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Equal partitioning of the multi-copy 2-micron plasmid of the budding yeast Saccharomyces cerevisiae requires association of the plasmid Rep1 and Rep2 proteins with the plasmid STB partitioning locus. Determining how the Rep proteins contribute has been complicated by interactions between the components. Here, each Rep protein was expressed fused to the DNA-binding domain of the bacterial repressor protein LexA in yeast harboring a replication-competent plasmid that had LexA-binding sites but lacked STB. Plasmid transmission to daughter cells was increased only by Rep2 fusion expression. Neither Rep1 nor a functional RSC2 complex (a chromatin remodeler required for 2-micron plasmid partitioning) were needed for the improvement. Deletion analysis showed the carboxy-terminal 65 residues of Rep2 were required and sufficient for this Rep1-independent inheritance. Mutation of a conserved basic motif in this domain impaired Rep1-independent and Rep protein/STB-dependent plasmid partitioning. Our findings suggest Rep2, which requires Rep1 and the RSC2 complex for functional association with STB, directly participates in 2-micron plasmid partitioning by linking the plasmid to a host component that is efficiently partitioned during cell division. Further investigation is needed to reveal the host factor targeted by Rep2 that contributes to the survival of these plasmids in their budding yeast hosts.

The use of the budding yeast Saccharomyces cerevisiae as a model genetic organism has been facilitated by the availability of a wide range of yeast shuttle vectors, plasmids that can be propagated in Escherichia coli and also in yeast, where they are stably maintained at low- or high-copy number, depending on the plasmid system. Here we provide an introduction to the low-copy (ARS/CEN) and multi-copy (2-μm-based) plasmids, the marker genes commonly used for plasmid selection in yeast, methods for transforming yeast and monitoring plasmid inheritance, and tips for working with yeast transformants.

The use of the budding yeast Saccharomyces cerevisiae as a model genetic organism has been facilitated by the availability of a wide range of yeast shuttle vectors, plasmids that can be propagated in Escherichia coli and also in yeast, where they are stably maintained at low- or high-copy number, depending on the plasmid system. Here we provide an introduction to the low-copy (ARS/CEN) and multi-copy (2-μm-based) plasmids, the marker genes commonly used for plasmid selection in yeast, methods for transforming yeast and monitoring plasmid inheritance, and tips for working with yeast transformants.

The yeast 2-μm plasmid is a remarkable genetic parasite, managing efficient maintenance at high-copy number with minimal impact on the host. Equal partitioning of the plasmid upon host cell division requires plasmid proteins Rep1 and Rep2 and the plasmid STB locus. The Rep proteins and the plasmid-encoded Raf protein also regulate plasmid gene transcription. In this study, protein interaction assays, sequence analyses and mutational approaches were used to identify domains and residues in Rep2 and Raf required for association with Rep1 and Rep2 and to delineate the Rep2 DNA-binding domain. Rep2 and Raf displayed similarities in interactions with Rep1 and Rep2, in having Rep1 promote their STB association in vivo, and in stabilizing Rep protein levels. Rep2 mutants impaired for self-association were competent for transcriptional repression while those deficient for Rep1 association were not. Surprisingly, Rep2 mutants impaired for either Rep1 interaction or self-association were able to maintain efficient plasmid inheritance provided Raf was present and competent for Rep protein interaction. Our findings provide insight into the Rep protein complexes required for partitioning and transcriptional repression, and suggest that in addition to its transcriptional function, Raf stabilization of Rep partitioning proteins contributes to the remarkable persistence of the 2-μm plasmid.

The yeast 2-micron plasmid is an almost perfect selfish DNA. The entire coding capacity of the plasmid is dedicated to ensuring its own inheritance, with no benefit to its host. Despite high copy number, the plasmid confers no phenotype. It manages this feat by possessing mechanisms for plasmid copy-number control and for partitioning. The former increases plasmid numbers when they fall, but is repressed at high copy number, while the latter ensures 2-micron copies are equally partitioned during host cell division. Although the plasmid amplification mechanism is well established, the partitioning system and the means by which the 2-micron plasmid partitioning proteins, Rep1 and Rep2, regulate plasmid copy number remain incompletely understood. This review focuses on recent efforts to determine the nature of Rep protein complexes formed at the plasmid stability locus (STB) and at plasmid gene promoters, the identity of DNA sequence elements required for Rep protein association, and the mechanism by which the Rep proteins manage their dual roles of plasmid partitioning and plasmid gene repression.

Equal partitioning of the multi-copy yeast 2-micron plasmid requires association of plasmid proteins Rep1 and Rep2 with tandem repeats at the plasmid STB locus. To identify sequence elements required for these associations we generated synthetic versions of a 63-bp section of STB, encompassing one repeat. A single copy of this sequence was sufficient for Rep protein association in vivo, while two directly arrayed copies provided partitioning function to a plasmid lacking all other 2-micron sequences. Partitioning efficiency increased with increasing repeat number, reaching that conferred by the native STB repeat array. By altering sequences in synthetic repeats, we identified the TGCA component of a TGCATTTTT motif as critical for Rep protein recognition, with a second TGCA sequence in each repeat also contributing to association. Mutation of TGCATTTTT to TGTATTTT, as found in variant 2-micron STB repeats, also allowed Rep protein association, while mutation to TGCATTAAT impaired inheritance without abolishing Rep protein recognition, suggesting an alternate role for the T-tract. Our identification of sequence motifs required for Rep protein recognition provides the basis for understanding higher-order Rep protein arrangements at STB that enable the yeast 2-micron plasmid to be efficiently partitioned during host cell division.

WOS: 000417698200025, PubMed ID: 29232376, General Control Non-derepressible 5 (GCN5) and Alteration/Deficiency in Activation 2 and 3 proteins (ADA2 and ADA3, respectively) are subunits of the Histone AcetylTransferase (HAT) module of SAGA-and ATAC-type co-activators. We previously reported four new interacting partners of human ADA3 identified by screening a human fetal brain cDNA library using yeast two hybrid technology. One of these partners was Apoptosis-Antagonizing Transcription Factor (AATF), also known as Che-1, an RNA polymerase II-binding protein with a number of roles in different cellular processes including regulation of transcription, cell proliferation, cell cycle control, DNA damage responses and apoptosis. Che-1/AATF is a potential therapeutic target for cancer treatments. In this study, we aimed to identify whether besides ADA3, other components of the HAT modules of SAGA and ATAC complexes, human ADA2 and GCN5 also interact with Che-1/AATF. Co-immunoprecipitation and co-localization experiments were used to demonstrate association of AATF both with two ADA2 isoforms, ADA2A and ADA2B and with GCN5 proteins in human cells and yeast two-hybrid assays to delineate domains in the ADA2 and GCN5 proteins required for these interactions. These findings provide new insights into the pathways regulated by ADA-containing protein complexes., Turkish Scientific and Technological Research Assembly [112T429]; Hungarian NRDIO [GINOP-2.3.2-15-2016-00020], This work was supported by the Turkish Scientific and Technological Research Assembly (112T429) (Dr. Sevil Zencir), and Hungarian NRDIO-GINOP-2.3.2-15-2016-00020 (Prof Imre M. Boros). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Factor VIII gene, F8, mutations cause haemophilia A (HA), an X-linked recessive disorder. Expression in heterozygous females has been ascribed to skewed X-chromosome inactivation (XCI). To investigate the cause of HA in three heterozygous females within an Atlantic Canadian kindred, the proband (severely affected girl, FVIII activity: 2%) and 17 relatives across three generations were studied. F8 genotype, FVIII activity, XCI ratio (XCIR) (paternal active X: maternal active X), karyotype, submegabase resolution tiling set array competitive genome hybridization (competitive genomic hybridization (SMRT)), and microsatellite analyses were utilized. A positive linear relationship between FVIII activity and percentage-activated normal X-chromosome was found in HA heterozygous females (R(2)=0.87). All affected, but no unaffected females, had an XCIR skewed toward activation of the mutant X-chromosome (proband 92:8, SD 2). Unexpectedly, high numbers of females have dramatically skewed XCIRs (>80:20 or

The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locus. The periodicity of cohesin association and dissociation is nearly identical for the plasmid and the chromosomes. The timely disassembly of cohesin is a prerequisite for plasmid segregation. Cohesin-mediated pairing and unpairing likely provides a counting mechanism for evenly partitioning plasmids either in association with or independently of the chromosomes.

The majority of chromosomes in Oreochromis niloticus, as with most fish karyotyped to date, cannot be individually identified owing to their small size. As a first step in establishing a physical map for this important aquaculture species of tilapia we have analyzed the location of the vertebrate telomeric repeat sequence, (TTAGGG)n, in O. niloticus. Southern blot hybridization analysis and a Bal31 sensitivity assay confirm that the vertebrate telomeric repeat is indeed present at O. niloticus chromosomal ends with repeat tracts extending for 4-10 kb on chromosomal ends in erythrocytes. Fluorescent in situ hybridization revealed that (TTAGGG)n is found not only at telomeres, but also at two interstitial loci on chromosome 1. These data support the hypothesis that chromosome 1, which is significantly larger than all the other chromosomes in the karyotype, was produced by the fusion of three chromosomes and explain the overall reduction of chromosomal number from the ancestral teleost karyotype of 2n=48 to 2n=44 observed in tilapia.

Both of the Saccharomyces cerevisiae 2μm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2μm-based stability vector with rep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2μm circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.

The efficient partitioning of the 2-μm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host encoded factors are likely to contribute to plasmid segregation. Direct observation of a 2-μm–derived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes. Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-μm plasmid and the chromosome are quite similar during the yeast cell cycle. Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus. Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle. Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules. In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem. Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates. They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation.

Niemann-Pick type D (NPD) disease is a severe degenerative disorder of the nervous system characterized by the accumulation of tissue cholesterol and sphingomyelin. Because of a founder effect, it is unusually common in southwestern Nova Scotia, Canada. We have confirmed that almost all patients from 20 affected sibships descended on both sides from a small group of Acadians who settled in this region in about the year 1767. Previously using classic linkage analysis of this large kindred, we defined the critical gene region to a 13-cM chromosome segment between D18S869 and D18S66. Seven ESTs have been positioned within this interval. Carstea et al. (Niemann Pick C disease gene: homology to mediators of cholesterol homeostasis. Science 1997: 277: 232-235) recently demonstrated that one of these ESTs is the Niemann-Pick type C (NPCI) gene, the gene disrupted in most patients with NPC disease, and we have shown that a G3097-->T mutation in the NPC1 gene is also responsible for NPD. Here we report the development of five new polymorphic microsatellite markers and the testing for complete linkage disequilibrium in our single large NPD kindred that allowed us to reduce the NPD critical region to a 1-cM (1.3-1.6 Mb) interval between D18S1398 and D18S1108. In contrast, Carstea et al., using classic linkage analysis, required more than 18 unrelated NPC families to reduce the NPC1 critical region to a 5-cM interval. Our work supports the finding that NPD is an allelic variant of NPC1, and illustrates the power of large kindreds, which are common in Atlantic Canada and other relatively isolated areas, for gene mapping and identification.

The 2-micron plasmid of the budding yeast Saccharomyces cerevisiae encodes copy-number amplification and partitioning systems that enable the plasmid to persist despite conferring no advantage to its host. Plasmid partitioning requires interaction of the plasmid Rep1 and Rep2 proteins with each other and with the plasmid-partitioning locus STB. Here we demonstrate that Rep1 stability is reduced in the absence of Rep2, and that both Rep proteins are sumoylated. Lysine-to-arginine substitutions in Rep1 and Rep2 that inhibited their sumoylation perturbed plasmid inheritance without affecting Rep protein stability or two-hybrid interaction between Rep1 and Rep2. One-hybrid and chromatin immunoprecipitation assays revealed that Rep1 was required for efficient retention of Rep2 at STB and that sumoylation-deficient mutants of Rep1 and Rep2 were impaired for association with STB. The normal co-localization of both Rep proteins with the punctate nuclear plasmid foci was also lost when Rep1 was sumoylation-deficient. The correlation of Rep protein sumoylation status with plasmid-partitioning locus association suggests a theme common to eukaryotic chromosome segregation proteins, sumoylated forms of which are found enriched at centromeres, and between the yeast 2-micron plasmid and viral episomes that depend on sumoylation of their maintenance proteins for persistence in their hosts.

WOS: 000316038500003, PubMed ID: 23395680, Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein-protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human glutaminase interacting protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans. (C) 2013 Elsevier Inc. All rights reserved., United States Department of AgricultureUnited States Department of Agriculture (USDA) [2003-35302-12930, 2011-65503-20030]; National Science FoundationNational Science Foundation (NSF) [IOS-0628064]; National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [DK082397]; NSERCNatural Sciences and Engineering Research Council of Canada [155268]; [TUBITAK 108T945], This research was financially supported by United States Department of Agriculture PECASE award 2003-35302-12930, and AFRI award 2011-65503-20030, National Science Foundation Grant IOS-0628064, and National Institutes of Health Grant DK082397 to Smita Mohanty, NSERC Grant 155268 to Melanie Dobson, and the Grant TUBITAK 108T945 to Zeki Topcu.

ADA (alteration/deficiency in activation) 3 is a conserved component of several transcriptional adaptor and HAT (histone acetyltransferase) complexes that regulate RNA polymerase IImediated gene expression. Within the HAT complexes ADA3 is associated with ADA2 and the HAT GCN5 (general control non-repressed 5). ADA3 plays roles in diverse cellular processes and also in malignancies by modulating GCN5 catalytic activity and/or by interactions with other regulators. To gain a better understanding of ADA3 function, we used a yeast two-hybrid approach to screen a human fetal cDNA library for proteins that interacted with hADA3 (human ADA3). We identified three novel hADA3-interacting partners, a transcriptional regulator, AATF (apoptosis-antagonizing transcription factor), and regulatory subunits of the PP1 (protein phosphatase 1) and PP2A (protein phosphatase 2A) [PPP1R7 (PP1 regulatory subunit 7) and PPP2R5D (PP2A 56 kDa regulatory subunit δ isoform) respectively]. Analysis of truncated versions of hADA3 indicated that the C-terminal ADA2-interacting domain was not required for these interactions. Fluorescent microscopy analysis and co-immunoprecipitation provided support for the co-localization and interaction of hADA3 with these proteins in human cells. Expression of the interacting proteins altered expression of an hADA3-regulated reporter gene, suggesting functional consequences for the interactions. The detected interactions of hADA3 might extend the spectrum of mechanisms by which ADA3 can contribute to the regulation of gene expression and shed light on processes mediated by these newly identified ADA3 partners. © 2013 Biochemical Society.

WOS: 000294317300024, PubMed ID: 21787750, The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-I, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, beta-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kit 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP. (C) 2011 Elsevier Inc. All rights reserved., USDA PECASE [2003-35302-12930]; NSFNational Science Foundation (NSF) [IBN-0628064]; USDAUnited States Department of Agriculture (USDA) [2011-65503-20030]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [DK082397]; NSERCNatural Sciences and Engineering Research Council of Canada [155268]; TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108T945], This research was financially supported by USDA PECASE Presidential Early Career Award for Scientists and Engineers award 2003-35302-12930, NSF Grant IBN-0628064, USDA Grant 2011-65503-20030 and NIH Grant DK082397 to Smita Mohanty, NSERC Grant 155268 to Melanie Dobson, and the Grant TUBITAK 108T945 to Zeki Topcu. We thank Dr. David L. Zoetewey for critical reading of this manuscript.

Total RNA was extracted daily from the beta-lactam antibiotic producing fungus A. chrysogenum strain CO728 during a 7 day cephalosporin C fermentation. IPNS mRNA species, with a size of about 1.5 kb, were detected by Northern blotting at high levels between days 2 and 4. The rapid appearance of IPNS mRNA in mycelial extracts up to day 2 suggests that IPNS is regulated at the transcriptional level. Primer extension and S1 endonuclease mapping studies indicate the existence of two major and at least two minor transcription initiation start sites. There was no change in the relative levels of the four transcripts during the period they could be detected. A region upstream of the IPNS structural gene (pcbC) has been sequenced and the transcription initiation sites appear as major and minor pairs on either side of one of the pyrimidine-rich blocks that punctuate the promoter sequence.

The somatosensory (SI) cortex of mice displays a patterned, nonuniform distribution of neurons in layer IV called the 'barrelfield' (ref. 1). Thalamocortical afferents (TCAs) that terminate in layer IV are segregated such that each barrel, a readily visible cylindrical array of neurons surrounding a cell-sparse center, represents a distinct receptive field. TCA arbors are confined to the barrel hollow and synapse on barrel-wall neurons whose dendrites are oriented toward the center of the barrel. Mice homozygous for the barrelless (brl) mutation, which occurred spontaneously in ICR stock at Université de Lausanne (Switzerland), fail to develop this patterned distribution of neurons, but still display normal topological organization of the SI cortex. Despite the absence of barrels and the overlapping zones of TCA arborization, the size of individual whisker representations, as judged by 2-deoxyglucose uptake, is similar to that of wild-type mice. We identified adenylyl cyclase type I (Adcy1) as the gene disrupted in brl mutant mice by fine mapping of proximal chromosome 11, enzyme assay, mutation analysis and examination of mice homozygous for a targeted disruption of Adcy1. These results provide the first evidence for involvement of cAMP signalling pathways in pattern formation of the brain.

Summary Niemann-Pick type D (NPD) disease is a progressive neurodegenerative disorder characterized by the accumulation of tissue cholesterol and sphingomyelin. This disorder is relatively common in southwestern Nova Scotia, because of a founder effect. Our previous studies, using classic linkage analysis of this large extended kindred, defined the critical gene region to a 13-cM chromosome segment between D18S40 and D18S66. A recently isolated gene from this region, NPC1, is mutated in the majority of patients with Niemann-Pick type C disease. We have identified a point mutation within this gene (G 3097 →T; Gly 992 →Trp) that shows complete linkage disequilibrium with NPD, confirming that NPD is an allelic variant of NPC1.

Previous reports concerning the location of D18S44 with respect to the centromere have been ambiguous. Also, it has not been possible, based on formerly reported markers, to show that contigs WC18.0 and WC18.1 overlap. However, the data presented here definitively show, using FISH technology, that D18S44 (located on WC18.0) maps to proximal 18q. Furthermore, inter-Alu fingerprinting shows a clear overlap between WC18.0 and WC18.1, thereby establishing a complete contig between D18S44 and markers from WC18.1.Key words: FISH mapping, inter-Alu, fingerprinting, human chromosome 18.

The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locus. The periodicity of cohesin association and dissociation is nearly identical for the plasmid and the chromosomes. The timely disassembly of cohesin is a prerequisite for plasmid segregation. Cohesin-mediated pairing and unpairing likely provides a counting mechanism for evenly partitioning plasmids either in association with or independently of the chromosomes.

Early retrotransposons (ETn) are murine transposable elements, bearing some structural similarity to integrated proviruses, and can be insertional mutagens. We have recently identified the causative mutation of the barrelless (Adcy1brl) phenotype as an integration of a 5.7-kb ETn in an intron of the adenylyl cyclase type I (Adcy1) gene. In the present study, Northern blot analysis shows that the ETn insertion results in loss of the normal Adcy1 transcript, a finding consistent with the loss-of-function Adcy1brl mutation, and generation of shorter transcripts. These aberrant transcripts are the products of abnormal RNA splicing and termination owing to the inserted sequence, and transcription initiation within the 3' long terminal repeat (LTR) of the ETn. The DNA sequences of the LTRs were compared in phylogenetic analyses with LTRs from 22 other ETn-related sequences. Three distinct families of ETn sequences can be identified on the basis of their LTRs. The ETn found in Adcy1brl is a member of a family that includes all classified ETn elements known to have recently transposed. Further, of the four known solitary (solo) LTRs, we have identified two that show evidence of recombination between LTRs from different ETn families.

We report the cloning and characterization of a long interspersed nucleotide element (LINE) from a cichlid fish, Oreochromis niloticus, and show the distribution of this element, called CiLINE2 for cichlid LINE2, in the chromosomes of this species. The identification of an open reading frame in CiLINE2 with amino acid sequence similarity to reverse transcriptases encoded by LINE-like elements in Caenorhabditis elegans, Platemys spixii, Schistosoma mansoni, Gallus gallus (CRI), Drosophila melanogaster (I factor), and Homo sapiens (LINE2), as well as the structure of the element, suggest it is a member of this family of non-long terminal repeat-containing retrotransposons. Search of a DNA sequence database identified sequences similar to CiLINE2 in four other fish species (Haplotaxodon microlepis, Oreochromis mossambicus, Pseudotropheus zebra, and Fugu rubripes). Southern blot hybridization experiments revealed the presence of sequences similar to CiLINE2 in all Tilapiini species analyzed from the genera Oreochromis, Tilapia, and Sarotherodon, and gave an estimated copy number of about 5500 for the haploid genome of O. niloticus. Fluorescent in situ hybridization showed that CiLINE2 sequences were organized in small clusters dispersed over all chromosomes of O. niloticus, with a higher concentration near chromosome ends. Furthermore, the long arm of chromosome 1 was strikingly enriched with this sequence. The distribution of LINE2-related elements might underlie the difference in chromosome banding patterns observed between cold-blooded vertebrates and mammals.

SummaryNiemann-Pick type II disease is an autosomal recessive disorder characterized by a defect in intracellular trafficking of sterols. We have determined the intron/exon boundaries of eight exons from the conserved 3′ portion of NPC1, the gene associated with most cases of the disease. SSCP analyses were designed for these exons and were used to identify the majority of mutations in 13 apparently unrelated families. Thirteen mutations were found, accounting for 19 of the 26 alleles. These mutations included eight different missense mutations (including one reported by Greer et al. [1998]), one 4-bp and two 2-bp deletions that generate premature stop codons, and two intronic mutations that are predicted to alter splicing. Two of the missense mutations were present in predicted transmembrane (TM) domains. Clustering of these and other reported NPC1 mutations in the carboxy-terminal third of the protein indicates that screening of these exons, by means of the SSCP analyses reported here, will detect most mutations. The carboxy-terminal half of the Npc1 protein shares amino acid similarity with the TM domains of the morphogen receptor Patched, with the largest stretch of unrelated sequence lying between two putative TM spans. Alignment of this portion of the human Npc1 protein sequence with Npc1-related sequences from mouse, yeast, nematode, and a plant, Arabidopsis, revealed conserved cysteine residues that may coordinate the structure of this domain. That 7 of a total of 13 NPC1 missense mutations are concentrated in this single Npc1-specific domain suggests that integrity of this region is particularly critical for normal functioning of the protein.

The human chromosome 20p telomere has been cloned on a yeast artificial chromosome (YAC). The telomere-associated DNA contains an interstitial tract of (TTAGGG) n telomeric repeats 60 kb in from the chromosome end. Frequent truncation of the YAC clone was observed due to resolution of the internal telomeric array into a telomere. The 20p internal telomeric repeat tract is flanked on its centromeric side by telomere-associated repeated sequences that have previously been found adjacent to terminal telomeric repeat arrays. The pseudotelomere structure of the 20p subtelomeric region is similar to the structure of some yeast subtelomeric regions where these sequences act as substrates for recombinational repair of chromosome ends that have lost their terminal telomeric repeat arrays. Sequences flanking the telomeric end of the internal (TTAGGG) n repeat array on 20p are found adjacent to three other subtelomeric (TTAGGG) n tracts on 4q, 18p, and an unknown chromosome end, respectively. These shared sequences provide evidence of exchange between nonhomologous chromosomes in humans.

The cloning and propagation of large fragments of DNA on yeast artificial chromosomes (YACs) has become a routine and valuable technique in genome analysis. Unfortunately, many YAC clones have been found to undergo rearrangements or deletions during the cloning process. The frequency of transformation-associated alterations and mitotic instability can be reduced in a homologous recombination-deficient yeast host strain such as a rad52 mutant. RAD52 is one member of an epistatic group of genes required for the recombinational repair of double-strand breaks in DNA. rad52 mutants grow more slowly and transform less efficiently than RAD + strains and are therefore not ideal hosts for YAC library construction. We have investigated the ability of both null and temperature-sensitive alleles of RAD54 , another member of the RAD52 epistasis group, to prevent rearrangements of human YAC clones containing tandemly repeated DNA sequences. Our results show that the temperature-sensitive rad54-3 allele blocks mitotic recombination between tandemly repeated DYZ3 satellite sequences and significantly stabilizes a human DYZ5 satellite-containing YAC clone. Yeast carrying the rad54-3 mutation can undergo meiosis, have growth and transformation rates comparable with RAD + strains, and therefore represent improved YAC cloning hosts.

We have used polyclonal antibodies against fusion proteins produced from cDNA fragments of a meiotic chromosome core protein, Cor1, and a protein present only in the synapsed portions of the cores, Syn1, to detect the occurrence and the locations of these proteins in rodent meiotic prophase chromosomes. The 234 amino acid Cor1 protein is present in early unpaired cores, in the lateral domains of the synaptonemal complex and in the chromosome cores when they separate at diplotene. A novel observation showed the presence of Cor1 axial to the metaphase I chromosomes and substantial amounts of Cor1 in association with pairs of sister centromeres. The centromere-associated Cor1 protein becomes dissociated from the centromeres at anaphase II and it is not found in mitotic metaphase centromeres. The extended presence of Cor1 suggests that it may have a role in chromosome disjunction by fastening chiasmata at metaphase I and by joining sister kinetochores, which ensures co-segregation at anaphase I. Two-colour immunofluorescence of Cor1 and Syn1 demonstrates that synapsis between homologous cores is initiated at few sites but advances rapidly relative to the establishment of new initiation sites. If the rapid advance of synapsis deters additional initiation sites between pairs of homologues, it may provide a mechanism for positive recombination interference. Immunogold epitope mapping of antibodies to four Syn1 fusion proteins places the amino terminus of Syn1 towards the centre of the synaptonemal complex while the carboxyl terminus extends well into the lateral domain of the synaptonemal complex. The Syn1 fusion proteins have a non-specific DNA binding capacity. Immunogold labelling of Cor1 antigens indicates that the lateral domain of the synaptonemal complex is about twice as wide as the apparent width of lateral elements when stained with electron-dense metal ions. Electron microscopy of shadow-cast surface-spread SCs confirms the greater width of the lateral domain. The implication of these dimensions is that the proteins that comprise the synaptic domain overlap with the protein constituents of the lateral domains of the synaptonemal complex more than was apparent from earlier observations. This arrangement suggests that direct interactions might be expected between some of the synaptonemal complex proteins.

A secreted invertase was purified 23-fold by ultrafiltration, ion-exchange, and gel filtration chromatography from the culture supernatant of 18 h sucrose-grown cultures of Aspergillus niger. The purified enzyme hydrolysed sucrose and raffinose but there was no detectable hydrolysis of inulin, melezitose or PNPG. Invertase activity was optimal at pH 5.5 and 50 degrees C. The molecular mass of reduced invertase was 115 kDa, as determined by SDS gel electrophoresis. The native molecular weight of between 225 kDa and 250 kDa, estimated by electrophoresis under non-denaturing conditions, suggests that the protein is a dimer of identical subunits. The suc1 gene encoding this protein was completely-sequenced. The translated sequence yields a protein of 566 amino acids with a calculated molecular mass of 61 kDa, suggesting that carbohydrates represent about 50% of the mass of the protein.

As an alternative to the production and use of monoclonal antisynaptonemal complex (SC) antibodies to isolate SC genes, we have explored the use of polyclonal anti-SC antibodies to identify SC genes from a cDNA expression library. The method proved relatively simple, reliable, and fast and has yielded two SC genes. A homologue of one of these genes from a different species has previously been isolated in another laboratory. © 1993 Wiley-Liss, Inc.

The aim of this chapter is to introduce the procedures currently used to isolate and manipulate yeast genes. We restrict our discussion to those procedures that are, at least in part, peculiar to the yeast Saccharomyces cerevisiae. We do not deal with procedures that are common to all other organisms, such as cDNA bank construction, hybrid-selected or arrested translation systems, nucleic acid probe strategies, or radioimmune detection strategies, as these have been covered in a variety of general texts (e.g., Maniatis et al., 1982; Glover, 1985).

[2 μm(+) and [2μm°] yeast were transformed to stable leucine prototrophy with the hybrid yeast - E. coli plasmid, pJDB219. This plasmid contains the entire sequence of the endogenous 2 μm yeast DNA plasmid in addition to the yeast nuclear LEU2 (+) gene and the Co1E1 derivative, pMB9. In the [2 μm(+)] transformants, a new wholly yeast LEU2 (+) plasmid, pYX, was generated, probably by a recombination event between pJDB219 and 2 μm DNA. The plamid, pYX, in the absence of 2 μm DNA, was found to exist in equimolar amounts of two forms, A and B, which probably arise by intramolecular recombination across the inverted repeat sequences of the 2 μm DNA portion of the plasmid. pJDB219 was found to require the presence of 2 μm DNA to undergo this intramolecular recombination. The results suggest that 2, μm DNA and pYX code for a gene product required in this recombination event which pJDB219 cannot produce.

Two plasmids containing Saccharomyces cerevisiae 2 µm DNA sequences and the S. cerevisiae LEU2 gene have been found to display incompatibility with 2 µm DNA; in the presence of the LEU2 plasmids, 2 µm DNA can be lost. The LEU2 plasmids can be lost spontaneously after (and before) 2 µm DNA loss has occurred, so that strains completely lacking 2 µm DNA sequences can be obtained routinely.

The 5' control region of the yeast phosphoglycerate kinase gene (PGK) was fused to the coding sequence of a human interferon-alpha. This PGK-interferon fusion was then introduced into yeast on a high copy number 2mu-based plasmid vector. Strains containing this plasmid produced a PGK-interferon-alpha fusion protein as 1-2% of cell protein and the expression of interferon activity was regulated by the availability of a fermentable carbon source. The system is capable of making as much as 15 mg of human interferon-alpha per litre of batch culture.

The Ty transposable elements of Saccharomyces cerevisiae form a heterogeneous family within which two broad structural classes (I and II) exist. The two classes differ by two large substitutions and many restriction sites. We show that, like class I elements a class II element, Tyl-17, also appears to contain at least two major protein coding regions, designated TYA and TYB, and the organisational relationship of these regions has been conserved. The TYA genes of both classes encode proteins, designated p1 proteins, with an approximate molecular weight of 50 Kd and, despite considerable variation between the TYA regions at the DNA level, the structures of these proteins are remarkably similar. These observations strongly suggest that the p1 proteins of Ty elements are functionally significant and that they have been subject to selection.

The 'promoter' fragment from the yeast phosphoglycerate kinase (PGK) gene has been used to direct the expression of human interferon-alpha-2 (IFN alpha 2) on a high-copy-number plasmid in yeast. The yields of IFN alpha 2 are only 1-3% of yeast total protein, whereas the maximum yield of PGK produced by the PGK gene on a high-copy-number plasmid is at least 50%. IFN alpha 2 is turned over more rapidly than PGK but in addition a major reason for the relatively low level of IFN alpha 2 is that IFN-specific RNA levels are much lower. This does not reflect differences in plasmid copy number or integrity, or differences in the 5' and 3' untranslated regions of the transcripts or DNA flanking regions. It appears that the presence of heterologous coding sequences, or the absence of specific yeast sequences causes a reduction in heterologous RNA levels in yeast.

The plant pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed using two positive selection systems, one based on the Escherichia coli hygromycin B phosphotransferase gene (hph) and the other on the Neurospora crassa β-tubulin gene bml which encodes resistance to the methyl benzimidazole carbamate fungicides. Both selection systems gave a transformation frequency of 1–20 transformants μg−1 DNA. The vector DNA was integrated into the genome and the number and sites of integration varied among the transformants. The hph transformants were mitotically stable and the transformed gene was transmitted through spores. In contrast the bml transformants were less stable.

The LEU2 regions present on yeast plasmid vectors come from two sources, a series of strains derived from S288c and strain M 127. The LEU 2 region from the S288c series contains a Tyl-17 element with its associated δ sequences and a small repetitive RNA gene while the LEU 2 region from M 127 which is present on pJDB248, lacks the Tyl-17 element, but carries a δ sequence and a small RNA gene. The various LEU 2 plasmids currently in use vary with respect to these sequences depending on which restriction fragment from the region is present on the recombinant molecule. In addition, strain M127 contains three LEU 2 homologous sequences that are represented by different Eco RI fragments and which segregate independently at meiosis. Therefore, there are at least four forms of the centromere-distal Eco RI fragment of the LEU 2 locus in the Saccharomyces cerevisiae gene pool; these are 7.1 kb, 1.9 kb, 1.48 kb and 1.15 kb long.

The [psi] factor is an extrachromosomally inherited genetic determinant of the yeast Saccharomyces cerevisiae for which no autonomous physical determinant has yet been identified. Using both physical and genetical techniques we demonstrate that the [psi] determinant does not reside on other previously described yeast extrachromosomally inherited determinants; namely mitochondrial DNA, L double-stranded RNA, M double-stranded RNA, and [2 μm] DNA. Stable [psi+] strains, lacking one or other of these determinants were constructed. It is concluded that the [psi] factor may represent a new yeast plasmid.

We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication Enhancer (RE) domain, has no inherent replication competence but is essential for ensuring maximum function of the RS domain. The nucleotide sequence of these domains reveals several conserved sequences one of which is strikingly similar to the yeast ARS consensus sequence.

The effect of the yeast 2 micron circle encoded REP1 and REP2 gene products on plasmid partitioning and copy number control was analyzed by removing the open reading frames from their normal sequence context and transcriptional control regions and directing their expression using heterologous promoters in [cir0] host strains. Both the REP1 and REP2 gene products are directly required at appropriate levels of expression to reconstitute the 2 microns circle partitioning system in conjunction with REP3 and the origin of replication. The level of expression of REP2 appears to be critical to re-establishing proper partitioning and may also play a role in monitoring and thereby regulating the plasmid copy number. Constitutive expression of the REP1 and REP2 open reading frames using heterologous expression signals can be used to reconstruct efficient plasmid partitioning even in the absence of FLP-mediated plasmid amplification and a functional D open reading frame.

The position of the yeast phosphoglycerate kinase (PGK) gene has been mapped on a 2.95kb Hind III fragment. We have determined the nucleotide sequence of the 5' flanking region and compared this sequence with those from 16 other yeast genes. PGK, like all other yeast genes has an adenine residue at position -3. It has two possible TATA boxes at positions -114 and -152 and a CAAT box at -129. In addition we have defined a structure at position -63 to -39 that is common to all yeast genes that encode an abundant RNA. This structure is a CT-rich block followed, about 10 nucleotides later, by the sequence CAAG.

Expression of heterologous genes from the PGK promoter on high copy number plasmids in yeast is relatively poor compared to the intact PGK gene because of low steady-state RNA levels. In this paper we show that low levels of heterologous RNA are not due to instability of mRNA but result from inefficient transcription due to a defect in RNA synthesis. A comparison of RNA levels from homologous and heterologous transcription units allowed the identification of a positive activator for transcription within the PGK coding region which is required for efficient expression of the PGK gene. Deletion of this region, the "downstream activator sequence", causes a six to ten fold reduction in transcriptional efficiency from the PGK 5' noncoding region.

Two new Ty determined proteins have been identified by placing a Ty transcriptional unit under the control of a high efficiency yeast expression vector. One of these proteins is the product of a post-translational processing event and it binds nucleic acids. A previously identified protein, pl (Tyl-15), has also been shown to bind nucleic acids and to be modified by phosphorylation.

Eukaryotic transposons such as the Ty element of yeast or the copia-like sequences of Drosophila show structural and functional similarities to both prokaryotic transposons and retroviral proviruses, but the prokaryotic transposons and retroviral proviruses use markedly different expression strategies which yield products having entirely different functions. To determine the phylogenetic relationship between eukaryotic transposons, prokaryotic transposons and retroviruses, we have sought to identify and characterize the proteins encoded by the yeast Ty element and to describe the strategies used to express these proteins. We show here that the yeast transposon produces a fusion protein by a specific frameshifting event that fuses two out-of-phase open reading frames (ORFs). The process is remarkably similar to that used by retroviruses such as Rous sarcoma virus (RSV) to produce Pr180gag-pol.

The structure of yeast phosphoglycerate kinase has been determined with data obtained from amino acid sequence, nucleotide sequence, and X-ray crystallographic studies. The substrate binding sites, as deduced from electron density maps, are compatible with known substrate specificity and the stereochemical requirements for the enzymic reaction. A carboxyl-imidazole interaction appears to be involved in controlling the transition between the open and closed forms of the enzyme.

We have constructed a high-efficiency expression vector to direct the synthesis of heterologous polypeptides in yeast. The vector is termed a sandwich expression vector as the heterologous gene is inserted between the 5' and 3' control regions of the efficiently expressed yeast PGK gene. We have used this vector to direct the expression of three derivatives of the calf chymosin cDNA gene; preprochymosin, prochymosin and chymosin. Prochymosin is synthesised to at least 5% of total yeast-cell protein and furthermore, it can be readily activated to produce an enzyme which has milk-clotting activity.