| 1. |
- Barsoum, Emad, 1979-, et al.
(författare)
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{alpha}3, a transposable element that promotes host sexual reproduction
- 2010
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Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 24:15, s. 33-44
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Tidskriftsartikel (refereegranskat)abstract
- Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called alpha3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5' and 3' flanking regions of the alpha3 gene were joined, forming a DNA circle, showing that alpha3 mobilized from the genome. The sequences encompassing the alpha3 gene circle junctions in the mating type alpha (MATalpha) locus were essential for switching from MATalpha to MATa, suggesting that alpha3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATalpha were important. Expression of Mts1 was repressed in MATa/MATalpha diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch.
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| 2. |
- Barsoum, Emad, 1979-
(författare)
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Mating type switching and transcriptional silencing in Kluyveromyces lactis
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- To explore the similarities and differences of regulatory circuits among budding yeasts, we characterized the role of unscheduled meiotic gene expression 6 (UME6) and a novel mating type switching pathway in Kluyveromyces lactis. We found that Ume6 was required for transcriptional silencing of the cryptic mating-type loci HMLα and HMRa. Ume6 acted directly at these loci by binding to the cis-regulatory silencers. Ume6 also served as a block to polyploidy and was required for repression of three meiotic genes, independently of the Rpd3 and Sin3 corepressors. Mating type switching from MATα to MATa required the α3 protein. The α3 protein was similar to transposases of the mutator like elements (MULEs). Mutational analysis showed that the DDE-motif in α3, which is conserved in MULEs was necessary for switching. During switching α3 mobilizes from the genome in the form of a DNA circle. The sequences encompassing the α3 gene circle junctions in the MATα locus were essential for switching from MATα to MATa. Switching also required a DNA binding protein, Mating type switch 1 (Mts1), whose binding sites in MATα were important. Expression of Mts1 was repressed in MATa/MATα diploids and by nutrients, limiting switching to haploids in low nutrient conditions. In a genetic selection for strains with increased switching rates we found a mutation in the RAS1 gene. By measuring the levels of the MTS1 mRNA and switching rates in ras1, pde2 and msn2 mutant strains we show that mating type switching in K. lactis was regulated by the RAS/cAMP pathway and the transcription factor Msn2. ras1 mutants contained 20-fold higher levels of MTS1 mRNA compared to wild type whereas pde2 and msn2 expressed less MTS1 mRNA and had decreased switching rates. Furthermore we found that MTS1 contained several potential Msn2 binding sites upstream of its ORF. We suggest that these observations explain the nutrient regulation of switching.
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| 3. |
- Barsoum, Emad, 1979-, et al.
(författare)
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Regulation of mating type switching in Kluyveromyces lactis by the RAS/cAMP pathway and the transcription factor Msn2
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- We explored the regulation of mating type switching in Kluyveromyces lactis. Using an assay dependent on loss of a URA3 gene inserted into the MATa locus, we determined that the switching rate of a wild type strain grown in rich media was ~6X10-4 events/generation. In a genetic selection for identifying strains with increased switching rates, we found a strain with an insertion in the K. lactis RAS1 gene, encoding a small GTPase with a central role in growth regulation. Compromised Ras1 function leads to a lower cAMP level suggesting a role for cAMP in promoting switching. Consistent with this idea, a strain lacking the PDE2 gene, which encodes an enzyme that degrades cAMP, resulted in decreased switching rates. To explore how cAMP regulated switching, we investigated the transcription of the MTS1 gene, encoding an inducer of switching. The ras1 mutant strain contained 20-fold higher levels of the MTS1 mRNA compared to wild type, but in the pde2 mutant strain MTS1 transcription was repressed 5-fold. In addition, strains lacking the MSN2 gene, which encodes a transcription factor that binds the stress response element (STRE), expressed less MTS1 mRNA and had decreased switching rates. We suggest a model in which nutrient limitation induces switching through cAMP and Msn2-dependent transcriptional induction of the MTS1 gene.
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| 4. |
- Barsoum, Emad, 1979-, et al.
(författare)
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Ume6 is required for the MATa/MATα cellular identity and transcriptional silencing in Kluyveromyces lactis
- 2010
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Ingår i: Genetics. - : The Genetics Society of America. - 0016-6731 .- 1943-2631. ; 184:4, s. 999-1011
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Tidskriftsartikel (refereegranskat)abstract
- To explore the similarities and differences of regulatory circuits among budding yeasts, we characterized the role of the unscheduled meiotic gene expression 6 (UME6) gene in Kluyveromyces lactis. We found that Ume6 was required for transcriptional silencing of the cryptic mating-type loci HMLα and HMRa. Chromatin immunoprecipitation (ChIP) suggested that Ume6 acted directly by binding the cis-regulatory silencers of these loci. Unexpectedly, a MATa ume6 strain was mating proficient, whereas a MATα ume6 strain was sterile. This observation was explained by the fact that ume6 derepressed HMLα2 only weakly, but derepressed HMRa1 strongly. Consistently, two a/α-repressed genes (MTS1 and STE4) were repressed in the MATα ume6 strain, but were expressed in the MATa ume6 strain. Surprisingly, ume6 partially suppressed the mating defect of a MATa sir2 strain. MTS1 and STE4 were repressed in the MATa sir2 ume6 double-mutant strain, indicating that the suppression acted downstream of the a1/α2-repressor. We show that both STE12 and the MATa2/HMRa2 genes were overexpressed in the MATa sir2 ume6 strain. Consistent with the idea that this deregulation suppressed the mating defect, ectopic overexpression of Ste12 and a2 in a MATa sir2 strain resulted in efficient mating. In addition, Ume6 served as a block to polyploidy, since ume6/ume6 diploids mated as pseudo a-strains. Finally, Ume6 was required for repression of three meiotic genes, independently of the Rpd3 and Sin3 corepressors.
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