5180 Nucleic Acids Research, 2010, Vol. 38, No. 15 QUEST software packages to collect and analyse the data (BD Biosciences). RESULTS Strains for the phenotypic analysis of the RPL35 genes Yeast L35 is an essential small r-protein of 120 amino acids with a predicted mass of 13.9 kDa and a predicted basic pI of 11.36 (Saccharomyces Genome Database, www.yeastgenome.org). As most yeast r-protein genes (33,68,69), L35 is encoded by two paralogous genes, RPL35A (SOS1, YDL191W) and RPL35B (SOS2, YDL136W), which both localized on the left arm of chromosome IV. As also quite common for yeast r-protein genes (70), their coding regions are interrupted by an intron. The coding regions of these two genes are nearly identical, diﬀering in only 3 nt out of 362, but re- sulting in no change in the amino acid sequences of the predicted L35A and L35B proteins. On the other hand, the two paralogous genes show no substantial sequence homology in their introns or in their 50 and 30-non-coding regions. Early studies indicated that both RPL35 genes are required for growth and normal accumulation of 60S r-subunits (50) but no further information is available on the role of L35 in ribosome biogenesis. To characterize in detail the contribution of L35 in ribosome biogenesis, we ﬁrst studied the phenotypic consequences of the deletion of either the RPL35A or the RPL35B gene. As shown in Figure 1A, the deletion of the RPL35A gene (Drpl35A strain) results in a severe growth defect while the deletion of the RPL35B gene (Drpl35B strain) has only a slight eﬀect on the growth rate. In agreement, while the generation time in YPD for the wild-type strain grown at 30 C is 1.4 h, the Drpl35A or Drpl35B mutant strains have a doubling time of 3.6 h and 1.7 h, respectively. Since genome-wide expression data suggest that RPL35A might contribute to 56% while RPL35B to 44% of total RPL35 mRNA levels (71), the functional disparity between the two deletion strains could not be totally explained by diﬀerences in mRNA expression Figure 1. Deletion of either RPL35A or RPL35B leads to a deﬁcit in 60S r-subunits. (A) Growth test of the rpl35A and rpl35B mutants compared to their isogenic wild-type control strain. Strains RBY138 (Drpl35A), RBY139 (Drpl35B) and BY4741 (Wild-type) were grown in YPD and diluted to an OD600 of 0.05. A 10-fold series of dilutions was performed for each strain and 5 ml drops were plated on YPD plates. Plates were incubated at 30 C for 4 days. (B) The above strains were grown in YPD at 30 C and harvested at an OD600 of 0.8, cell extracts were prepared and 10 A260 of each extract were resolved in 7–50% sucrose gradients. The A254 was continuously measured. Sedimentation is from left to right. The peaks of free 40S and 60S r-subunits, 80S free couples/monosomes and polysomes are indicated. Half-mers are labelled by arrows. (C) Equivalent amounts of the cell extracts described above were subjected to western blot analysis with antibodies against the r-proteins L35, L1 and S8.