Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by -mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.

SummaryThe ribosome has a distinct lateral protuberance called the stalk; in eukaryotes it is formed by the acidic ribosomal P-proteins which are organized as a pentameric entity described as P0-(P1-P2) 2 . Bilateral interactions between P0 and P1/P2 proteins have been studied extensively, however, the region on P0 responsible for the binding of P1/P2 proteins has not been precisely defined. Here we report a study which takes the current knowledge of the P0 -P1/P2 protein interaction beyond the recently published information. Using truncated forms of P0 protein and several in vitro and in vivo approaches, we have defined the region between positions 199 and 258 as the P0 protein fragment responsible for the binding of P1/P2 proteins in the yeast Saccharomyces cerevisiae . We show two short amino acid regions of P0 protein located at positions 199-230 and 231-258, to be responsible for independent binding of two dimers, P1A-P2B and P1B-P2A respectively. In addition, two elements, the sequence spanning amino acids 199-230 and the P1A-P2B dimer were found to be essential for stalk formation, indicating that this process is dependent on a balance between the P1A-P2B dimer and the P0 protein.

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The predominant cell wall melanin of Wangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced directly as a pentaketide by an iterative type I polyketide synthase (PKS). In contrast, the bluish-green pigment in Aspergillus fumigatus is produced after the enzyme Ayg1p converts the PKS product, the heptaketide YWA1, to T4HN. Previously, we created a new melanin-deficient mutant of W. dermatitidis, WdBrm1, by random molecular insertion. From this strain, the altered gene WdYG1 was cloned by a marker rescue strategy and found to encode WdYg1p, an ortholog of Ayg1p. In the present study, two gene replacement mutants devoid of the complete WdYG1 gene were derived to eliminate the possibility that the phenotype of WdBrm1 was due to other mutations. Characterization of the new mutants showed that they were phenotypically identical to WdBrm1. Chemical analyses of mutant cultures demonstrated that melanin biosynthesis was blocked, resulting in the accumulation of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (AT4HN) and its oxidative product 3-acetylflaviolin in the culture media. When given to an albino W. dermatitidis strain with an inactivated WdPKS1 gene, AT4HN was mostly oxidized to 3-acetylflaviolin and deacetylated to flaviolin. Under reduced oxygen conditions, cell-free homogenates of the albino converted AT4HN to D2HN. This is the first report of evidence that the hexaketide AT4HN is a melanin precursor for T4HN in W. dermatitidis.Melanins are dark pigments widely produced by fungi and other organisms. In fungi, they are frequently found in the cell wall. While not essential for growth and development, these complex polymers seem to enhance the survival and competitive abilities of fungi in certain environments. They are composed of various types of phenolic monomers and are often complexed with protein and, less often, carbohydrates (12,22,29). The melanins in fungi are named according to their composition and the way they are synthesized and include dihydroxyphenylalanine melanin, catechol melanin, ␥-glutaminyl-4-hydroxybenzene melanin, and 1,8-dihydroxynaphthalene (D2HN) melanin (23, 26). The best characterized of these fungal melanins is probably D2HN melanin, which is synthesized by related polyketide pathways ( Fig. 1 and 2A and B). The D2HN melanin pathways start with one acetyl-coenzyme A (acetyl-CoA) molecule and four malonyl-CoA molecules, or solely with malonyl-CoA molecules, which undergo a head-totail joining and cyclization catalyzed by an iterative type I polyketide synthase (PKS) to initially form 1,3,6,8-tetrahydroxynaphthalene (T4HN) (16). From T4HN, multiple sequential enzyme-catalyzed steps produce D2HN, which is then polymerized to form melanin by a poorly characterized oxidase/laccase reaction (5,7,22).In Colletotrichum lagenarium, T4HN is made directly from malonyl-CoA by PKS1p, as shown in Fig. 2B (16). In contrast, in the bluis...

The GTPase center of the large ribosomal subunit, being a landing platform for translation factors, and regarded as one of the oldest structures in the ribosome, is a universally conserved structure in all domains of life. It is thought that this structure could be responsible for the major breakthrough on the way to the RNA/protein world, because its appearance would have dramatically increased the rate and accuracy of protein synthesis. The major part of this center is recognized as a distinct structural entity, called the stalk. The main functional part of the stalk in all domains of life is composed of small L12/P proteins, which are believed to form an evolutionarily conserved group. However, some data indicate that the bacterial and archaeo/eukaryal proteins are not related to each other structurally, and only a functional relationship may be clearly recognized. To clarify this point, we performed a comprehensive comparative analysis of the L12/P proteins from the three domains of life. The results show that bacterial and archaeo/eukaryal L12/P-proteins are not structurally related and, therefore, might not be linked evolutionarily either. Consequently, these proteins should be regarded as analogous rather than homologous systems and probably appeared on the ribosomal particle in two independent events in the course of evolution.

The regulation of the Saccharomyces cerevisiae GAL genes in response to galactose as a source of carbon has served as a paradigm for eukaryotic transcriptional control over the last 50 years. Three proteins-a transcriptional activator (Gal4p), an inhibitor (Gal80p), and a ligand sensor (Gal3p)-control the switch between inert and active gene expression. The molecular mechanism by which the recognition of galactose within the cell is converted into a transcriptional response has been the subject of considerable debate. In this study, using a novel and powerful method of localizing active transcription factors within the nuclei of cells, we show that a short-lived complex between Gal4p, Gal80p, and Gal3p occurs soon after the addition of galactose to cells to activate GAL gene expression. Gal3p is subsequently replaced in this complex by Gal1p, and a Gal4p-Gal80p-Gal1p complex is responsible for the continued expression of the GAL genes. The transient role of the ligand sensor indicates that current models for the induction and continued expression of the yeast GAL genes need to be reevaluated.

The eukaryotic 60S ribosomal subunit has a set of very acidic proteins (P-proteins), which form a distinct lateral protuberance called the stalk structure. This protein complex is directly involved in the elongation step of polypeptide synthesis. In our study on acidic ribosomal P-proteins from the human opportunistic pathogen Candida albicans, we isolated and characterized one of the genes, called CARP2A, and its product, the P2A protein. The CARP2A gene is intron-less, present in a single copy per haploid genome, and transcriptionally active. The open reading frame of the studied gene contains information for a sequence of 108 amino acids. Based on this, the molecular mass and isoelectric point of the P2A protein were theoretically calculated to be 10.85 kDa and 3.7, respectively. The characteristic feature of the CARP2A gene transcript is the presence of a GUG start codon, which is rare in eukaryotic organisms and not previously reported in yeast. To our knowledge this is the first report showing the presence of a naturally occurring non-AUG start codon on mRNA in yeast species. The nucleotide sequence of CARP2A has been deposited in the GenBank data library under Accession No. AF317661.

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