In contrast to the deregulated hepatocellular division that is a feature of many hepatic diseases and malignancies, physiologic liver growth during embryonic development and after partial hepatectomy (PH) in adults is characterized by tightly controlled cell proliferation. We used forward genetic screening in zebrafish to test the hypothesis that a similar genetic program governs physiologic liver growth during hepatogenesis and regeneration after PH. We identified the uhrf1 gene, a cell cycle regulator and transcriptional activator of top2a expression, as required for hepatic outgrowth and embryonic survival. By developing a methodology to perform PH on adult zebrafish, we found that liver regeneration in uhrf1 ^+/− adult animals is impaired. uhrf1 transcript levels dramatically increase after PH in both mice, and zebrafish and top2a is not up-regulated in uhrf1 ^+/− livers after PH. This indicates that uhrf1 is required for physiologic liver growth in both embryos and adults and illustrates that zebrafish livers regenerate.

Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C‐terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C‐terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro , and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo . Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high‐level Paf1C recruitment in otherwise wild‐type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR‐interaction domain (PCID) in Cdc73 decreases Paf1C‐dependent histone methylation in cells containing non‐phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR‐independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high‐level Paf1C recruitment.

In the present study we describe the isolation and functional analysis of a sphingolipid biosynthetic gene, IPT1 , of Candida albicans . The functional consequence of the disruption of both alleles of IPT1 was confirmed by mass analysis of its sphingolipid composition. The disruption of both alleles or a single allele of IPT1 did not lead to any change in growth phenotype or total sphingolipid, ergosterol, or phospholipid content of the mutant cells. The loss of mannosyl diinositol diphosphoceramide [M(IP) ₂ C] in the ipt1 disruptant, however, resulted in increased sensitivity to drugs like 4-nitroquinoline oxide, terbinafine, o -phenanthroline, fluconazole, itraconazole, and ketoconazole. The increase in drug susceptibilities of ipt1 cells was linked to an altered sphingolipid composition, which appeared to be due to the impaired functionality of Cdr1p, a major drug efflux pump of C. albicans that belongs to the ATP binding cassette superfamily. Our confocal and Western blotting results demonstrated that surface localization of green fluorescent protein-tagged Cdr1p was affected in ipt1 disruptant cells. Poor surface localization of Cdr1p resulted in an impaired ability to efflux fluconazole and rhodamine 6G. The effect of mannosyl inositol phosphoceramide accumulation in the ipt1 mutant and the absence of M(IP) ₂ C from the ipt1 mutant on the efflux of drug substrates was very selective. The efflux of methotrexate, a specific substrate of CaMdr1p, another major efflux pump of major facilitator superfamily, remained unaffected in ipt1 mutant cells. Interestingly, changes in sphingolipid composition affected the ability of mutant cells to form proper hyphae in various media. Taken together, our results demonstrate that an altered composition of sphingolipid, which is among the major constituents of membrane rafts, affects the drug susceptibilities and morphogenesis of C. albicans .