5 Appendix
The D-element silencer screen in the “silencing cassette”- plasmid

To search for silencing active sequences within the D element of HML-E, we initially used a plasmid carrying the “silencing cassette” (pAE370) that had been previously developed [Grunweller and Ehrenhofer-Murray, 2002]. Using this cassette it is possible to assess the capability of DNA sequences to confer silencing to reporter genes. The silencing cassette consists of two components: (1) the URA3 gene, whose expression can be monitored by growth of transformants on uracil-lacking medium and whose repression can be monitored by growth of transformants on medium containing the drug 5-FOA, and (2) the mating-type gene a1, whose expression leads to a non-mating phenotype in a MATα strain that can be measured in mating assays. The effect of silencing on URA3 is sensitized by utilizing strain AEY565 that lacks the trans-activator of URA3, Ppr1 [Roy, et al., 1990]. The silencing cassette is flanked on one side by the HMR-I silencer (Fig. 5.1), which on its own does not confer silencing, but is capable of supporting silencing by weak silencers. On the other side of the cassette it is possible to insert potential silencers and test their silencing activity. For our purpose of screening HML-D we wanted to compare the silencing activity of HML-E with that of HML-E lacking small fragments of HML-D.

	Figure 5.1: Schematic representation of the silencing cassette.
	X indicates the location for the tested sequences. Arrows indicate, that the silencing properties of a DNA sequence can depend on its direction relatively to the reporter genes.

The properties of the silencing cassette are such that in the absence of a silencer, transformants are completely Ura⁺ and FOA sensitive, indicating full expression of URA3, despite the presence of HMR-I (Fig. 5.2A line 2). In the presence of a silencer (Fig. 5.2A, WT line 1), transformants are FOA resistant but at the same time URA3 ⁺, indicating that URA3 is still expressed in a portion of the [Grunweller and Ehrenhofer-Murray, 2002]. In our experiment HML-E did also confer silencing to URA3 in the above mentioned fashion regardless of its orientation towards the reporter gene (Fig. 5.2, line 3 and data not shown). However if the HML-E silencer lacking the D element was inserted into the plasmid URA3 repression in the transformants was not discernable from that of the complete HML-E silencer (Fig. 5.2, line 3, 4). This was also observed in a tester strain of the opposite mating type (pAE2225, data not shown). Thus we were not able to visualize subtle effects on silencing capacity at HML-E.

In a second approach we wanted to exploit the observation of [Mahoney,et al., 1991] that removal of any one of the silencer elements at HML-E results only in little derepression at HMLα whereas removal of any two of the silencer elements leads to total loss of silencing. We reasoned that if silencing at the silencer cassette is compromised by a deletion of the ACS site of HML-E any further removal of silencer elements, for example HML-D should lead to total loss of silencing. However deleting the ACS site alone completely abolished the ability of the HML-E silencer to confer silencing to the silencing cassette regardless of the orientation (Fig. 5.2A, lines 5-8).

	Figure 5.2: HML-E properties in the silencing cassette
	(A) HML-E silencing was independent of D but dependent on the ACS. Serial dilutions of transformants with plasmids carrying the indicated sequence elements at the silencer cassette were plated and incubated at 30°C for 4 days (+ura) and 7 days (-ura, +ura +5-FOA) respectively. ΔX: no sequence element was ligated into X. ΔA: The ACS site was deleted. Plasmids were 1: pAE374, 2: pAE369, 3: pAE421, 4: pAE442, 5: pAE735, 6: pAE736, 7: pAE739, 8: pAE740; (B) HML-E silencing was abolished in an orc2-1 strain. Control: CEN6-LEU2 plasmid as a control for plasmid maintenance. Plasmids were 1: pRS315, 2: pAE369, 3: pAE419, 4: pAE440, 5: pAE421, 6: pAE442.

This was in contrast to HML-E deletion experiments done previously [Mahoney,et al., 1991], where an ACS deletion alone was not sufficient to cause full derepression at HMLα. However the deletions there had been introduced genomically and the necessity for a silencer element could be altered on a plasmid.

Binding of ORC to the ACS is important for the establishment of silencing but mutant alleles of ORC with reduced silencing properties exist. We reasoned that performing the HML-D screen in an ORC mutant strain might circumvent the strong effect of an complete ACS deletion. The orc2-1 allele represents such a mutation. orc2-1 haploids are severly but not completely mating defective, indicating that this mutation is strongly affecting silencing at the HM loci [Foss,et al., 1993]. We transformed AEY565 that harboured an orc2-1 mutation with the wild-type HML-E and HML-E ΔD silencing-cassette plasmids and measured URA3 silencing. However, as in an HML-E ΔACS plasmid, silencing was completely abolished in this strain background. Therefore we considered the plasmid based silencer cassette as inappropriate for our plan to search for a silencing active core element within D. We proceeded with a screening method, where mutations or deletion were genomically introduced into the D element as outlined in chapter 3.1.