation and DNA repair.

MGA2 or SPT23 is required for transcription of the delta9 fatty acid desaturase gene, OLE1, and nuclear membrane integrity in Saccharomyces cerevisiae.

MGA2 and SPT23 are functionally and genetically redundant homologs in Saccharomyces cerevisiae. Both genes are implicated in the transcription of a subset of genes, including Ty retrotransposons and Ty-induced mutations. Neither gene is essential for growth, but mga2 spt23 double mutants are inviable. We have isolated a gene-specific activator, SWI5, and the Delta9 fatty acid desaturase of yeast, OLE1, as multicopy suppressors of an mga2Delta spt23 temperature-sensitive mutation (spt23-ts). The level of unsaturated fatty acids decreases 35-40% when the mga2Delta spt23-ts mutant is incubated at 37 degrees. Electron microscopy of these cells reveals a separation of inner and outer nuclear membranes that is sometimes accompanied by vesicle-like projections in the intermembrane space. The products of Ole1p catalysis, oleic acid and palmitoleic acid, suppress mga2Delta spt23-ts and mga2Delta spt23Delta lethality and restore normal nuclear membrane morphology. Furthermore, the level of the OLE1 transcript decreases more than 15-fold in the absence of wild-type Mga2p and Spt23p. Our results suggest that Mga2p/Spt23p control cell viability by stimulating OLE1 transcription.

Facilitation of benzodiazepine discontinuation by melatonin: a new clinical approach.

BACKGROUND: Benzodiazepines are the most frequently used drug for the treatment of insomnia. Prolonged use of benzodiazepine therapy is not recommended. However, many patients, particularly older patients, have difficulties discontinuing therapy. Melatonin, a hormone that is produced at night by the pineal gland, promotes normal sleep in humans and augments sleep induction by benzodiazepine therapy. OBJECTIVE: To assess whether the administration of melatonin could facilitate the discontinuation of benzodiazepine therapy in patients with insomnia. METHODS: Thirty-four subjects receiving benzodiazepine therapy were enrolled in the 2-period study. In period 1, patients received (double-blinded) melatonin (2 mg in a controlled-release formulation) or a placebo nightly for 6 weeks. They were encouraged to reduce their benzodiazepine dosage 50% during week 2, 75% during weeks 3 and 4, and to discontinue benzodiazepine therapy completely during weeks 5 and 6. In period 2, melatonin was administered (single-blinded) for 6 weeks to all subjects and attempts to discontinue benzodiazepine therapy were resumed. Benzodiazepine consumption and subjective sleep-quality scores were reported daily by all patients. All subjects were then allowed to continue melatonin therapy and follow-up reassessments were performed 6 months later. RESULTS: By the end of period 1, 14 of 18 subjects who had received melatonin therapy, but only 4 of 16 in the placebo group, discontinued benzodiazepine therapy (P = .006). Sleep-quality scores were significantly higher in the melatonin therapy group (P = .04). Six additional subjects in the placebo group discontinued benzodiazepine therapy when given melatonin in period 2. The 6-month follow-up assessments revealed that of the 24 patients who discontinued benzodiazepine and received melatonin therapy, 19 maintained good sleep quality. CONCLUSION: Controlled-release melatonin may effectively facilitate discontinuation of benzodiazepine therapy while maintaining good sleep quality.

Hybrid Ty1/HIV-1 elements used to detect inhibitors and monitor the activity of HIV-1 reverse transcriptase.

We previously demonstrated that hybrid retrotransposons composed of the yeast Ty1 element and the reverse transcriptase (RT) of HIV-1 are active in the yeast Saccharomyces cerevisiae. The RT activity of these hybrid Ty1/HIV-1 (his3AI/AIDS RT; HART) elements can be monitored by using a simple genetic assay. HART element reverse transcription depends on both the polymerase and RNase H domains of HIV-1 RT. Here we demonstrate that the HART assay is sensitive to inhibitors of HIV-1 RT. (-)-(S)-8-Chloro-4,5,6, 7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk][1, 4]-benzodiazepin-2(1H)-thione monohydrochloride (8 Cl-TIBO), a well characterized non-nucleoside RT inhibitor (NNRTI) of HIV-1 RT, blocks propagation of HART elements. HART elements that express NNRTI-resistant RT variants of HIV-1 are insensitive to 8 Cl-TIBO, demonstrating the specificity of inhibition in this assay. HART elements carrying NNRTI-resistant variants of HIV-1 RT can be used to identify compounds that are active against drug-resistant viruses.

Utilization of microhomologous recombination in yeast to generate targeting constructs for mammalian genes.

We have developed a new procedure utilizing microhomologous recombination in yeast to generate targeting constructs for producing targeted mutations in mice. This procedure is rapid and efficient, and should be directly applicable to all mammalian genes. Moreover, only minimal information about the locus being targeted is required. The feasibility of this approach was demonstrated by producing another allele of the mouse Tg737 polycystic kidney gene.

Posttranslational regulation of Ty1 retrotransposition by mitogen-activated protein kinase Fus3.

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.

Posttranslational inhibition of Ty1 retrotransposition by nucleotide excision repair/transcription factor TFIIH subunits Ssl2p and Rad3p.

rtt4-1 (regulator of Ty transposition) is a cellular mutation that permits a high level of spontaneous Ty1 retrotransposition in Saccharomyces cerevisiae. The RTT4 gene is allelic with SSL2 (RAD25), which encodes a DNA helicase present in basal transcription (TFIIH) and nucleotide excision repair (NER) complexes. The ssl2-rtt (rtt4-1) mutation stimulates Ty1 retrotransposition, but does not alter Ty1 target site preferences, or increase cDNA or mitotic recombination. In addition to ssl2-rtt, the ssl2-dead and SSL2-1 mutations stimulate Ty1 transposition without altering the level of Ty1 RNA or proteins. However, the level of Ty1 cDNA markedly increases in the ssl2 mutants. Like SSL2, certain mutations in another NER/TFIIH DNA helicase encoded by RAD3 stimulate Ty1 transposition. Although Ssl2p and Rad3p are required for NER, inhibition of Ty1 transposition is independent of Ssl2p and Rad3p NER functions. Our work suggests that NER/TFIIH subunits antagonize Ty1 transposition posttranslationally by inhibiting reverse transcription or destabilizing Ty1 cDNA.

A Ty1 integrase nuclear localization signal required for retrotransposition.

Ty1 retrotransposition in Saccharomyces cerevisiae requires integrase (IN)-mediated insertion of Ty1 cDNA into the host genome. The transposition components are assembled in the cytoplasm and must cross the nuclear envelope to reach the genomic target, since, unlike animal cell nuclear membranes, the yeast cell nuclear membrane remains intact throughout the cell cycle. We have identified a bipartite nuclear localization signal (NLS) in IN required for Ty1 transposition (Ty1 IN) that directs IN to the nucleus. Mutations in the NLS that specifically abolish nuclear localization inactivate transpositional integration but do not affect reverse transcription, protein processing, or catalytic activity in vitro. No additional Ty1-encoded proteins are required for IN nuclear localization. Intragenic complementation experiments suggest that Ty1 IN functions as a multimer and contains two distinct domains, one required for integration and the other for nuclear localization. Nuclear targeting of the preintegration complex by an IN NLS may prove to be a general strategy used by retrotransposons and retroviruses that infect nondividing cells.

Genetic redundancy between SPT23 and MGA2: regulators of Ty-induced mutations and Ty1 transcription in Saccharomyces cerevisiae.

SPT23 was isolated as a dosage-dependent suppressor of Ty-induced mutations in Saccharomyces cerevisiae. SPT23 shows considerable sequence homology with MGA2, a gene identified as a dosage-dependent suppressor of a snf2-imposed block on STA1 transcription in S. cerevisiae var. diastaticus. Although single mutations in either of these genes have only modest effects on cell growth, spt23 mga2 double mutants are inviable. Unlike SPT23, multicopy expression of a truncated form of MGA2 suppresses a narrow subset of Ty-induced mutations. SPT23/MGA2 and the SNF/SWI genes affect transcription of certain target genes in similar ways. Spt23p appears to be a rate-limiting component required for functional HIS4 expression of his4-912delta, a promoter insertion mutation induced by the Ty1-912 long terminal repeat. Furthermore, both Spt23p and Mga2p can activate transcription when fused to the Gal4p DNA-binding domain, as previously observed with Snf2p and Snf5p. A 50-amino-acid region in the N terminus of the predicted Spt23p protein is necessary and sufficient for the transactivation and necessary for suppression of Ty1-induced mutations and the essential function of Spt23p. Cell fractionation and cytological experiments suggest that Spt23p is associated with the nucleus. Our results suggest that SPT23/MGA2 affects transcription of a subset of genes in yeast, perhaps by changing chromatin accessibility.

Improvement of sleep quality by controlled-release melatonin in benzodiazepine-treated elderly insomniacs.

Benzodiazepines are widely used in the elderly population for the initiation of sleep. However, very frequently, complaints about poor sleep maintenance persist despite benzodiazepine treatment. Melatonin, a hormone produced by the pineal gland at night, is involved in the regulation of the sleep/wake cycle. Melatonin production decreases with age and can also be inhibited by benzodiazepines. We have recently reported on the association between insomnia and impaired melatonin output in the elderly. In the present study we have investigated the efficacy of melatonin replacement therapy in improving sleep in 21 elderly subjects who have been taking benzodiazepines and had low melatonin output. In a randomized, double-blind, crossover designed study the subjects were treated for three weeks with 2 mg per night of controlled-release melatonin and for 3 weeks with placebo, 2 h before desired bedtime with a 1-week washout period between treatment periods. Subjects' sleep was assessed by wrist actigraphy. Melatonin treatment significantly increased sleep efficiency and total sleep time and decreased wake after sleep onset, sleep latency, number of awakenings and fragmental index, as compared to placebo. The results of our study indicate that melatonin replacement therapy can improve sleep quality in the elderly and that the beneficial effects are augmented in the presence of benzodiazepines.