The human genome is generally organized into stable chromosomes, and only tumor cells are known to accumulate kilobase (kb)-sized extrachromosomal circular DNA elements (eccDNAs). However, it must be expected that kb eccDNAs exist in normal cells as a result of mutations. Here, we purify and sequence eccDNAs from muscle and blood samples from 16 healthy men, detecting ~100,000 unique eccDNA types from 16 million nuclei. Half of these structures carry genes or gene fragments and the majority are smaller than 25 kb. Transcription from eccDNAs suggests that eccDNAs reside in nuclei and recurrence of certain eccDNAs in several individuals implies DNA circularization hotspots. Gene-rich chromosomes contribute to more eccDNAs per megabase and the most transcribed protein-coding gene in muscle, TTN (titin), provides the most eccDNAs per gene. Thus, somatic genomes are rich in chromosome-derived eccDNAs that may influence phenotypes through altered gene copy numbers and transcription of full-length or truncated genes.

Examples of extrachromosomal circular DNAs (eccDNAs) are found in many organisms, but their impact on genetic variation at the genome scale has not been investigated. We mapped 1,756 eccDNAs in the Saccharomyces cerevisiae genome using Circle-Seq, a highly sensitive eccDNA purification method. Yeast eccDNAs ranged from an arbitrary lower limit of 1 kb up to 38 kb and covered 23% of the genome, representing thousands of genes. EccDNA arose both from genomic regions with repetitive sequences ≥15 bases long and from regions with short or no repetitive sequences. Some eccDNAs were identified in several yeast populations. These eccDNAs contained ribosomal genes, transposon remnants, and tandemly repeated genes (HXT6/7, ENA1/2/5, and CUP1-1/-2) that were generally enriched on eccDNAs. EccDNAs seemed to be replicated and 80% contained consensus sequences for autonomous replication origins that could explain their maintenance. Our data suggest that eccDNAs are common in S. cerevisiae, where they might contribute substantially to genetic variation and evolution.circular DNA | chromosomal instability | gene deletion | homologous recombination | DNA replication origins

SummaryAmino acid transporters of the yeast plasma membrane (permeases) belong to a family of integral membrane proteins with pronounced structural similarity. We present evidence that a member of this family, encoded by the open reading frame (ORF) YDR160w (SSY1), is required for the expression of a set of transporter genes. Thus, deletion of the SSY1 gene causes loss of leucine-inducible transcription of the amino acid permease genes BAP2, TAT1 and BAP3 (ORF YDR046c) and the peptide transporter, PTR2. D-leucine can generate the signal without entering the cell. We propose that Ssy1p is situated in the plasma membrane and is involved in sensing leucine in the medium.

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All known amino-acid permeases (AAPs) in Saccharomyces cerevisiae belong to a single family of homologous proteins. Genes of 15 AAPs were overexpressed in different strains, and the ability to take up one or more of the 20 common L-alpha-amino acids was studied in order to obtain a complete picture of the substrate specificity for these permeases. Radiolabelled amino-acid uptake measurements showed that Agp1p is a general permease for most uncharged amino acids (Ala, Gly, Ser, Thr, Cys, Met, Phe, Tyr, Ile, Leu, Val, Gln and Asn). Gnp1p, which is closely related to Agp1p, has a somewhat less-broad specificity, transporting Leu, Ser, Thr, Cys, Met, Gln and Asn, while Bap2p and Bap3p, which are also closely related to Agp1p, are able to transport Ile, Leu, Val, Cys, Met, Phe, Tyr and Trp. All four permeases are transcriptionally induced by an extracellular amino acid, but differ in expression with respect to the nitrogen source. On a non-repressive nitrogen source, AGP1 is induced, while GLN1, BAP2 and BAP3 are not. Except for Dip5p, which is a transporter for Glu, Asp, Gln, Asn, Ser, Ala and Gly, the rest of the permeases exhibit narrow specificity. Tat2p can take up Phe, Trp and Tyr; Put4p can transport Ala, Gly and Pro; while Can1p, Lyp1p and the previously uncharacterized Alp1p are specific for the cationic amino acids. These findings modify the prevalent view that S. cerevisiae only contains one general amino-acid permease, Gap1p, and a number of permeases that are specific for a single or a few amino acids.

To study adaptive evolution in defined environments, we performed evolution experiments with Saccharomyces cerevisiae (yeast) in nitrogen-limited chemostat cultures. We used DNA microarrays to identify copy-number variation associated with adaptation and observed frequent amplifications and deletions at the GAP1 locus. GAP1 encodes the general amino acid permease, which transports amino acids across the plasma membrane. We identified a self-propagating extrachromosomal circular DNA molecule that results from intrachromosomal recombination between long terminal repeats (LTRs) flanking GAP1. Extrachromosomal DNA circles (GAP1 circle ) contain GAP1, the replication origin ARS1116, and a single hybrid LTR derived from recombination between the two flanking LTRs. Formation of the GAP1 circle is associated with deletion of chromosomal GAP1 (gap1Δ) and production of a single hybrid LTR at the GAP1 chromosomal locus. The GAP1 circle is selected following prolonged culturing in Lglutamine-limited chemostats in a manner analogous to the selection of oncogenes present on double minutes in human cancers. Clones carrying only the gap1Δ allele were selected under various non-amino acid nitrogen limitations including ammonium, urea, and allantoin limitation. Previous studies have shown that the rate of intrachromosomal recombination between tandem repeats is stimulated by transcription of the intervening sequence. The high level of GAP1 expression in nitrogen-limited chemostats suggests that the frequency of GAP1 circle and gap1Δ generation may be increased under nitrogen-limiting conditions. We propose that this genomic architecture facilitates evolvability of S. cerevisiae populations exposed to variation in levels and sources of environmental nitrogen. adaptive evolution | genome plasticity | intrachromosomal recombination | double minute | retrotransposon A daptive evolution is the process by which a population becomes better suited to its environment through mutation and selection. The pace at which a population adapts to a novel environment is determined by the rate at which beneficial mutations arise in the population. In both experimental and natural populations, mutations underlying adaptive evolution include nucleotide changes (1, 2), transposition events (3, 4), and copy-number variants including gene amplifications and deletions (5, 6). Although the relative importance of these different classes of genomic variation for adaptive evolution is not known, their spontaneous rates differ dramatically. In the single-celled eukaryote Saccharomyces cerevisiae (budding yeast) the rate of point mutation is 10 −9 -10 −10 per base pair/generation (7), whereas rates of retrotransposition are estimated to be on the order of 10 −8 events/generation (8). Gene deletion events are estimated to occur at a very high rate of around 10 −5 events/generation (9). Conversely, gene amplification events, which are much more difficult to assay accurately, have been estimated to occur at rates as low as 10 −10 events/generation (10). Repetitiv...

Analysis of S. cerevisiae cultures with generation times varying between 2 and 35 hours shows that the expression of half of all yeast genes is affected by the specific growth rate.

Through genome-wide transcript analysis of a reference strain and two recombinant Saccharomyces cerevisiae strains with different rates of galactose uptake, we obtained information about the global transcriptional response to metabolic engineering of the GAL gene regulatory network. One of the recombinant strains overexpressed the gene encoding the transcriptional activator Gal4, and in the other strain the genes encoding Gal80, Gal6, and Mig1, which are negative regulators of the GAL system, were deleted. Even though the galactose uptake rates were significantly different in the three strains, we surprisingly did not find any significant changes in the expression of the genes encoding the enzymes catalyzing the first steps of the pathway (i.e., the genes encoding Gal2, Gal1, Gal7, and Gal10). We did, however, find that PGM2, encoding the major isoenzyme of phosphoglucomutase, was slightly up-regulated in the two recombinant strains with higher galactose uptake rates. This indicated that PGM2 is a target for overexpression in terms of increasing the flux through the Leloir pathway, and through overexpression of PGM2 the galactose uptake rate could be increased by 70% compared to that of the reference strain. Based on our findings, we concluded that phosphoglucomutase plays a key role in controlling the flux through the Leloir pathway, probably due to increased conversion of glucose-1-phosphate to glucose-6-phosphate. This conclusion was supported by measurements of sugar phosphates, which showed that there were increased concentrations of glucose-6-phosphate, galactose-6-phosphate, and fructose-6-phosphate in the strain construct overexpressing PGM2.In the yeast Saccharomyces cerevisiae the flux through the galactose utilization pathway is threefold lower than the rate of glucose utilization (35). From an industrial point of view galactose utilization is relevant due to the presence of galactose in several industrial media, such as cheese whey (see reference 43 for a review), molasses (40), and lignocellulose (53). Therefore, there is interest in increasing galactose utilization through the use of metabolic engineering (3,30,34). Besides the industrial relevance, the gene system involved in galactose metabolism in yeast (the GAL genes) has served as a eukaryotic model system for gene regulation and thus is one of the bestcharacterized systems of eukaryotic transcriptional control (18,20). Moreover, the importance of the S. cerevisiae GAL system is further emphasized by its role as a model for the human hereditary disease galactosemia (42), which is caused by a disorder in galactose metabolism.Galactose utilization in S. cerevisiae occurs through the Leloir pathway (Fig. 1), in which galactose is converted to glucose-1-phosphate via galactose-1-phosphate. Glucose-1-phosphate is then converted to glucose-6-phosphate, which may enter both the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway. The genes encoding the enzymes of the Leloir pathway are tightly regulated; they are repressed by glucose and...