Abstract: Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast.

Abstract: Expression of yeast RuvB-like gene analogues of bacterial RuvB is self-regulated, as episomal overexpression of RVB1 and RVB2 decreases the expression of their chromosomal copies by 85%. Heterozygosity for either gene correlates with lower double-strand break repair of inverted-repeat DNA and decreased survival after UV irradiation, suggesting their haploinsufficiency, while overexpression of the bacterial RuvAB complex improves UV survival in yeast. Rvb2p preferentially binds artificial DNA Holiday junctions like the bacterial RuvAB complex, whereas Rvb1p binds to duplex or cruciform DNA. As both proteins also interact with chromatin, their role in recombination and repair through chromatin remodelling, and their evolutionary relationship to the bacterial homologue, is discussed.

Abstract: In Saccharomyces cerevisiae, aneuploidy is well tolerated and stable. We analysed whether the induced loss of a disomic chromosome favours endo-reduplication of the remaining chromosome or the cells prefer to retain the acquired euploidy. Chromosome VIII disomes and trisomes were tagged with GFP (green fluorescent protein), DsRed (red fluorescent protein) and BFP (blue fluorescent protein) integrated at the thr1 locus, using our newly designed STIK (specific targeted integration of kanamycin resistance-associated, non-selectable DNA) plasmid system. A knockout cassette for centromere 8 was constructed with the hygromycin-B marker, which was transformed into the strains. The transformants lost sensitivity to hygromycin, thereby indicating the event of centromere replacement. Quantitative PCR and Southern analysis were performed for chromosome VIII copy number determination by probing the markers located on both the right (ARG4 and THR1) and left (GUT1) arm whereas, for chromosome V, markers such as HIS1, located on right arm, and URA3, on left arm, were used. The loss of an extranumerary chromosome VIII in a disome and trisome leads to stable euploidy. Furthermore, in a wild-type diploid, deletion of a copy of chromosome VIII, leads to monosomy, and restoration of euploidy after 22 generations, by reduplication of chromosome VIII, and consequent loss of heterozygosis (LOH). However, chromosome V knockouts in chromosome VIII trisome, still showed LOH and duplication of chromosome V, with return to the original aneuploid condition. These results suggest that yeast cells could control the integrity of their genetic complement acting at the individual chromosome level.

Abstract: Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II-directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.

Abstract: Several experimental in vivo systems exist that generate reciprocal translocations between engineered chromosomal loci of yeast or Drosophila, but not without previous genome modifications. Here we report the successful induction of chromosome translocations in unmodified yeast cells via targeted DNA integration of the KAN(R) selectable marker flanked by sequences homologous to two chromosomal loci randomly chosen on the genome. Using this bridge-induced translocation system, 2% of the integrants showed targeted translocations between chromosomes V-VIII and VIII-XV in two wild-type Saccharomyces cerevisiae strains. All the translocation events studied were found to be non-reciprocal and the fate of their chromosomal fragments that were not included in the translocated chromosome was followed. The recovery of discrete-sized fragments suggested multiple pathway repair of their free DNA ends. We propose that centromere-distal chromosome fragments may be processed by a break-induced replication mechanism ensuing in partial trisomy. The experimental feasibility of inducing chromosomal translocations between any two desired genetic loci in a eukaryotic model system will be instrumental in elucidating the molecular mechanism underlying genome rearrangements generated by DNA integration and the gross chromosomal rearrangements characteristic of many types of cancer.

Abstract: The efficiency of gene targeting within different segments of genes in yeast was estimated by transforming yeast cells with double-stranded integrative plasmids, bearing functional gene domains [promoter (P), ORF (O) and terminator (T)] derived from the common genetic markers HIS3, LEU2, TRP1 and URA3. Transformation experiments with circular plasmids carrying a single gene domain demonstrated that the 5' and 3' flanking DNA regions (P and T) of the HIS3 and URA3 genes are preferred as sites for plasmid integration by several fold over the corresponding ORFs. Moreover, when plasmids bearing combinations of two or three regions were linearized to target them to a specific site of integration, three of the ORFs were found to be less preferred as sites for plasmid integration than their corresponding flanking regions. Surprisingly, in up to 50% of the transformants obtained with plasmids that had been linearized within coding sequences, the DNA actually integrated into neighbouring regions. Almost the same frequencies of ORF mis-targeting were obtained with plasmid vectors containing only two functional domains ("PO" or "OT") of the gene URA3, demonstrating that this event is not the consequence of competition between homologous DNA regions distal to the ORF. Therefore, we suggest that coding sequences could be considered to be "cold spots" for plasmid integration in yeast.

Abstract: The advent of genomics has greatly influenced fundamental and applied microbiology. This has become paradigmatic in the case of Bacillus subtilis, a primary model bacterium for research and biotechnology. Indeed, mining its genome has provided more fruitful information than classical approaches would have yielded in a longer period of time. Through advanced analysis of its genome and transcriptome, fundamental discoveries dealing with the informational architecture of the B. subtilis chromosome, as well as with the elucidation of its pathway-level regulation of gene expression, have been achieved. The possibility of performing a complete metabolic manipulation of the secretory pathway of Bacillus is promising important biotechnological fallouts. Similar emphasis exists for the possibility of controlling the cell in the formation of biofilms with specific physical and chemical characteristics. At the theoretical level, the new concept of genetic superinformation has been formulated and its analytical approach implemented, while the understanding of the minimal genetic requirements for the existence of a reproducing bacterial cell is being tackled. In summary, the impact of the B. subtilis genome has philosophically revolutionised the way that basic knowledge is translated into applied microbiology and biotechnology, making this bacterium the workhorse of post-genomic microbiology.

Abstract: The entire genomic DNA sequence of the Gram-positive bacterium Bacillus subtilis reported in the SubtiList database has been subjected in this work to a complete bioinformatic analysis of the potential formation of secondary DNA structures such as hairpins and bending. The most significant of these structures have been mapped with respect to their genomic location and compared to those structures already known to have a physiological role, such as the rho-independent transcription terminators. The distribution of these structures along the bacterial chromosome shows two major features: (i). the concentration of the most curved DNA in the intergenic regions rather than within the ORFs, and (ii). a decreasing gradient of large hairpins from the origin towards the terC end of chromosomal DNA replication. Given the increasing biological relevance of secondary DNA structures, these findings should facilitate further studies on the evolution, dynamics and expression of the genetic information stored in bacterial genomes.

Abstract: Sophisticated genome manipulation requires the possibility to modify any intergenic or intragenic DNA sequence at will, without leaving large amounts of undesired vector DNA at the site of alteration. To this end, a series of vectors was developed from a previous gene knockout plasmid system to integrate nonselectable foreign DNA at any desired genomic location in yeast, with a minimum amount of residual plasmid DNA. These vectors have two mutated Flp recognition targets (FRT) sequences flanking the KanMX4 gene and multiple sites for subcloning the DNA fragment to be integrated. The selectable marker can be recycled by Flp site-specific excision between the identical FRTs, thereby allowing the integration of further DNA fragments. With this system, the NLS-tetR-GFP and DsRed genes were successfully integrated at the thr1 locus, and the RVB1 gene was tagged at the C-terminus with the V5-epitope-6-histidine tag. This plasmid system provides for a new molecular tool to integrate any DNA fragment at any genome location in [cir+] yeast strains. Moreover, the system can be extrapolated to other eukaryotic cells in which the FLP/FRT system functions efficiently.

Abstract: A comparison of the selective fitness of four 2-microm-based shuttle-plasmids carrying the yeast genes HIS3, LEU2, TRP1, and URA3 was performed. The effect of each marker on long-term growth rate and plasmid maintenance was measured. In selective medium, the LEU2 and URA3 plasmids were maintained at the lowest and the highest levels, respectively, while the HIS3 and TRP1 plasmids were maintained at an intermediate level. In synthetic complete medium, plasmid loss rate was lower for the genes TRP1 and URA3 than for the other two markers, and a similar pattern was observed for cells growing in rich medium. These results were confirmed by competition experiments among transformants with different plasmids in complete and rich media, indicating a different degree of fitness for the markers used. A potential correlation of the energy cost of plasmid maintenance with the secondary DNA structure and the level of expression of the selective markers is also investigated.

Abstract: To investigate the possibility of inducing specific chromosome loss by centromere deletion in eukaryotic cells, the yeast diploid strain ZG1, carrying three pairs of heterozygous marker genes (CAN1(S)/can1(R), URA3/Deltaura3, hphMX4/HIS1), widely spread on both arms of chromosome V, was constructed. One of the two centromeres V of ZG1 was replaced by the LEU2 gene via the well-established PCR-mediated knockout technique. After DNA transformation, putative yeast colonies that showed loss of heterozygosity (LOH) for the three markers of chromosome V (CAN1(S) URA3 hphMX4) were identified among the colonies selected for leucine prototrophy. Phenotypic tests, colony-PCR and Southern blot analysis of these cells demonstrated the physical loss of the CAN1(s), URA3, and hphMX4 marker genes from the genome. Further tetrad analysis results were consistent with this conclusion; however, four-spore viability indicated a normal chromosome number of these transformants. To verify the diploidy of the selected chromosome V, the HIS1 gene was deleted with a standard KanMX4 knockout DNA cassette. The resulting heterogeneity of the HIS1/KanMX4 markers, together with quantitative PCR and densitometric analysis on chromosome V, confirmed its diploid complement, thereby indicating that an endoreduplication event had taken place. Restitution of diploidy also occurred in MAD2-deleted strains undergoing higher rates of spontaneous chromosome V loss, indicating a more general phenomenon that is undetectable by phenotypic analysis alone.

Abstract: As part of the EULEISH international genome project, a region of 74,674 nucleotides from chromosome 21 of Leishmania major Friedlin was subcloned and sequenced; and 31 new coding sequences were predicted. Of particular interest was a unique coding strand switching region covering 1.6 kb of DNA; and this was subjected to further investigation. Bioinformatic analysis of this region revealed an unusually high AT composition, a lack of putative hairpins and a strong curvature of the DNA in agreement with the structural characteristics of similar regions of other Leishmania chromosomes. These observations and a comparison with the secondary DNA structure of four other Leishmania chromosomes and chromosomes of different organisms could suggest a functional role of this region in transcription and mitotic division.

Abstract: The mechanism of excretion into bile of hepatospecific magnetic resonance imaging (MRI) contrast media employed labeled Gd-reagents EOB.DTPA, BOPTA, B 20790 (iopanoate-linked), and B 21690 (glycocholate-linked) for measurement in rat liver canalicular plasma membrane vesicles and yeast vacuoles. The presence of ATP gave threefold greater transport of B 20790 and B 21690 than of EOB.DTPA and BOPTA. In yeast vacuoles the ATP stimulatory effect was eightfold with B 20790 and fivefold greater for B 21690, whereas in YCF1- or YLLO115w-deleted yeast cells the transport was significantly reduced and absent from double mutants, YCF1 and YLLO15w. The transport was similar in wild-type and deletant cells for B 21690; taurocholate gave 85% inhibition. These data suggest that bilary secretion of structurally related MRI agents depend on molecular structure. The findings are suggestive as of possible value for clinical diagnosis of inherited hyperbilirubinemias and other liver disorders.

Abstract: Site-specific recombination within the Saccharomyces cerevisiae 2-micron DNA plasmid is catalyzed by the Flp recombinase at specific Flp Recognition Target (FRT) sites, which lie near the center of two precise 599-bp Inverted Repeats (IRs). However, the role of IR DNA sequences other than the FRT itself for the function of the Flp reaction in vivo is not known. In the present work we report that recombination efficiency differs depending on whether the FRT or the entire IR serves as the substrate for Flp. We also provide evidence for the involvement of the IR in RAD52-dependent homologous recombination. In contrast, the catalysis of site-specific recombination between two FRTs does not require the function of RAD52. The efficiency of Flp site-specific recombination between two IRs cloned in the same orientation is about one hundred times higher than that obtained when only the two FRTs are present. Moreover, we demonstrate that a single IR can activate RAD52-dependent homologous recombination between two flanking DNA regions, providing new insights into the role of the IR as a substrate for recombination and a new experimental tool with which to study the molecular mechanism of homologous recombination.

Abstract: Since bilirubin-like pigments are present in the environment as degradation products of heme-containing proteins, yeast could have developed a detoxifying system to transport these compounds into their vacuoles. Vacuoles from Saccharomyces cerevisiae showed an ATP-dependent, saturative transport of unconjugated bilirubin (UCB) that was reduced by 60% and 40% in YCF1 and YLL015w-deleted cells, respectively; the double deletant showed no UCB uptake. Conversely, the transport of bile acids (taurocholate) was comparable in wild and deleted stains. These data identify YCF1 and YLL015w, named BPT1 (Bile Pigment Transporter), as the genes responsible for ATP-dependent UCB transport in yeast. Since YCF1 and YLL015w are rather homologous with multidrug resistant proteins (MRPs), they also suggest the involvement of this class of transporters in the ATP-dependent transport of unconjugated bilirubin.

Abstract: A molecular FRT (Flp recombinase recognition target)-based cassette system for multiple gene disruption in the yeast Saccharomyces cerevisiae was developed. FRT DNA sequences were designed with different core mutations and subsequently cloned in direct orientation upstream and downstream of a marker gene to serve as template for the amplification of a set of different gene disruption cassettes. After each disruption, the marker can be easily eliminated from its integration site by in vivo site-specific recombination between the two identical, mutated FRT sequences flanking the marker, leaving behind one FRT sequence with a particular point mutation. Since recombination between two FRTs with a different core mutation is extremely rare, the possibility of chromosome rearrangements, due to site-specific recombination between residual FRTs, is very low. In strains containing 2-microm ([cir+]) the site-specific reaction is catalysed by the endogenous Flp gene product, whereas in strains without 2-microm ([cir0]), the FLP gene is carried on the cassette, together with the marker gene. This system can be applied for haploid and diploid [cir+] and [cir0] strains.

Abstract: By in silicio analysis, we have discovered that there are seven open reading frames (ORFs) in Saccharomyces cerevisiae whose protein products show a high degree of amino acid sequence similarity to the aryl alcohol dehydrogenase (AAD) of the lignin-degrading fungus Phanerochaete chrysosporium. Yeast cultures grown to stationary phase display a significant aryl alcohol dehydrogenase activity by degrading aromatic aldehydes to the corresponding alcohols. To study the biochemical and the biological role of each of the AAD genes, a series of mutant strains carrying deletion of one or more of the AAD-coding sequences was constructed by PCR-mediated gene replacement, using the readily selectable marker kanMX. The correct targeting of the PCR-generated disruption cassette into the genomic locus was verified by analytical PCR and by pulse-field gel electrophoresis (PFGE) followed by Southern blot analysis. Double, triple and quadruple mutant strains were obtained by classical genetic methods, while the construction of the quintuple, sextuple and septuple mutants was achieved by using the marker URA3 from Kluyveromyces lactis, HIS3 from Schizosaccharomyces pombe and TRP1 from S. cerevisiae. None of the knock-out strains revealed any mutant phenotype when tested for the degradation of aromatic aldehydes using both spectrophotometry and high performance liquid chromatography (HPLC). Specific tests for changes in the ergosterol and phospholipids profiles did not reveal any mutant phenotype and mating and sporulation efficiencies were not affected in the septuple deletant. Compared to the wild-type strain, the septuple deletant showed an increased resistance to the anisaldehyde, but there is a possibility that the nutritional markers used for gene replacement are causing this effect.

Abstract: The Providencia rettgeri and Escherichia coli pac genes encoding heterodimeric penicillin G amidases (PAC) were successfully expressed in Saccharomyces cerevisiae. Furthermore, these recombinant enzymes are secreted from the yeast cell into the medium which is in contrast to bacterial hosts, where the enzymes are retained in the periplasm. Contrary to the P. rettgeri PAC-encoding gene, the E. coli pac is poorly expressed in yeast. The highest yield of P. rettgeri PAC was obtained with a multi-copy plasmid, resulting in of 1500units per liter. This yield is higher by an order of magnitude than that obtained in the best recombinant bacterial expression system. The recombinant P. rettgeri enzyme is only partially and selectively O-glycosylated. Only every sixth or seventh alpha-subunit is glycosylated, while the beta-subunit is not glycosylated at all. N-Glycosylation has not been detected.

Abstract: Transposon Tn5 genomic mutants of plant-growth-promoting Pseudomonas putida strain WCS358 have been isolated which no longer utilize ferulic and coumaric acids as sole sources of carbon and energy. Genetic studies confirmed previous biochemical data showing that ferulic acid is degraded via vanillic acid, and coumaric acid via hydroxybenzoic acid. The genes involved in these enzymic steps were cloned and characterized. Two proteins designated Fca (26.5 kDa) and Vdh (50.3 kDa) were identified as responsible for the conversion of ferulic acid to vanillic acid; the proteins are encoded by the fca and vdh genes which are organized in an operon structure in the chromosome. The Vdh protein is 69% identical at the amino acid level to the Vdh protein recently identified in Pseudomonas sp. strain HR199 and converts vanillin to vanillic acid. Homology studies revealed that the Vdh proteins exhibited significant identity to aldehyde dehydrogenases from different organisms whereas Fca belonged to the enoyl-CoA hydratase family of proteins. Two proteins, designated VanA (39.9 kDa) and VanB (34.3 kDa), encoded by two genes, vanA and vanB, are organized in an operon in the chromosome. They were found to be responsible for the demethylation of vanillic acid to protocatechuic acid. The VanA proteins showed no homology to any other known protein, while VanB belonged to the ferredoxin family of proteins. This two-component enzyme system demethylated another phenolic monomer, veratric acid, thus indicating broad specificity. Studies of the regulation of the vanAB operon demonstrated that the genes were induced by the substrate, vanillic acid; however, the strongest induction was observed when cells were grown in the presence of the product of the reaction, protocatechuic acid.

Abstract: Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55 degrees C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis contant (Km) and Vmax for alpha-naphthyl acetate were 1.54 mM and 360 micromol min-1 mg of protein-1, respectively.

Abstract: Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for manipulating DNA both in vitro and in living organisms. In this work we describe the isolation of yeast Saccharomyces cerevisiae segregants which have lost the highly stable 2 microm DNA plasmid, exploiting the site-specific recombination system of 2 microm itself. We efficiently isolated [cir0] segregants from two haploid yeast strains and also a diploid. Moreover, the effect of mutations in the core region of the FRT (Flp Recognition Target) sequence was investigated in vivo, studying the result of the recombination event between several mutated and wild-type FRT sequences. From our result it seems that the identity between the core regions of two FRT sites is necessary but not sufficient, indicating that the core sequence itself has a relevant function in the recombination mechanism in vivo.

Abstract: The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VII has 572 predicted open reading frames (ORFs), of which 341 are new. No correlation was found between G+C content and gene density along the chromosome, and their variations are random. Of the ORFs, 17% show high similarity to human proteins. Almost half of the ORFs could be classified in functional categories, and there is a slight increase in the number of transcription (7.0%) and translation (5.2%) factors when compared with the complete S. cerevisiae genome. Accurate verification procedures demonstrate that there are less than two errors per 10,000 base pairs in the published sequence.

Abstract: The nucleotide sequence of a 40.5 kb DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae was determined and analysed. Twenty-eight open reading frames (ORFs) longer than 300 nucleotides were identified. Eight of the them correspond to the following known yeast genes: EMP24, GCN1, SPO8, COX13, CDC55, RPS26, COX4 and LSR1, also called GTS1. Twelve ORFs are new, among them eight show homology with other genes while four have no homology with any sequence in the databases. Eight additional ORFs are internal to or partially overlapping with other ORFs.

Abstract: A 15 kb DNA fragment from the Bacillus subtilis chromosome between citB and ppsC has been sequenced, and new ORFs encoding putative enzymes involved in lipopolypeptide synthesis, which complete a partial operon previously reported, and a new set of enzymes responsible for lipid metabolism have been identified. From the analysis of DNA sequence homology of the fragment it was deduced that these new peptide synthetase genes are part of an operon for the biosynthesis of the fungicide fengycin.

Abstract: A wild-type Acetobacter pasteurianus was subjected to chemical mutagenesis for the induction and isolation of a cellulose overproducing strain. A mutagenized strain capable of synthesizing double amounts of cellulose compared to the wild type was obtained. Cellulose, both from the wild-type and the mutagenized strain, was extracted and purified for chemical characterization and investigation of its physico-chemical properties. The comparison of the two microbial polysaccharides shows that the putative mutation of A. pasteurianus strain had no effect on some cellulose features such as chemical structure, polymorphic form, crystallinity.

Abstract: Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Abstract: In the yeast Saccharomyces cerevisiae the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment, while epistatic mutations in the same pathway, i.e. ade8, preclude this phenomenon, resulting in normal white colonies. The shift in color from red to white (or vice versa) with a combination of appropriate wild-type and mutant alleles of the adenine-pathway genes has been widely utilized as a non-selective phenotype to visualise and quantify the occurrence of various genetic events such as recombination, conversion and aneuploidy. It has provided an invaluable tool for the study of gene dosage and plasmid stability. In competition experiments between disrupted ade2, ade8-18 transformants carrying either a functional or non-functional episomal ADE8 gene, we verified that white ade8 ade2 cells show a remarkable selective advantage over red ade2 cells, with important implications on the use of this assay for the monitoring of genetic events. The accumulation of the red pigment in ade2 cells is likely to be the cause for impaired growth in these cells.

Abstract: We report the sequence of an 8.8 kb segment of DNA from the left arm of chromosome VII of Saccharomyces cerevisiae. The sequence reveals seven open reading frames (ORFs) G1651, G1654, G1660, G1663, G1666, G1667 and G1669 greater than 100 amino acids in length and the tRNALys1 gene. ORF G1651 shows 100% identity with the ROK1 protein which is a putative RNA helicase of the 'DEAD box' protein family. ORF G1654 exhibits a motif highly conserved in ATP/GTP binding proteins generally referred to as 'P-loop'. From FastA analysis, G1660 and G1666 were found to be previously sequenced genes, respectively SUA5 and PMR1. The three other ORFs identified are partially (G1663) or completely (G1667 and G1669) overlapping with the PMR1 sequence on the complementary strand. This feature, together with their low codon adaptation indexes and the absence of significant homology with known proteins suggest that they do not correspond to real genes.

Abstract: Bacillus pumilus PS213 isolated from bovine ruminal fluid was able to transform ferulic acid and p-coumaric acid to 4-vinylguaiacol and 4-vinylphenol, respectively, by nonoxidative decarboxylation. The enzyme responsible for this activity has been purified and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extract from a culture induced by ferulic acid or p-coumaric acid shows three bands that are not present in the crude extract of an uninduced culture, while the purified enzyme shows a single band of 23 kDa; the molecular mass calculated by size exclusion chromatography is 45 kDa. Enzyme activity is optimal at 37 degrees C and pH 5.5 and is not enhanced by any cation. Kinetic studies indicated a Km of 1.03 mM and a Vmax of 0.19 mmol.min-1/mg.liter-1 for ferulic acid and a Km of 1.38 mM and a Vmax of 0.22 mmol.min-1/mg.liter-1 for p-coumaric acid.

Abstract: Cell suspensions of Acinetobacter calcoaceticus strain DSM 586 and DSM 590 were able to grow on benzoic, p-hydroxybenzoic and vanillic acid as sole carbon source. Testing the utilization of trans-ferulic and p-coumaric acid, we found that the sole A. calcoaceticus DSM 586 efficiently degraded the lignocellulose related monomers. Cells induced with trans-ferulic acid were able to oxidize trans-ferulic, p-coumaric, vanillic, p-hydroxybenzoic and protocatechuic acid at rates higher than the uninduced culture. The same activity was found in the p-coumaric acid induced culture. Two aromatic compounds, vanillic and p-hydroxybenzoic acid, were isolated from culture filtrates of trans-ferulic and p-coumaric acid grown cells, respectively, and further characterized by high performance liquid chromatography. 1H- and 13C-nuclear magnetic resonance and ultraviolet spectrophotometry. Cell extracts of trans-ferulic or p-coumaric acid induced cultures were shown to rapidly convert protocatechuic acid to beta-carboxymuconic acid. Moreover, A. calcoaceticus DSM 586 produced high levels of protocatechuic 3,4-dioxygenase compared to cathecol 1,2-dioxygenase and gentisate 1,2-dioxygenase in the degradation of trans-ferulic or p-coumaric acid. Based upon these results, a reaction sequence for the complete degradation of trans-ferulic and p-coumaric acid in A. calcoaceticus DSM 586 is proposed.

Abstract: We report the sequence of an 11.1 kb fragment located on the left arm of chromosome VII of Saccharomyces cerevisiae. By sequence analysis we have detected six open reading frames (ORFs) longer that 300 bp, which cover 87% of the entire sequence. ORF G1645 is 100% identical to the KEM1 gene, also identified as DST2, XRN1, SEP1 and RAR5, while G1648 is 100% identical to the NSP49 or NUP49 gene. ORF G1642 shares some identity with a hypothetical protein of Caenorhabditis elegans, while the other four ORFs show no significant homology to known proteins.

Abstract: When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.

Abstract: The Bacillus pumilus gene encoding a ferulic acid decarboxylase (fdc) was identified and isolated by its ability to promote ferulic acid decarboxylation in Escherichia coli DH5 alpha. The DNA sequence of the fdc gene was determined, and the recombinant enzyme produced in E. coli was purified and characterized.

Abstract: Temperature-sensitive Saccharomyces cerevisiae cdc6 mutants, under restrictive conditions, show an increase in recombination frequency, as well as chromosome and circular minichromosome loss. The role of the essential CDC6 gene was tested in trans and in cis to study circular plasmid stability. It was possible to demonstrate that the product of the CDC6 gene, acting in trans, is important for centromeric, episomal, and also 2-microns plasmid maintenance, while the gene sequence itself has no effect in cis on the stability of the plasmids tested. A high percentage of phenotypic revertants for the cdc6 mutation loses 2 microns upon shifting to the restrictive temperature and, under semipermissive conditions, the endogenous plasmid becomes very unstable, favoring a more efficient curing procedure. A positive correlation between centromeric plasmid size and stability was demonstrated even for small circular plasmids.

Abstract: We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues. ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight-repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S. cerevisiae and the Pumilio protein of Drosophila. ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology.

Abstract: We have employed the analysis of spontaneous forward mutations that confer the ability to utilize L-alpha-aminoadipate as a nitrogen source (alpha-Aa+) to discern the events that contribute to mitotic segregation of spontaneous recessive mutations by diploid cells. alpha-Aa- diploid cells yield alpha-Aa+ mutants at a rate of 7.8 +/- 3.6 x 10(-9). As in haploid strains, approximately 97% (30/31) of alpha-Aa+ mutants are spontaneous lys2-x recessive mutations. alpha-Aa+ mutants of diploid cells reflect mostly the fate of LYS2/lys2-x heterozygotes that arise by mutation within LYS2/LYS2 populations at a rate of 1.2 +/- 0.4 x 10(-6). Mitotic recombination occurs in nonrandom association with forward mutation of LYS2 at a rate of 1.3 +/- 0.6 x 10(-3). This mitotic recombination rate is tenfold higher than that of a control LYS2/lys2-1 diploid. Mitotic segregation within LYS2/lys2-x subpopulations yields primarily lys2-x/lys2-x diploids and a minority of lys2-x aneuploids. Fifteen percent of lys2-x/lys2-x diploids appear to have arisen by gene conversion of LYS2 to lys2-x; 85% of lys2-x/lys2-x diploids appear to have arisen by mitotic recombination in the CENII-LYS2 interval. lys2-1/lys2-1 mitotic segregants of a control LYS2/lys2-1 diploid consist similarity of 18% of lys2-1/lys2-1 diploids that appear to have arisen by gene conversion of LYS2 to lys2-1 and 82% of lys2-1/lys2-1 diploids that appear to have arisen by mitotic recombination in the CENII-LYS2 interval.(ABSTRACT TRUNCATED AT 250 WORDS)

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Abstract: A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

Abstract: The best candidate for a high-copy-number and mitotic stability expression system in yeast is the endogenous 2 microns plasmid. Nevertheless, derivatives of the 2 microns plasmid typically exhibit lower copy numbers and require selection for adequate maintenance within cells. We report the construction and utilization of an efficient heterologous gene expression system containing a 4.5-kb inducible expression cassette inserted into the 2 microns plasmid and selected in cells utilizing a carrier plasmid which is subsequently lost via FRT/Flp recombination. The non-selectable 2 micron plasmid, containing the cassette, was found to be stably maintained in cells, without selection, at high copy number. The dynamics of resolution and partitioning of this plasmid were analyzed during the course of 50 generations of growth under non-selective conditions. The heterologous lacZ reporter gene coding for beta-galactosidase (beta Gal) is driven by the hybrid, galactose-inducible promoter GAL10::pMF alpha 1. Upon induction, beta Gal was secreted into the periplasm and culture supernatant at levels which could be detected directly from Coomassie blue-stained SDS-PAGE. Furthermore, plasmid-containing cells could be maintained directly on rich YPD medium and identified either by utilizing XGal or by observing inhibition of colony growth on YPGal solid medium. The cassette was designed for direct, high-level, inducible expression of cloned genes downstream from the MF alpha 1 signal sequence, with or without a C-terminal lacZ fusion. This vector represents the first demonstration of a non-selectable, mitotically stable, episomal plasmid system capable of expressing recombinant proteins at high levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Xylan-degrading enzymes were induced when Phanerochaete chrysosporium was grown at 30 degrees C in shake flask media containing xylan, Avicel PH 102, or ground corn stalks. The highest xylanase activity was produced in the corn stalk medium, while the xylan-based fermentation resulted in the lowest induction. Analytical and preparative isoelectric focusing were used to characterize xylanase multienzyme components. Preparative focusing was performed only with the cultures grown on Avicel and corn stalk. Of over 30 protein bands separated by analytical focusing from the Avicel and corn stalk media, three main groups (I, II, and III) of about five isoenzymes each showed xylanase activity when a zymogram technique with a xylan overlay was used. Enzyme assays revealed the presence of 1,4-beta-endoxylanase and arabinofuranosidase activities in all three isoenzyme groups separated by preparative isoelectric focusing. beta-Xylosidase activity appeared in the first peak and also as an independent peak between peaks II and III. Denatured molecular masses for the three isoenzyme groups were found to be between 18 and 90 kDa, and pI values were in the range of 4.2 to 6.0. beta-Xylosidase has an apparent molecular mass of 20, 30, and 90 kDa (peak I) and 18 and 45 kDa (independent peak), indicating a trimer and dimer structure, respectively, with pI values of 4.2 and 5.78, respectively. Three more minor xylanase groups were produced on corn stalk medium: a double peak in the acidic range (pI 6.25 to 6.65 and 6.65 to 7.12) and two minor peaks in the alkaline range (pI 8.09 to 8.29 and 9.28 to 9.48, respectively). The profile of xylanases separated by isoelectric focusing (zymogram) of culture filtrate from cells grown on corn stalk media was more complex than that of culture supernatants from cells grown on cellulose. The pH optima of the three major xylanase groups are in the range of pH 4 to 5.5.

Abstract: The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

Abstract: The 2 mu DNA plasmid of the yeast Saccharomyces cerevisiae does not confer any known selectable phenotype to the host cell carrying it. Selection of cells transformed with purified 2 mu DNA therefore cannot be achieved, and the intracellular presence of 2 mu can only be assessed by molecular analysis of the DNA complement. In addition, 2 mu alone does not replicate in bacterial hosts, thus rendering its amplification by conventional methods impossible. We have isolated a shuttle plasmid, pBH-2L, generated by in vivo site-specific recombination between the endogenous 2 mu DNA plasmid and pRL, a pBR322 derivative containing the yeast LEU2 gene and one 2 mu repeat sequence associated with the origin of replication. This new shuttle plasmid has the property, when transformed into yeast, of undergoing site-specific recombinational resolution between its two direct repeat sequences. This releases 2 mu plasmid and pRL as individual molecules. The latter can undergo progressive mitotic loss during growth in nonselective medium, ultimately leaving leucine auxotrophic transformants that contain only 2 mu DNA plasmid. This system can be utilized to introduce 2 mu DNA alone into cells lacking it, thereby providing a novel means to study the biology and the molecular genetics of the plasmid and its potential practical applications as a vector.

Abstract: The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

Abstract: The purpose of this work is to identify and quantitate in vivo 2 mu plasmid FLP-independent recombination in yeast, using a nonselective assay system for rapid detection of phenotypic expression of the recombination events. A tester plasmid was constructed such that in vivo recombination between 2 mu direct repeat sequences produces the resolution of the plasmid into two circular DNA molecules. This recombinational event is detected as a phenotypic shift from red to white colonies, due to the mitotic loss of the plasmid portion containing the yeast ADE8 gene in a recipient ade1 ade2 ade8 genetic background. In the absence of the 2 mu FLP recombinase and/or its target DNA sequence, recombination is not abolished but rather continues at a high frequency of about 17%. This suggests that the FLP-independent events are mediated by the chromosomally-encoded general homologous recombination system. We therefore conclude that the totality of 2 mu DNA recombination events occurring in FLP+ cells is the contribution of both FLP-mediated and FLP-independent events.

Abstract: We have studied the mechanism of DNA transformation of whole yeast cells in Saccharomyces cerevisiae with particular emphasis on the role of the cell wall complex in DNA uptake. Two new aspects of the process have been investigated in order to evaluate its specificity. Such aspects are: (i) effect of monovalent vs. divalent cations during incubation with the transforming DNA and (ii) timing of DNA adsorption and uptake. We found that the specificity for cation requirement is a strain-dependent characteristic influenced by the presence of transforming DNA in the cell suspension. This finding is supported by reports from several laboratories that some yeast strains show mutually exclusive transformability with monovalent vs. divalent cations. While irreversible adsorption of plasmid DNA molecules is induced by both heat shock and polyethylene-glycol (PEG), DNA uptake seems to occur only after the removal of PEG. In the course of this study we have developed a new, alternative method of whole cell DNA transformation with CaCl2 able to transform strains that do not respond to other methods.

Abstract: Both nonreciprocal and reciprocal mitotic recombination are enhanced by the recessive mutant spo11-1, which was previously shown to affect meiosis by decreasing recombination and increasing nondisjunction. The mitotic effects are not distributed equally in all chromosomal regions. The genotypes of mitotic recombinants in spo11-1/spo11-1 diploid cells provide further evidence that widely spaced chromosomal markers undergo coincident conversion in mitosis.