DNA polymerases α and δ play essential roles in the replication of chromosomal DNA in eukaryotic cells. DNA polymerase α (Pol α)-primase is required to prime synthesis of the leading strand and each Okazaki fragment on the lagging strand, whereas DNA polymerase δ (Pol δ) is required for the elongation stages of replication, a function it appears capable of performing on both leading and lagging strands, at least in the absence of DNA polymerase ε (Pol ε).in vivo and in vitro. Pol1 interacts with the C-terminal domain of Cdc27, at a site distinct from the previously identified binding sites for Cdc1 and PCNA. Comparative protein sequence analysis identifies a protein sequence motif, called the DNA polymerase interaction motif (DPIM), in Cdc27 orthologues from a wide variety of eukaryotic species, including mammals. Mutational analysis shows that the DPIM in fission yeast Cdc27 is not required for effective DNA replication, repair or checkpoint function.Here it is shown that the catalytic subunit of Pol α, Pol1, interacts with Cdc27, one of three non-catalytic subunits of fission yeast Pol δ, both The absence of any detectable phenotypic consequences arising from mutation of the DPIM suggests that despite its evolutionary conservation, the interaction between the two polymerases mediated by this motif is a non-essential one. Three conserved DNA polymerase enzymes whose activities are essential for complete chromosomal DNA replication have been identified through biochemical studies in mammalian systems  and combSchizosaccharomyces pombe, the Pol α-primase and Pol δ complexes are both heterotetrameric in structure [Each of the three essential polymerases is a multi-subunit entity, comprising a large catalytic subunit and a number of smaller subunits that are presumed to play either regulatory or structural roles . Little tructure . The cattructure . Both cotructure ,10, althtructure ,12, and tructure . Ortholotructure ,15.Perhaps surprisingly, there have been few reports of physical interactions between the various polymerase enzymes believed to be present at the eukaryotic replication fork. One exception to this comes from large-scale functional analysis of the budding yeast proteome where an interaction was uncovered between the catalytic subunit of the Pol α-primase complex, Pol1, and the C-subunit of the Pol δ complex in budding yeast, Pol32 . Interacin vitro, using purified recombinant proteins and peptides. Interaction is also seen between the human Cdc27 and Pol1 homologues, p66 and p180. The binding site for Pol1 maps to the extended C-terminal domain of the Cdc27 protein and requires the presence of a short protein sequence motif, which we have designated the DPIM, for DNA polymerase interaction motif. This short sequence is conserved in Cdc27 orthologues from a wide variety of eukaryotic species. Despite this evolutionary conservation, mutational inactivation of the Pol1 binding motif does not affect Cdc27 function in vivo. The implications of these results are discussed.In this paper, it is shown that the orthologues of the budding yeast Pol1 and Pol32 proteins in fission yeast, Pol1 and Cdc27 respectively, also interact in the two-hybrid system. It is also shown that these proteins are capable of interacting directly with one another, To test for interaction between fission yeast Pol1 and Cdc27, the two-hybrid system was used. Full-length Cdc27 fused to the activation domain (AD) of the yeast Gal4 protein was tested for its ability to interact with amino acids 278–527 of fission yeast Pol1 fused to the DNA binding domain of the bacterial transcription factor LexA . Amino acids 278–527 correspond to the smallest region of budding yeast Pol1 shown to interact with Pol32 . ReporteCip1-like PCNA binding sequence, the PIP box. To map the Pol1 binding site, a series of thirteen truncated Cdc27 proteins fused to the Gal4 AD were tested against LexA-Pol1 in the two-hybrid system. The results of this analysis are shown in Figure Previously, minimal Cdc1 and PCNA binding regions on Cdc27 have been mapped ,12,18. Cin vitro. Purified GST-Cdc27-273-352 fusion protein [In order to test whether the interaction between Pol1 and Cdc27 was a direct one, purified recombinant Cdc27 and Pol1 proteins were assayed for their ability to interact  protein  was testXenopus [DNA polymerase interaction motif (or DPIM) with the consensus sequence D(D/E)-G -- (V/I)(T/S). The sequences flanking the DPIM are generally highly charged in character and some sequence conservation (particularly of charged amino acids) is apparent in these regions. Mutagenesis and peptide binding studies (described below) suggest that, in addition to the DPIM, some of these sequences may also play a role in binding Pol1 (see Discussion).Previously, Cdc27 homologues from fission yeast ,18, buddXenopus  have beeXenopus  and ψ-BLXenopus ,24 were To examine whether the Pol1-Cdc27 (Pol1-Pol32) interaction observed in the yeasts was also conserved in higher eukaryotes, the catalytic subunit of human Pol α and the human Cdc27 orthologue p66/KIA00039 were assayed for interaction using the two-hybrid system. Amino acids 291–540 of the human Pol α catalytic subunit, corresponding to the minimum Cdc27 binding region (amino acids 278–527) in fission yeast Pol1, were expressed as a LexA fusion alongside Gal4 activation domain fusions of either the entire C-terminal domain of human p66 (amino acids 253–466) or the C-terminal 111 amino acids only (356–466). Interactions were tested by β-galactosidase assay. Both p66 constructs bound to Pol α Table , indicatin vivo by Cdc27. Similar reductions were seen with the Cdc27-P4 and Cdc27-P6 mutants, where the mutated residues are located N-terminal and C-terminal to the DPIM respectively. The conserved amino acids of the DPIM are therefore essential for Pol1 binding by Cdc27, although sequences flanking the conserved motif also play a role. Only three of the eight mutant proteins  were able to interact with LexA-Pol1 (278–527), though the strength of the interaction was reduced to 30–40% of the wild-type value for Cdc27-P3 and Cdc27-P5 , and to ~ 10% of wild-type for Cdc27-P7 . Immunoblotting showed that all the mutant proteins were present in yeast protein extracts at the same level as the wild-type Gal4-Cdc27 fusion protein (data not shown).Next, the importance for Pol1 binding of the conserved amino acids in the DPIM was examined. Eight mutant Cdc27 proteins  were constructed and tested as LexA fusions in the two-hybrid system . Removal of fifty amino acids from the N-terminus, or twenty amino acids from the C-terminus, of the Pol1 (278–527) protein was found to abolish the interaction with Cdc27 altogether Table .pol1 allele, pol1-ts13, had been reported [pol1+ by deletion of 9 bp from the ORF, resulting in loss of three amino acids  from within the minimal Cdc27 binding domain defined above. In this study, the ability of a Pol1-TS13(278–527) bait to bind to Cdc27 was tested using the two-hybrid system. No interaction could be detected at a range of growth temperatures . Indeed, no suppression was observed of any of three pol1 alleles that were analysed in this way, the others being pol1-1 [pol1-H4 [Previously, the isolation of a temperature-sensitive mutant reported . Sequenc3X-Cdc27  and trang pol1-1  and pol1in vivo role of the DPIM in Cdc27, four of the eight DPIM mutant alleles (cdc27-P1 through cdc27-P4) were cloned into plasmid pREP3X, 3' to the repressible nmt1 promoter [cdc27+/cdc27::his7+ diploid strain. Transformant colonies were then induced to sporulate and the spores plated on media containing thiamine, to repress the nmt1 promoter. Under these conditions, residual low level expression from the nmt1 promoter ensures that the level of Cdc27 protein present in the cell is comparable to that seen in wild-type cells [cdc27Δ haploid cells; indeed, no phenotypic defects were apparent (data not shown).To assay the promoter ,29, and pe cells . Analysicdc27+ gene was precisely replaced with the cdc27-Q1 mutant allele. In the Cdc27-Q1 mutant protein, the central five amino acids of the DPIM are replaced with alanine, resulting in loss of Cdc27-Pol1 interaction in the two-hybrid system . PCR analysis of genomic DNA using primers specific for wild-type cdc27+ and cdc27-Q1 sequences , with a generation time indistinguishable from wild-type (110 minutes at 32°C in YE medium). At 32°C, cdc27-Q1 cells underwent division at ~ 14.1 μm (compared to wild-type at ~ 14.4 μm). Further analysis showed that the cdc27-Q1 cells were indistinguishable from wild-type in all respects examined, including responses to the DNA replication inhibitor hydroxyurea (HU) and the DNA damaging agents methylmethane sulphonate (MMS), camptothecin (CPT), bleomycin sulphate (BMS) and UV light . The kinetics of cell division arrest in response to HU were also examined, but again no difference was detectable between cdc27-Q1 and wild-type strains (data not shown). That cell number increase is arrested in cdc27-Q1 cultures following treatment with HU, and that both wild-type and cdc27-Q1 cells become highly elongated under these conditions, is indicative of the presence of a functional DNA replication checkpoint in these cells. Taken together, these results strongly suggest that the Pol1-Cdc27 interaction does not play an essential role either during S-phase or in various DNA repair pathways in fission yeast.Phenotypic analysis of cdc27-Q1 background were also investigated. cdc27-Q1 was crossed to strains carrying mutations in the other three subunits of Pol δ  and in the helicase-endonuclease Dna2 and its associated protein Cdc24 (dna2-C2 and cdc24-M38). [cdc27-Q1 were indistinguishable from the single mutant. The cdc27-Q1 mutation was also combined with rad3Δ [cds1Δ [cdc27-Q1 rad3Δ and cdc27-Q1 cds1Δ double mutants, both of which were viable. The Rad3 and Cds1 proteins are key components of various DNA structure checkpoints in fission yeast [cdc27-Q1 double mutants were viable indicates that cdc27-Q1 cells do not require the presence of a functional checkpoint for viability. When wild-type cells are treated with hydroxyurea, activation of Cds1 results in cell cycle arrest and replication fork stabilisation. In the absence of Cds1, however, replication forks are believed to collapse, resulting in loss of viability [rad3Δ and cds1Δ double mutants were tested for their sensitivity to HU and CPT, but as before, no differences were observed between single and double mutants with cdc27-Q1 (data not shown).The consequences of introducing additional mutations into the 24-M38). ,30-33. Tth rad3Δ  and cds1on yeast . That thiability . The radIn fission yeast, DNA polymerase δ is a multisubunit complex comprising a large catalytic subunit that is required for chromosomal replication as well as three smaller subunits, two of which are also essential for cell viability ,18,36. Uin vivo and in vitro with Pol1, the catalytic subunit of Pol α. The Pol1 binding site on Cdc27 has been mapped by in vivo and in vitro approaches and a region of 40 amino acids, from amino acids 293 – 332, has been shown to be sufficient for binding. Protein sequence alignments of Cdc27 homologues across this 40 amino acid region identify a short protein sequence motif (D -- G --VT) that is highly conserved and which is essential for Pol1 binding. This DNA polymerase interaction motif (DPIM) is flanked by relatively highly charged sequences. Ten basic amino acids are found flanking the DPIM in the fission yeast Cdc27 protein sequence, nine of which are located N-terminal to the central conserved motif. Similarly, there are ten acidic amino acids, all of which lie either within the conserved DPIM or C-terminal to it. Our mutagenesis data clearly implicates several of these charged groups in the binding to Pol1 .The Cdc27 protein has an elongated shape, with a frictional ratio of 1.85 . The samRecently, the results of a deletion analysis of Pol32, the budding yeast orthologue of Cdc27, were reported , includiS. pombe cdc27-Q1 cells were indistinguishable from wild-type. No genetic interactions were observed between cdc27-Q1 and various other DNA replication or checkpoint mutants, including the key checkpoint kinase Rad3.In this paper, cells expressing the DPIM mutant protein Cdc27-Q1 were shown to be no more sensitive then wild-type to HU, MMS, CPT, UV and BMS (see Results), while analysis of budding yeast cells expressing Pol32 lacking the DPIM (270–309 deletion) showed them to be no more sensitive than wild-type to HU and UV, and to show normal rates of mutagenesis following UV exposure . Indeed,cdc27-Q1 mutation might be expected to be synthetically lethal or sick with a mutant that disrupted the overlapping redundant function. With the genetic tools available in yeast, this is a hypothesis that is readily testable.In conclusion, while the interaction between Pol α-primase and Pol δ mediated via Cdc27 could play an important role in coordinating the events of lagging strand synthesis, we have yet to obtain any evidence that this is the case. This raises two possibilities. First, that the observed interaction does not play an important role in chromosomal replication. We believe that this is unlikely to be the case, given the high degree of conservation of the DPIM sequence across evolution, in a region of the Cdc27 protein that is very poorly conserved at the primary sequence level. The second possibility is that there are multiple redundant interactions within the lagging strand machinery. In this case, an important role for the Cdc27-Pol1 interaction might only be uncovered when another protein-protein interaction is perturbed, in which case, the in vivo.In fission yeast, interaction between Pol α and Pol δ is mediated, at least in part, by direct binding of the Pol δ C-subunit Cdc27 to the Pol α catalytic subunit Pol1, and requires the presence of a short sequence motif (DPIM) in the C-terminal region of Cdc27. The DPIM is conserved in all known Cdc27 orthologues. Despite this, it has not been possible to identify any phenotypic consequences associated with deletion of the DPIM sequence, raising the possibility that the observed interaction does not play a crucial role pol1-H4 [pol1-1 [pol1-ts13 [polα-ts13 but is correctly renamed here to bring it in line with accepted nomenclature standards (see  for further details). S. pombe media and techniques were essentially as described [S. pombe was carried out by electroporation [S. cerevisiae CTY10-5d  was used [S. cerevisiae was cultured in YPDA and SD medium.All fission yeast strains were as described previously, except for pol1-H4  and pol1 [pol1-1 , which wol1-ts13 , which wescribed , with thporation , whereasporation . For twowas used . S. cereS. cerevisiae lexA op-lacZ strain CTY10-5d, as described previously [Two-hybrid interactions were monitored using the Gal4 transcription activation domain (prey) plasmid pACT2 (Clontech), the LexA DNA binding domain (bait) plasmid pBTM116, and eviously .pol1+ gene from S. pombe cosmid SPAC3H5, see , into plasmid pTZ19R) using oligonucleotides POL15 (5'-GTGTGGTTTGGGATCCCCCTATCACCAATGACACCTTTA-3') and POL13-2 (5'-GTGTGGTTTGGGATCCTACATCACCGTCATTGGAGGCGT-3'), restricting the PCR product with BamHI (sites underlined), cloning to pTZ19R (Fermentas) and sequencing to confirm the absence of errors. The 763 bp BamHI fragment was then transferred to pBTM116, to generate pBTM116-Pol1(278–527). Plasmid pBTM116-Pol1-TS13(278–527) was produced in a similar manner, except that the starting PCR template was genomic DNA prepared from pol1-ts13 cells [pol1+ ORF, resulting in the loss of coding capacity for amino acids L470, S471 and R472. Plasmids expressing C-terminally truncated Pol1(278–527) proteins were constructed by PCR amplification using oligo POL15 in conjunction with POL25-3 (5'-GTGTGGTTTGGGATCCTAAGGACCCATAACTCTTCTACT-3'), POL13-487 (5'-GTGTGGTTTGGGATCCTAAAAATTTGGTTGTTGTATTTT-3'), POL1-497 (5'-GTGTGGTTTGGGATCCTACCGGCACCAACTAGCATTTTT-3') or POL1-507 (5'-GTGTGGTTTGGGATCCTAGTTCTGAGGTGACGAACATCC-3'), cloning directly to pBTM116 following BamHI cleavage of the PCR product, and sequencing. The resulting constructs encoded the following proteins as Lex A fusions: Pol1(278–477), Pol1(278–487), Pol1(278–497) and Pol1(278–507). A construct expressing an N-terminally truncated Pol1(278-527) protein, designated Pol1(328–527), was generated by amplification using POL15-2 (5'-GTGTGGTTTGGGATCCCCGGCTCATTGTGTCTATTTGGC-3') with POL13-2 (above). Amplification with POL15-2 and POL25-3 generated a construct with the potential to express a LexA-Pol1 protein truncated at both ends: LexA-Pol1(328–477).The Pol1 bait plasmid pBTM116-Pol1-(278–527) was constructed by amplifying sequences encoding amino acids 278–527 from plasmid pTZ19R-Pol1 (prepared by subcloning a 5925 bp SmaI-PstI fragment encompassing the entire GGATCCCCTCAGAACAAGCAGTGAAAGAA-3') or P66-52 (5'-GTGTGGTTTGGGATCCCGTCTCCACCTCTTGAACCAGTG-3') with P66-3 (5'-GTGTGGTTTGGGATCCTTGGTCTTCACCCTTGACCACTC-3'). The PCR products were then restricted with BamHI, cloned to pACT2 and sequenced. The resulting plasmids encode, as Gal4 AD fusions, either the entire C-terminal domain of p66/KIAA0039  or a shorter region . Sequences encoding the interacting domain of the catalytic domain of human Pol α were amplified from plasmid pBR322-Pol α  using oligos HPOL1-5 (5'-GTGTGGTTTGGGATCCCCAAAGGGACCGTGTCCTACTTA-3') and HPOL1-3 (5'-GTGTGGTTTGGGATCCTACATCACGACAAGCGGTGGTGG-3'), restricted with BamHI, cloned to pBTM116 and sequenced. The resulting plasmid encodes amino acids 291–540 of the human protein as a LexA fusion.Sequences encoding the C-terminal region from human p66/KIAA0039 were amplified by PCR from plasmid pET19b-p66  using either oligo P66-51 (5'-GTGTGGTTTGGGATCCCCACCGAAGCAAAATCTGCTGCA-3') and PMUT3 (5'-GTTGTGGGTGGGATCCTACTTCTTAGTTGCAATGTTTAC-3'). Mutant Q1 was generated by PCR amplification with same primers but using pHBLA-Cdc27-Q1 (below) as template. Plasmids expressing mutants P5-P7 were generated by overlap extension PCR using PMUT5 and PMUT3 together with the following mutagenic oligonucleotides (shown with mutated sequence underlined in top strand oligo): CDC27-P5F (5'-AAAGAAAAGTTGCAGCGTACGCGACAACGAAAG-3'), CDC27-P5R (5'-CTTTCGTTGTCGCGTACGCTGCAACTTTTCTTT-3'), CDC27-P6F (5'-TTGGTTACTAAGGCAGCAGCAGTCTGGGAATCA-3'), CDC27-P6R (5'-TGATTCCCAGACTGCTGCTGCCTTAGTAACCAA-3'), CDC27-P7F (5'-GAATCATTTTCTGCAGCTGCAAACATCTCAACT-3'), CDC27-P7R (5'-AGTTGAGATGTTTGCAGCTGCAGAAAATGATTC-3').The Cdc27 prey plasmids were constructed in pACT2 (Clontech) and have been previously described, with the exception of mutants Cdc27-273-352-P1 through -P7, and Cdc27-273-352-Q1. The first four of these (P1-P4) were generated by PCR amplification from the pREP3X-Cdc27-P1 to -Cdc27-P4 plasmids described below. Oligonucleotides for PCR, with BamHI sites underlined: PMUT5 -encoding BamHI fragment described above was cloned into pQE32 (Qiagen). The resulting plasmid, pQE32-Pol1 (278–527), was transformed into E. coli M15 (pREP4) and recombinant protein expression induced in 500 ml cultures (OD600 nm of 0.6) by addition of IPTG to a final concentration of 1 mM. Following incubation for 4 hours at 37°C, the cells were pelleted and the pellet resuspended in 40 ml of ice-cold buffer A  containing EDTA-free Complete™ inhibitors (Roche Applied Science) and 1 mM PMSF. The cells were lysed by sonication, then centrifuged at 25000 g for 15 minute at 4°C. The soluble supernatant was added to 4 ml of 50% (v/v) Ni-NTA agarose (Qiagen) in buffer A, mixed on a wheel at 4°C for one hour, then packed into a disposable chromatography column at 4°C. The column was drained of the flow-through and subsequently washed with 100 ml of buffer B  containing Complete™ inhibitors and PMSF, then 100 ml of buffer A containing Complete™ inhibitors and PMSF, before the bound H6-Pol1 protein was eluted using 4 ml of buffer C . Following elution, samples were analysed by SDS-PAGE and the peak fractions pooled and dialysed overnight in PBS at 4°C. Protein concentration was determined by BCA assay (Pierce) versus BSA standards.The Pol1 (278–527) domain was expressed in GST and GST-Cdc27-273-352 fusion proteins were prepared as described previously  from plapol1+ locus in an otherwise wild-type leu1-32 ura4-D18 h- strain with sequences encoding thirteen copies of the 9E10 (c-myc) mAb epitope, such that the encoded Pol1 protein (termed Pol1-13Myc) carries the 9E10 epitopes at its C-terminus. Oligonucleotides for amplification from pFA6a-13Myc-kanMX6 [CGGATCCCCGGGTTAATTAA-3', with plasmid-specific sequence underlined) and POL1-13MYC-3 (5'-GGCAATTCCCAAGTCTTTGAAACAGGTATTCCCATCAACATTTCTTGTACTGCATGAGCAAATATCTGTTCGAGGTGTCGAATTCGAGCTCGTTTAAAC-3'). Following transformation of ~ 10 μg of PCR product, correct G418-resistant integrants were identified by PCR amplification of genomic DNA using primer FCGPOL1-5 (5'-ATGTCGTGGAAGCGTTCATT-3') within the pol1+ ORF and KAN269R (5'-GATCGCAGTGGTGAGTAACCATGCATCATC-3') within the kanMX6 cassette, and by Western blotting using the mouse anti-Myc mAb 9E10 (Roche).PCR-based gene targeting was used to tag the chromosomal c-kanMX6  were as 7 cells in mid-exponential growth in EMM medium. Cells were harvested, washed once in STOP buffer  and disrupted in buffer A  supplemented with Complete™ protease inhibitors (Roche), using a bead beater (Hybaid Ribolyser). Protein concentrations were determined at 595 nm using the BioRad protein assay reagent according to the manufacturer's instructions, with BSA as standard.Peptides were synthesized by Mimotopes . All peptides were synthesized to contain the sequence biotin-SGSG at the N-terminus. Peptide sequences: SpA AAPDEPQEIIKSVSGGKRRG; SpB PQEIIKSVSGGKRRGKRKVK; SpC KSVSGGKRRGKRKVKKYATT; SpD GKRRGKRKVKKYATTKDEEG: SpE KRKVKKYATTKDEEGFLVTK; HsA PKTEPEPPSVKSSSGENKRK; HsB EPPSVKSSSGENKRKRKRVL; HsC KSSSGENKRKRKRVLKSKTY; HsD ENKRKRKRVLKSKTYLDGEG; HsE RKRVLKSKTYLDGEGCIVTE. For binding assays, the peptides were initially bound to 10 μl Streptavidin-agarose beads in PBS in a final volume of 100 μl at room temperature for 1 hour with gentle agitation. After binding, the beads were washed 3 times with 100 μl PBS. Protein extracts (100 μg of fission yeast extract) were added to the beads, and incubated at 4°C for 1 hr with gentle agitation. The beads were pelleted by centrifugation at 2000 rpm for 3 minutes, and subsequently washed extensively with NP40 buffer  NP-40), prior to final resuspension with 20 μl of SDS loading buffer. Following SDS-PAGE, Pol1-13Myc was visualised by immunoblotting with the mouse anti-Myc mAb 9E10 (Roche). Fission yeast protein extracts were prepared from 8 × 10cdc27+ cDNAs  from pTZ19R-Cdc27-cDNA [GCCGCAGCAGGATTCTTGGTTACTAAGG-3'), CDC27-P2 (5'-CGACAACGAAAGATGAAGAAGCCGCCTTGGTTACTAAGGAAGAAG-3'), CDC27-P3 (5'-TCAAATCCGTATCCGGTGGAGCCGCAGCAGGGAAAAGAAAAGTTAAAAAG-3'), CDC27-P4 (5'-CCGGTGGAAAGAGACGTGGGGCCGCAGCAGTTAAAAAGTACGCGACAAC-3'). The resulting mutant alleles were tested for function by transforming a cdc27+/cdc27::his7+leu1-32/leu1-32 ura4-D18/ura4-D18 his7-366/his7-366 ade6-M210/ade6-M216 h-/h+ diploid [Plasmids pREP3X-Cdc27-P1 to -Cdc27-P4 were constructed by cloning mutagenised c27-cDNA  into pRE diploid , transfecdc27+ gene was first cloned into pTZ19R to make pHB-Cdc27. This vector was then modified by addition of the S. cerevisiae LEU2 gene and S. pombe ars1, to make pHBLA-Cdc27. The cdc27+ gene was then subjected to oligonucleotide-directed in vitro mutagenesis using the QuikChange method (Stratagene) with oligonucleotides CDC27-QC1  and CDC27-QC2 (5'-CTTCTTCCTTAGTAACCAAGgcggccgcTGCAGCTTTCGTTGTCGCGTAC-3'), to create plasmid pHBLA-Cdc27-Q1. This was then was transformed into a cdc27+/cdc27::ura4+ diploid, and cdc27::ura4+ (pHBLA-Cdc27-Q1) haploids obtained following sporulation and regrowth. These were then plated on YE plates containing 1 mg/ml 5-FOA. 5-FOA resistant colonies were identified, purified and characterised by PCR amplification of genomic DNA ; CDC27-Q1M-DIAG2 ; CDC27-H (5'-ACTGGTAGAATTGCGTTCGCGCTC-3'); CDC27-B (5'-TCTAGGATCAGAGTGAACTGATTG-3'); CDC27-SEQ2005 (5'-AGGTTGTACTAACATTAACAG-3'). The resulting strain, cdc27-Q1 leu1-32 ura4-D18 his7-366 ade6-M216 h- was then analysed alongside the wild-type leu1-32 ura4-D18 his7-366 ade6-M216 h-, as described below.A 3.1 kb HindIII-BamHI genomic DNA fragment carrying the oworkers ), to ens6 cells/ml) in YE medium and ~ 2000 cells plated on YE medium supplemented with varying concentrations of hydroxyurea , methylmethanesulphonate , bleomycin sulphate , or camptothecin . Cells were also plated on plates containing sub-lethal doses of both HU and CPT, specifically 7.5 mM HU with either 5.5 or 6 μM CPT, or 10 mM HU with either 5.5 or 6 μM CPT. To analyse the effects of HU treatment in liquid culture, HU was added to EMM medium to a final concentration of 12 mM. Cell number per ml of culture was monitored using a Coulter Z1 electronic particle counterTo test sensitivity to UV, 1000 cells were plated on EMM plates, allowed to dry for 20 minutes, then irradiated using either a Stratalinker UV source (Stratagene) over the range 0 – 250 J/m2. Following UV treatment, plates were placed immediately in the dark to avoid photoreversal. For all treatments, the efficiency of colony formation was determined after 4 days growth at 32°C. Growth rate  was determined by cell counting using a particle counter. Cell length at cell division was determined using a graduated eyepiece.Cells were grown to mid-exponential phase ; PCNA (proliferating cell nuclear antigen).In Edinburgh, SM conceived of the study, performed some of the experimental work and prepared and revised the final manuscript, while FG performed the remainder of the experimental work. In Dundee, EW and JRGP designed and carried out the peptide binding studies. All four authors read and approved the manuscript.


Bisdioxopiperazine anti-cancer agents are inhibitors of eukaryotic DNA topoisomerase II, sequestering this protein as a non-covalent protein clamp on DNA. It has been suggested that such complexes on DNA represents a novel form of DNA damage to cells. In this report, we characterise the cytotoxicity and DNA damage induced by the bisdioxopiperazine ICRF-187 by a combination of genetic and molecular approaches. In addition, the well-established topoisomerase II poison m-AMSA is used for comparison.Saccharomyces cerevisiae single-gene deletion strains, homologous recombination was identified as the most important DNA repair pathway determining the sensitivity towards ICRF-187. However, sensitivity towards m-AMSA depended much more on this pathway. In contrast, disrupting the post replication repair pathway only affected sensitivity towards m-AMSA. Homologous recombination (HR) defective irs1SF chinese hamster ovary (CHO) cells showed increased sensitivity towards ICRF-187, while their sensitivity towards m-AMSA was increased even more. Furthermore, complementation of the XRCC3 deficiency in irs1SF cells fully abrogated hypersensitivity towards both drugs. DNA-PKcs deficient V3-3 CHO cells having reduced levels of non-homologous end joining (NHEJ) showed slightly increased sensitivity to both drugs. While exposure of human small cell lung cancer (SCLC) OC-NYH cells to m-AMSA strongly induced γH2AX, exposure to ICRF-187 resulted in much less induction, showing that ICRF-187 generates fewer DNA double strand breaks than m-AMSA. Accordingly, when yeast cells were exposed to equitoxic concentrations of ICRF-187 and m-AMSA, the expression of DNA damage-inducible genes showed higher levels of induction after exposure to m-AMSA as compared to ICRF-187. Most importantly, ICRF-187 stimulated homologous recombination in SPD8 hamster lung fibroblast cells to lower levels than m-AMSA at all cytotoxicity levels tested, showing that the mechanism of action of bisdioxopiperazines differs from that of classical topoisomerase II poisons in mammalian cells.By utilizing a panel of Our results point to important differences in the mechanism of cytotoxicity induced by bisdioxopiperazines and topoisomerase II poisons, and suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms. Type II topoisomerases are essential nuclear enzymes found in all living organisms . Their bTopoisomerase II is also a major drug target in human cancer therapy, where a number of clinically active drugs such as the epipodophyllotoxins VP-16 and VM-26, the aminoacridine m-AMSA, and antracyclines such as doxorubicin, daunorubicin and epirubicin are widely used. These drugs have collectively been called topoisomerase II poisons due to their mechanism of action on topoisomerase II. Rather than inhibiting the basic catalytic activity of the enzyme, these drugs perturb the topoisomerase II catalytic cycle resulting in an increase in the level of a transient reaction intermediate, where DNA is cleaved and covalently attached to DNA .Catalytic inhibitors of topoisomerase II have a different mode of action. These drugs exemplified by merbarone, aclarubicin, F11782 and the bisdioxopiperazines work by inhibiting topoisomerase II at other stages in the reaction cycle where DNA is not cleaved as reviewed in ,6. Amongi) Expression of bisdioxopiperazine-sensitive topoisomerase II in cells also expressing bisdioxopiperazine-resistant topoisomerase II confers dominant sensitivity to these drugs methanesulphonanilide); MMR, Mismatch Repair; NER, Nucleotide Excision Repair; NHEJ, Non-Homologous End Joining; PCR, Polymerase Chain Reaction; PRR, Post Replication Repair; SCLC, Small Cell Lung Cancer; SC-URA, Synthetic medium lacking uracil; SSA, Single Strand Anealing; YPD, Medium containing Yeast extract, Peptone and Dextrose.Lars H. Jensen: Participated in planning the experiments, performed yeast transformations and clonogenic assays, and prepared the manuscript. Marielle Dejligbjerg: Performed γH2AX western blots, primer design, real-time PCR experiments, and data quantitation. Lasse T. Hansen: Performed mammalian clonogenic assays and recombination assays. Morten Grauslund: Performed RNA purification and microarray analysis. Peter B. Jensen: Participated in planning and monitoring the study. Maxwell Sehested: Participated in the initiation and conduction of the study. All authors read and approved the final manuscript.Clonogenic sensitivity of mutant single-gene deletion yeast strains towards ICRF-187 and m-AMSA. Clonogenic sensitivity of a panel of human topoisomerase II α-transformed haploid yeast deletion strains towards equitoxic (to wt cells) concentrations of ICRF-187 and m-AMSA. Error-bars represent SEM of 3 – 10 independent experiments.Click here for filet2–4 cells were inoculated into YPD medium and grown overnight at 34°C, 150 rpm. The cultures were then diluted into 50 ml YPD medium to obtain an OD600 of 0.2. After growing the cells for 2 hours to assure exponential growth, equitoxic concentrations of ICRF-187 and m-AMSA were applied and the cells were grown for an additional 2 hours before RNA was isolated. The figure depicts the clonogenecity of drug treated and untreated cells. Exposure of the cells to the two drugs resulted in a reduction of their clonogenecity of approx. 50 %. Error bars-represent SEM of three independent experiments.Level of killing of yeast cells used in transcriptional profiling experiments. Fresh colonies of pMJ1-transformed JN362AClick here for fileTranscriptional response towards ICRF-187. A list of yeast genes whose average expression in two independent experiments is induced or repressed more than 1.5 fold by exposure to ICRF-187.Click here for fileTranscriptional response towards m-AMSA. A list of yeast genes whose average expression in two independent experiments is induced or repressed more than 1.5 fold by exposure to m-AMSA.Click here for fileTopoisomerase II activity levels in hamster cell lines. The levels of topoisomerase II catalytic activity in crude extracts from wt and recombination defective hamster cell lines. Error-bars represent SEM of two independent experiments. No difference in the level of topoisomerase II catalytic (DNA strand passage activity) is observed.Click here for file


K562 leukaemia cells were selected for resistance using 0.5 microM etoposide (VP-16). Cloned K/VP.5 cells were 30-fold resistant to growth inhibition by VP-16 and 5- to 13-fold resistant to m-AMSA, adriamycin and mitoxantrone. K/VP.5 cells did not overexpress P-glycoprotein; VP-16 accumulation was similar to that in K562 cells. VP-16-induced DNA damage was reduced in cells and nuclei from K/VP.5 cells compared with K562 cells. Topoisomerase II protein was reduced 3- to 7-fold and topoisomerase II alpha and topoisomerase II beta mRNAs were each reduced 3-fold in resistant cells. After drug removal, VP-16-induced DNA damage disappeared 1.7 times more rapidly and VP-16-induced DNA-topoisomerase II adducts dissociated 1.5 times more rapidly in K/VP.5 cells than in K562 cells. ATP (1 mM) was more effective in enhancing VP-16-induced DNA damage in nuclei isolated from sensitive cells than in nuclei from resistant cells. In addition, ATP (0.3-5 mM) stimulated VP-16-induced DNA-topoisomerase II adducts to a greater extent in K562 nuclei than in K/VP.5 nuclei. Taken together, these results indicate that resistance to VP-16 in a K562 subline is associated with a quantitative reduction in topoisomerase II protein and, in addition, a distinct qualitative alteration in topoisomerase II affecting the stability of drug-induced DNA-topoisomerase II complexes.


We have studied the genetic alterations acquired during selection of a cloned human leukaemic cell line (CEM/VP-1) that is 15-fold more resistant to the anticancer topoisomerase II-inhibitor etoposide than parental CCRF-CEM cells. CEM/VP-1 cells exhibit an 'atypical MDR' phenotype: cross resistance to other topo II inhibitors  and expression of a drug-resistant topo II activity. Cytogenetic and molecular studies revealed that the cell line carried multiple genetic changes affecting TOP2 genes encoding both topo II alpha and beta isoforms. CEM/VP-1 was diploid, 47,XX,+20, and appears to have been preferentially selected from a 1% diploid subpopulation present in the tetraploid parental cells. The same chromosomal abnormalities were present in resistant and sensitive cells except for an acquired 3p- change most likely deleting one TOP2 beta allele. PCR/DNA sequence analysis and allele-specific hybridisation showed that one of two TOP2 alpha alleles expressed in CEM/VP-1 cells had acquired a Lys-797-->Asn codon change. This mutation lies close to the catalytic Tyr-804 residue of the protein and may interfere with drug-induced trapping of the cleavable complex. Alternatively, it could exert a loss of function phenotype. CEM/VP-1 cells did not exhibit codon 449 or 486 TOP2 alpha mutations in the ATP binding domain reported in two other resistant cell lines. Diploid selection and multiple changes observed in CEM/VP-1 cells appear to be consequences of the recessive phenotype of at-MDR. These results may be useful in approaching the mechanisms of clinical resistance.


The cytoxicity of both intercalating (m-AMSA) and non-intercalating  topoisomerase II-targeting drugs is thought to occur via trapping DNA topoisomerase II on DNA in the form of cleavable complexes. First, analysis of cleavable complexes (detected as DNA double-strand breaks) by pulsed-field gel electrophoresis confirmed the correlation between cleavable complex formation and cytotoxicity of three topoisomerase-targeting drugs in HeLa S3 cells (the order of effects being VM26 > m-AMSA > VP16). In contrast to many antineoplastic agents, hyperthermic treatments were found to protect cells against the toxicity of all three topoisomerase II drugs. Hyperthermia treatment does not alter drug accumulation but reduces the ability of the drug-topoisomerase II complex to form the cleavable complexes. Nuclear protein aggregation induced by heat at the sites of topoisomerase II-DNA interaction may explain such an effect. In thermotolerant cells, the toxic effects of VP16 but not m-AMSA were reduced. For both drugs, however, the status of thermotolerance did not affect cleavable complex formation by the drugs. Thus, protection against VP-16 toxicity seems not to be associated with heat-induced activation of the P-gp 170 pump or altered topoisomerase II-DNA interactions. Rather, a protective (heat shock protein mediated?) mechanism against non-intercalating topoisomerase II drugs seems to occur at a stage after DNA-drug interaction. Finally, heat treatment before topoisomerase II drug treatment reduced toxicity and cleavable complex formation in thermotolerant cells to about the same extent as in non-tolerant cells, consistent with the presumption of nuclear protein aggregation being responsible for this effect.


Camptothecins are DNA topoisomerase I-directed anti-tumour drugs with a novel mechanism of action. Topotecan (TPT), a hydrophilic derivative of camptothecin, is currently undergoing phase II clinical trials in small-cell lung cancer (SCLC). Human SCLC OC-NYH cells were made more than 6-fold resistant to topotecan by stepwise drug exposure and resistance was stable for 70 passages without drug. NYH/TPT cells had half the topoisomerase I level and activity of wild-type cells. However, no difference in camptothecin or topotecan inhibition of topoisomerase I-mediated DNA relaxation was found, indicating that the enzyme itself was unchanged in the resistant cell. In NYH/TPT cells, topoisomerase II alpha and beta levels were increased approximately 2-fold. Accordingly, the topoisomerase II-directed drug etoposide (VP-16) induced an increased number of DNA single-strand breaks in NYH/TPT cells. However, sensitivity to different topoisomerase II-targeting agents in NYH/TPT cells varied from increased to decreased, indicating a role for as yet unidentified factors acting on the pathway to cell death after topoisomerase II-induced DNA damage has occurred. Of 20 anti-cancer agents tested, only hydroxyurea showed marked collateral hypersensitivity in NYH/TPT cells.


Multidrug-resistant (MDR) cell lines often have a compound phenotype, combining reduced drug accumulation with a decrease in topoisomerase II. We have analysed alterations in topoisomerase II in MDR derivatives of the human lung cancer cell line SW-1573. Selection with doxorubicin frequently resulted in reduced topo II alpha mRNA and protein levels, whereas clones selected with vincristine showed normal levels of topo II alpha. No alterations of topo II beta levels were detected. To determine the contribution of topo II alterations to drug resistance, topo II activity was analysed by the determination of DNA breaks induced by the topo II-inhibiting drug 4'-(9-acridinylamino)methane-sulphon-m-anisidide (m-AMSA) in living cells, as m-AMSA is not affected by the drug efflux mechanism in the SW-1573 cells. The number of m-AMSA-induced DNA breaks correlated well (r = 0.96) with in vitro m-AMSA sensitivity. Drug sensitivity, however, did not always correlate with reduced topo II mRNA or protein levels. In one of the five doxorubicin-selected clones m-AMSA resistance and a reduction in m-AMSA-induced DNA breaks were found in the absence of reduced topo II protein levels. Therefore, we assume that post-translational modifications of topo II also contribute to drug resistance in SW-1573 cells. These results suggest that methods that detect quantitative as well as qualitative alterations of topo II should be used to predict the responsiveness of tumours to cytotoxic agents. The assay we used, which measures DNA breaks as an end point of topo II activity, could be a good candidate.


These agents were observed to inhibit HeLa DNA topoisomerase II activity ~ 200 μM and L1210 topoisomerase II activity ≥ 100 μM. These agents did not cause DNA protein linked breaks themselves, but upon incubation for 14-24 hr did enhance the ability of VP-16 to cause cleavable complexes. The heterocyclic amineboranes inhibited DNA synthesis and caused DNA strand scission. They were additive with VP-16 in affording these results as well as inhibiting colony growth of L1210 cells after co-incubation for 1 hr. The agents inhibited


The objective of this study was to determine whether arachidonate metabolites are involved in the vasoconstrictive effects of angiotensin II in rats. In the isolated perfused heart, dexamethasone (4 mg/kg) significantly suppressed the maximal decreases in coronary flow induced by angiotensin II and vasopressin (reference drug). In the heart, the nonselective lipoxygenase inhibitor nordihydroguaiaretic acid  markedly suppressed the angiotensin II-induced decreases in coronary flow. NDGA (10 μM) inhibited both angiotensin II- and methoxamine- (reference drug) induced contractions in aortic rings with (in the presence of L-NAME) and without endothelium. In the heart, the leukotriene synthesis inhibitor MK-886 (0.3 μM) significantly reduced the maximal effects to angiotensin II, but the leukotriene antagonist FPL 55712 (0.1 and 0.3 μM) had no effect. We conclude that in the isolated perfused rat heart angiotensin II-induced decreases in coronary flow are in part mediated by Hpoxygenase products, which might be derived from the 5-Hpoxygenase pathway, but are probably not leukotrienes. Furthermore, endothelium independent Hpoxygenase products mediate part of the contractile responses to angiotensin II in the isolated rat aorta.


Drug resistance to anti-tumour agents often coincides with mutations in the gene encoding DNA topoisomerase II alpha. To examine how inactive forms of topoisomerase II can influence resistance to the chemotherapeutic agent VP-16 (etoposide) in the presence of a wild-type allele, we have expressed point mutations and carboxy-terminal truncations of yeast topoisomerase II from a plasmid in budding yeast. Truncations that terminate the coding region of topoisomerase II at amino acid (aa) 750, aa 951 and aa 1044 are localised to both the cytosol and the nucleus and fail to complement a temperature-sensitive top2-1 allele at non-permissive temperature. In contrast, the plasmid-borne wild-type TOP2 allele and a truncation at aa 1236 are nuclear localised and complement the top2-1 mutation. At low levels of expression, truncated forms of topoisomerase II render yeast resistant to levels of etoposide 2- and 3-fold above that tolerated by cells expressing the full-length enzyme. Maximal resistance is conferred by the full-length enzyme carrying a mutated active site (Y783F) or a truncation at aa 1044. The level of phosphorylation of topoisomerase II was previously shown to correlate with drug resistance in cultured cells, hence we tested mutants in the major casein kinase II acceptor sites in the C-terminal domain of yeast topoisomerase II for changes in drug sensitivity. Neither ectopic expression of the C-terminal domain alone nor phosphoacceptor site mutants significantly alter the host cell's sensitivity to etoposide.


Complexes of the diuretic benzothiadiazine derivative chlorothiazide  with V(IV); Fe(II); Co(II); Ni(II); Cu(II), Ag(I) and U(VI) wereprepared and characterized by elemental analysis, spectroscopic, thermogravimetric, magnetic andconductimetric measurements. The complexes behave as effective inhibitors for two isozymes (I and II) ofcarbonic anhydrase (CA).


In order to evaluate the effect of metal ions upon chelation, eephradine and its complexes have been screened for their antibacterial activity against bacterial strains, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.Some Co(II), Cu(II), Ni(II) and Zn(II) complexes of antibacterial drug cephradine have been prepared and characterized by their physical, spectral and analytical data. Cephradine acts as bidentate and the complexes have compositions, [M(L)


These three structures are defined by metal to proteinstoichiometric ratios, which we believe specifically determine the coordination geometry adoptedby the metal in the metal binding site at that metal to protein molar ratio. Tetrahedral geometry isassociated with the thiolate coordination of the metals in the M7-MT species, for M = Zn(II), Cd(II),and possibly also Hg(II), trigonal coordination is proposed in the M11-12-MT species, for M = Ag(I),Cu(I), and possibly also Hg(II), and digonal coordination is proposed for the metal in the M17-18-MTspecies for M = Hg(II), and Ag(I). The M7-MT species has been completely characterized for M =Cd(II) and Zn(II). 113Cd NMR spectroscopic and x-ray crystallographic data show that mammalianCd7-MT and Zn7-MT have a two domain structure, with metal-thiolate clusters of the form M4(Scys)11 (the α domain) and M3(Scys)9 (the β domain). A similar two domain structure involving Cu6(Scys)11 (α) and Cu6(Scys)9 (β) copper-thiolate clusters has been proposed for the Cu12-MT species.Copper-, silver- and gold-containing metallothioneins luminesce in the 500-600 nm region fromexcited triplet, metal-based states that are populated by absorption into the 260-300 nm region ofthe metal-thiolate charge transfer states. The luminescence spectrum provides a very sensitiveprobe of the metal-thiolate cluster structures that form when Ag(I), Au(I), and Cu(I) are added tometallothionein. CD spectroscopy has been used in our laboratory to probe the formation ofspecies that exhibit well-defined three-dimensional structures. Saturation of the optical signalsduring titrations of MT with Cu(I) or Ag(I) clearly show formation of unique metal-thiolate structuresat specific metal:protein ratios. However, we have proposed that these M=7, 12 and 18 structuresform within a continuum of stoichiometries. Compounds prepared at these specific molar ratioshave been examined by X-ray Absorption Spectroscopy (XAS) and bond lengths have beendetermined for the metal-thiolate clusters through the EXAFS technique. The stoichiometric ratiodata from the optical experiments and the bond lengths from the XAS experiments are used topropose structures for the metal-thiolate binding site with reference to known inorganicmetal-thiolate compounds.Metallothionein is a ubiquitous protein with a wide range of proposed physiological roles, includingthe transport, storage and detoxification of essential and nonessential trace metals. The aminoacid sequence of isoform 2a of rabbit liver metallothionein, the isoform used in our spectroscopicstudies, includes 20 cysteinyl groups out of 62 amino acids. Metallothioneins in general representan impressive chelating agent for a wide range of metals. Structural studies carried out by anumber of research groups (using


Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumonae. The activity data show the metal complexes to be more active than the parent freeligands against one or more bacterial species.Biologically active complexes of Co(II), Ni(II), Cu(II) and Zn(II) with novel ONO, NNO andSNO donor pyrazinoylhydrazine-derived compounds have been prepared and characterized on the basis ofanalytical data and various physicochemical studies. Distorted octahedral structures for all the complexeshave been proposed. The synthesized ligands and their complexes have been screened for their antibacterialactivity against bacterial species


Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and the results are reported.Some acylhydrazine derived ONO donor Schiff bases and their Co(II) and Ni(II) complexes have been prepared having the same metal ion (cation) but different anions. These synthesized metal(II) complexes have been characterized on the basis of their elemental analyses, magnetic moment, molar conductance, and IR and electronic spectral data. All of the Schiff base ligands function as tridentates and the deprotonated enolic form is preferred for coordination. In order to evaluate the effect of anions on the bactericidal activity, these synthesized complexes, in comparison to the uncomplexed Schiff bases have been screened against bacterial species.,


The cytotoxicity of the complexes under investigation decreases in theorder 3 > 2 > 1 which is in accord with structure-activity relationships with other platinum(II) and platinum(IV) complexes: Both trans complexes (2 and 3) display a higher in vitropotency than the corresponding cis isomer (I), with the trans-R,R isomer (3) being the most active in thisseries. In comparison to the analogous platinum(II) complexes withbis(phosphonomethyl)aminoacetic acid as osteotropic carrier ligand, the cytotoxicity of 1-3 was found to be1.5 – 2 fold higher, which is explainable by a different coordination mode of the phosphonic acid ligands(acetato versus phosphonato).In order to develop platinum complexes with selective activity in primary and secondary bonemalignancies and with the aim to optimize antitumor activity, platinum(II) complexes withaminotris(methylenephosphonic acid) as bone-seeking (osteotropic) ligand have been synthesized,characterized and tested in the cisplatin-sensitive ovarian carcinoma cell line CH1. As non-leaving diamineligands, which are decisive for the cellular processing of DNA adducts,


The agents blocked L1210 leukemic cell DNA and RNA syntheses by inhibitingmultiple enzyme activities for nucleic acid synthesis, e.g. PRPP amido transferase, IMP dehydrogenase, DNApolymerase α, thymidine kinase, and TMP kinase. The copper (II) complex 3 demonstrated improved abilityto inhibit L1210 partially purified DNA topoisomerase II compared to the parent compound while the sodiumsalt was inactive at 100 μM.Sodium N-[(trimethylamineboryl)-carbonyl]-L-phenylalanine


Organotin(IV) complexes were more active than tin(II) complexes.Some organotin(IV) and tin(II) complexes of composition R


Complexes containing the anions of 5-benzoylamido-1,3,4-thiadiazole-2-sulfonamide and 5-(3-nitro-benzoylamido)-1,3,4-thiadiazole-2-sulfonamid as ligands, and V(IV); Cr(III); Fe(III); Co(II); Ni(II); Cu(II) and Ag(I) were synthesized and characterized by standard procedures . The original sulfonamides and their metal complexes are strong inhibitors of two carbonic anhydrase (CA) isozymes, CA I and II.


Male SHR and Wistar Kyoto rats (WKY) aged 16–18 weeks were given l-arginine (10 g/L in drinking water) for 1 week. Separate control groups received no supplementation. The SHR control had a significantly lower plasma l-arginine than WKY control, but this was increased to a comparable level following l-arginine. Atrial cGMP was lower in the SHR control compared with the WKY control , but increased to 4.1 ± 0.5 pmol/mg protein  with l-arginine. Evoked [3H]norepinephrine release in isolated spontaneously beating right atria from the SHR control  was 28% higher than the WKY control , but was reduced to 258 ± 11% with l-arginine feeding . Soluble guanylyl cyclase (sGC) inhibition caused a greater increase of evoked norepinephrine release in the l-arginine fed SHR compared with the non-fed SHR. l-arginine feeding did not reduce evoked norepinephrine release in the WKY. In-vitro heart rate response to exogenous norepinephrine (0.1–5 μmol/L) was similar between l-arginine fed (n = 13) and non-fed SHR (n = 10), suggesting that l-arginine supplementation worked pre-synaptically. Myocardial tyrosine hydroxylase protein was decreased in SHR following l-arginine supplementation, providing a link to reduced synthesis of norepinephrine. In conclusion, l-arginine supplementation corrects local cardiac noradrenergic hyperactivity in the SHR, probably via increased pre-synaptic substrate availability of NOS–sGC–cGMP pathway and reduced tyrosine hydroxylase levels.Spontaneously hypertensive rats (SHR) are known to have cardiac noradrenergic hyperactivity due to an impaired nitric oxide (NO)–cGMP pathway. We hypothesized that dietary  Its presence is associated with adverse clinical outcome, even in apparently normal subjects l-arginine (l-arg) is the sole substrate of the enzyme NOS in the synthesis of NO l-arg normally greatly exceed the level that is required for maximal enzyme kinetics of NOS; the Michaelis constant (Km) of l-arg for NOS is 2.9 μmol/L whereas the intracellular level of l-arg in-vivo is 0.8–2 mmol/L, with plasma concentration in the region of 60–350 μmol/L l-arg supplementation has been shown to improve endothelial function, reduce atherosclerosis progression, and alter autonomic function; all consistent with enhanced NO synthesis l-arginine paradox, and can probably be best explained by compartmentalisation of NOS and a relative l-arg deficiency rather than an absolute one.l-arg supplementation could reduce local cardiac noradrenergic hyperactivity in the SHR by increasing pre-synaptic substrate availability of the NOS–sGC–cGMP pathway, and provided further mechanistic link with demonstration of reduction in cardiac tyrosine hydroxylase levels.In the present study, we showed that oral 22.1l-arg supplemented groups were given l-arg  in drinking water (10 g/L water) for 1 week. Control groups received no supplementation but otherwise have free access to water and rat chow. The experiments conformed to the Animals (Scientific Procedures) Act 1986 (UK) and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .Male SHR aged 16–18 weeks and Wistar Kyoto rats (WKY) were sourced from Harlan, UK, and kept under standard laboratory conditions. 2.2The heart rate and arterial blood pressure were measured invasively as a terminal procedure  on some rats. Briefly the rat was ventilated under anaesthesia (3% isoflurane and 100% oxygen) and its left carotid artery was cannulated with a 3F portex cannula. The cannula was then connected to a pressure transducer and data was acquired using a Biopac M100 system connected to a Dell P4 computer and run on the AcqKnowledge 3.7.3 software. After an equilibration period of at least 10 min at lower isoflurane concentration (1.5%), the heart rate and blood pressure readings were taken. Following completion of the hemodynamic measurement, the animals were euthanised with intraperitoneal injection of pentobarbitone. Blood samples were taken via intraventricular puncture and the whole heart removed. Blood samples were immediately spun at 5000 rpm for 10 min. Red blood cell and plasma (in heparinised tube) were separated and stored, as was serum. These were snapped frozen in liquid nitrogen before being stored at − 80 °C for long term.2.32), water-jacketed organ bath containing 3 ml Tyrode solution  where the atrium was pinned flat on a silver stimulating electrode. The method for determining the local NE release was based on one previously described with modification 3H] NE  and ascorbic acid . To facilitate the uptake and exchange of [3H] NE into the transmitter store in pre-synaptic terminal, the atrium was stimulated at 5 Hz  for 10 s every 30 s, for 30 min. After that, excess radioactive NE was washed from the preparation by superfusion with Tyrode solution for 45 min at a rate of 3 ml/min. Superfusion was then stopped and the bath solution was replaced every 3 min. 0.5 ml sample from each solution change was added to 4.5 ml scintillation liquid  and the amount of radioactivity  was measured using a liquid scintillation counter . At the 16th minute, the atrium was stimulated at 5 Hz for 1 min . The solution was changed at the 27th minute to one containing a drug . A second stimulation was applied at the 49th minute for 1 min . At the end of the experiment, the atrium was immersed overnight in 4 U/ml papain (Sigma) and the radioactivity contained in the extract was determined (z). [3H]NE outflow was expressed as a proportional increase using the formula below:The spontaneously beating right atrium (RA) was isolated and transferred to a preheated (37 ± 0.2 °C), continuously oxygenated  was used as denominator. However this newer method appears to be more accurate as it takes into account the reduction of radioactivity content during the course of the experiment, and obviates the variability introduced by overnight digestion and subsequent measurement of radioactivity in the tissue extract.This method of calculating the  radioimmunoassay kit from Amersham Biosciences as previously described 125I] by employing similar method.Atrial cGMP level was determined using the Biotrak cGMP 2.7Left ventricles were cut into small pieces on ice and put into a tube containing 200 μl of buffer  and 1 mm glass beads (Biospec). The tissues were homogenised for 90 s  and centrifuged  for 10 min at 4 °C. The supernatants were recovered and used for protein analysis and Western blotting.15 μg of denatured protein from each sample was loaded into each well of a Bio-Rad Criterion XT 4–12% polyacrylamide gel. Molecular weight marker , positive control and standards (where available) were also loaded. The proteins were separated by electrophoresis and transferred to a polyvinylidene fluoride membrane . The non-specific binding sites were blocked with 5% non-fat skim milk in phosphate buffered saline (Fisher) containing 0.05% Tween-20 (Sigma) (PBST). The membrane was then probed with a primary antibody  overnight at 4 °C. On the following day, the membrane was rinsed twice and then washed 3 times with PBST (10 min each wash) before incubation with a suitable secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. The membrane was rinsed twice again and washed 3 times with PBST (10 min each wash). The relevant protein signal was amplified with luminol based chemiluminescence  and detected using light-sensitive film . The film was digitised and the relative densities were determined with a computer software . The results were normalised to β-actin that served as loading control .2.8detect® assay kit and protocol from Stratagene. This assay is based on the conversion of l-arg to l-citrulline by NOS in a stoichiometric way. l-citrulline can be separated from l-arg using an ion exchange column/resin. By using a radiolabelled l-arg , the rate of its conversion to l-citrulline was determined by measuring the radioactivity of the separated labelled fraction of l-citrulline. By performing the assay in the absence and presence of nNOS inhibitor (N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]N′-nitroguanidine, 20 μmol/L, Sigma), the activities of nNOS could be estimated. The assay was carried out in the presence of 5 μmol/L arginase inhibitor, N-hydroxy-nor-l-arginine  as l-arg can be converted to l-citrulline via alternative pathway involving arginase The activity of NOS in left ventricles was determined using NOS2.9t test was used to compare the results before (S1) and after application of inhibitor (S2) in the evoked NE release experiments. Statistical significance was accepted at p < 0.05.Data are presented as mean ± S.E.M. For comparisons among more than 2 groups, one way analysis of variance (ANOVA) was used to compare the means with post-hoc Tukey's HSD test to assess individual significance. Paired 33.1l-arg feeding (n = 13) and non-supplemented SHR  was observed. Plasma l-arg level was significantly lower (p < 0.01) in the SHR/not-sup group  compared with the WKY/not-sup group  but it increased to a comparable level following l-arg supplementation  following l-arg oral supplementation , but increased to 4.1 ± 0.5 pmol/mg protein  was 28% higher than the non-supplemented WKY , but was reduced with l-arg feeding  (t change B. Propor < 0.01) .3.43H]NE release in the l-arg fed SHR (n = 6) compared with the non-fed SHR (n = 7) (p < 0.01 when compared with respective S1 in paired t test) such that the two became no longer significantly different (Addition of a soluble guanylyl cyclase (sGC) inhibitor, 1H-oxadiazoloquinoxaline-1-one , to the organ bath suffusate prior to second stimulation (S2) caused a greater increase of evoked [ifferent .3.53H]NE release (S2) significantly in supplemented SHR , supplemented  and non-supplemented WKY  (p < 0.05 when compared with respective S1 in paired t test), but not in non-supplemented SHR .Highly selective nNOS inhibitor, N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]N′ nitroguanidine , increased proportional evoked [3.6l-arg fed (n = 13) and non-fed SHR (n = 10), and was not affected by 20 min incubation with ODQ (10 μmol/L)  was similar between  μmol/L) .3.7p < 0.05, n = 6 in each). l-arg feeding reduced the tyrosine hydroxylase in SHR (p < 0.05) but not in WKY (n = 6 in each) . The nNOS activity measured in-vitro based on l-citrulline conversion was similar between non-supplemented WKY , non-supplemented SHR  and supplemented SHR  (p = NS).We found that non-supplemented SHR had a significantly higher myocardial tyrosine hydroxylase protein compared with non-supplemented WKY (in each) . We did 4l-arg supplementation reduced the peripheral sympathetic hyperactivity in the SHR by attenuating evoked NE release from the right atria. Secondly, this effect is mediated at least in part via an increase in the l-arg–NO–cGMP signalling (by augmenting l-arg availability), and the associated reduction in myocardial tyrosine hydroxylase protein levels. Finally, the effect of l-arg supplementation on peripheral cardiac noradrenergic neurotransmission is a pre-synaptic one, as evidenced by lack of differences between treated and untreated SHR in in-vitro heart rate responses to exogenous norepinephrine.There are three novel findings from this present study. First, we demonstrated that l-arg studies have examined the effect on platelet aggregation, endothelial function and atheroma formation l-arg concentration. Dietary l-arg supplementation circumvented these potential issues as the increase in plasma l-arg level is relatively lower in the physiological range where this non-NO effect has not been reported to occur. In this study, we showed that oral l-arg supplementation could decrease the peripheral cardiac sympathetic hyperactivity in the SHR to a comparable level seen in the WKY. In addition, l-arg supplementation increased atrial cGMP to levels similar to that of the WKY. l-arg is the sole substrate of the enzyme NOS, with NO as the immediate but labile product. NO in turn activates sGC to synthesise cGMP, the key secondary messenger of this signalling system l-arg–NO–cGMP pathway plays an important neuromodulatory role in cardiac autonomic nervous system; augmenting the vagal arm and inhibiting the sympathetic counterpart l-arg treated SHR than non treated SHR, suggesting that this pathway had been augmented by l-arg supplementation. Similarly, the highly selective nNOS inhibitor increased the evoked NE release (S2) in the supplemented SHR but not in the non-supplemented SHR when compared with S1. Although the possibility of a non-NO effect could not be excluded completely, taken together, these results support the notion that l-arg feeding had augmented the l-arg–NO–cGMP pathway with a resultant decrease in evoked NE release. We cannot however, rule out the possibility that the reduction in evoked NE release may be an indirect result of enhanced parasympathetic neurotransmission by the augmented NOS–cGMP pathway. In addition, the conclusion arrived here is based on ex-vivo data, and therefore it remains to be established whether this is applicable to the in-vivo setting.Most published l-arg supplementation. To our knowledge, this is the first study to demonstrate this feature, although it is known that the SHR has higher tyrosine hydroxylase contents in key organs when compared with normotensive controls l-arg supplementation in the SHR. One possible explanation is cGMP activating phosphodiesterase-2 (PDE-2) resulting in increased hydrolysis of cAMP l-arg supplementation in either WKY or SHR groups. This would suggest that modulation of cAMP levels was not involved. Nonetheless, we cannot completely rule this out because localised changes of compartmentalised cAMP, a feature which has been well described One of the most intriguing and significant findings in this study is the reduction of myocardial tyrosine hydroxylase level in the SHR following l-arg levels should not be rate limiting to NO synthesis based on its pharmacokinetics l-arg supplementation has been shown to improve endothelial function, reduce atherosclerosis progression, and augment cardiac vagal function; all consistent with enhanced NO synthesis l-arg), and a relative l-arg deficiency in diseased states from elevated endogenous NOS inhibitor such as asymmetric dimethylarginine (ADMA), rather than an absolute one l-arg level was lower in the SHR when compared with the WKY, but this was increased to a comparable level following l-arg supplementation. In addition, we also found that l-arg supplementation had little effect on the WKY (either evoked NE release or cGMP levels). This was in keeping with other studies that showed that negative results were more likely to be obtained when healthy subjects were fed l-arg l-arg in the WKY following supplementation. Increased breakdown by hepatic arginase (whose activity is dependent on l-arg concentration) and increased renal excretion  may prevent further increase in plasma l-arg level beyond a certain set point l-arg in WKY, although neither this nor other studies have looked at this pharmacokinetic question specifically in rats.l-arg supplementation did not alter myocardial nNOS, eNOS or sGC protein levels in the SHR which may seem inconsistent with the increase in the cGMP brought about by l-arg supplementation. However, l-arg supplementation served only to provide additional substrate for NO production which in turn stimulated sGC to generate more cGMP. nNOS activity was also unchanged in the l-arg treated SHR, when measured in-vitro; consistent with the overall concept on the role of l-arg supplementation. We did not measure the amount of NO produced in-vivo because of its labile nature, but had shown that l-arg feeding increased the more stable secondary messenger cGMP in the SHR.l-arg supplementation did not affect the heart rate response to exogenous NE in the SHR. In other words, the effect of l-arg supplementation on cardiac noradrenergic neurotransmission is a pre-synaptic one and did not appear to change the post-synaptic beta adrenergic receptor driven excitability. Gene transfer of nNOS with an adenovirus to the heart has been found to attenuate evoked NE release, a pre-synaptic feature, via upregulation of NOS–sGC pathway In the double atrial experiments, we observed that l-arg, through the NO–cGMP pathway, probably plays a modulatory role during sympathetic nerve stimulation rather than under basal resting condition.Basal heart rate or arterial blood pressure did not differ between supplemented and non-supplemented SHR when measured under anaesthesia. From published literatures 4.1l-arg has been used widely as a potential therapeutic tool in both human and animal studies. Two recent clinical trials on patients with myocardial infarction have yielded conflicting results l-arg following supplementation and did not measure markers of NO production. This raised the question whether any effects observed could be confidently attributed to the l-arg treatment. Our study of l-arg supplementation on the SHR is significantly different, because it demonstrates both an increase in plasma l-arg as well as augmentation of nNOS–sGC–cGMP pathway with a reduction in evoked NE release. Moreover the SHR is from a homogenous population; the same is unlikely for the human subjects. Whether this intervention could restore autonomic balance and thus improve outcome in patients with hypertension will require a randomised clinical trial in a well controlled well selected clinical population. Accumulating evidence suggest that certain groups such as those with elevated endogenous NOS inhibitor ADMA and impaired l-arg–NO metabolism may be more likely to benefit from l-arg supplementation


N-(2-furanylmethylene)-2-aminothiadiazole have been prepared and characterized by their physical, spectral and analytical data. The synthesized Schiff-bases act as tridentate ligands during the complexation reaction with Co(II) and Ni2]Xn (where M=Co(II) or Ni(II), L=, X=NO3−, SO42−, C2O42− or CH3CO2− and n=1 or 2) andshow an octahedral geometry. In order to evaluate the effect of anions upon chelation, the Schiff-base and itscomplexes have been screened for antibacterial activity against bacterial strains e.g., Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.Co(II) and Ni(II) complexes with a Schiff base,


Ternary Ni(II) complexes of hydrazine and eight heterocyclic sulfonamides possessing carbonicanhydrase (CA) inhibitory properties, were prepared and characterized by elemental analysis, spectroscopic,magnetic, thermogravimetric, and conductimetric measurements. The complexes behave as strong inhibitorsfor two isozymes (I and II) of carbonic anhydrase.


Some of them inhibited the growth of several fungi species (Aspergillus and Candida spp.)Complexes containing 1,3,5-tris-(8-hydroxyquinolino)-trichlorocyclotriphosphazatriene, a newcyclophosphazene ligand, and Co(II), Cu(II) and Ni(II) were prepared. The new complexes, having the generalformula [MLCl


The effect of insulin-like growth factor II (IGF-II) on tumour development in the mouse mammary gland was studied. To promote extra IGF-II expression in the mammary gland, sheep beta-lactoglobulin regulatory elements were attached to the coding regions of the mouse Igf-2 gene and injected into the pronuclei of mouse zygotes. Mammary tumours developed in each of the four independent lines of mice which expressed transgene IGF-II in the gland. Tumours from two of the lines grew after transplantation to both male and female hosts. Primary tumours contained stromal and epithelial regions, but the tumours were dominated by mammary adenocarcinoma after transplantation. The tumours expressed high levels of Igf-2 mRNA transcribed from the integrated transgenes.


To identify the causative mutation of canine progressive retinal atrophy (PRA) segregating as an adult onset autosomal recessive disorder in the Basenji breed of dog.Basenji dogs were ascertained for the PRA phenotype by clinical ophthalmoscopic examination. Blood samples from six affected cases and three nonaffected controls were collected, and DNA extraction was used for a genome-wide association study using the canine HD Illumina single nucleotide polymorphism (SNP) array and PLINK. Positional candidate genes identified within the peak association signal region were evaluated.10(P) value of 4.65 was obtained for 12 single nucleotide polymorphisms on three chromosomes. Homozygosity and linkage disequilibrium analyses favored one chromosome, CFA25, and screening of the S-antigen (SAG) gene identified a non-stop mutation (c.1216T>C), which would result in the addition of 25 amino acids (p.*405Rext*25).The highest -LogSAG mutation in dogs affected with retinal degeneration establishes this canine disease as orthologous to Oguchi disease and SAG-associated retinitis pigmentosa in humans, and offers opportunities for genetic therapeutic intervention.Identification of this non-stop  SAG), also known as arrestin, encodes a major soluble rod outer segment protein that exemplifies a family of inhibitory proteins that bind tyrosine-phosphorylated receptors to block their interaction with specific G-proteins to terminate a signal chain. In the retina, arrestin binds to activated rhodopsin [HGMD). This disease has an unusual but characteristic clinical feature, the Mizuo-Nakamura phenomenon, in which an unusual golden-yellow discoloration of the fundus disappears in the dark-adapted condition and reappears shortly after exposure to light [The gene S-antigen  comprises a group of genetically inherited diseases affecting dogs of various breeds. Similar to RP in humans, PRA is characterized by photoreceptor degeneration causing progressive vision loss, culminating in blindness. This is a highly heterogeneous group of diseases with more than 12 different causative gene mutations already identified in canine populations, causing either early or late onset disease, and inherited as autosomal dominant, autosomal recessive, or X-linked [for a review, see prcd), late onset retinal degeneration affecting multiple breeds caused by a point mutation in the PRCD gene [prcd excluded allelism between these two similar diseases [PRCD genotyping. Complicating this broadly recognizable clinical phenotype has been considerable heterogeneity in the disease manifestation, and uncertainty about whether this clinical heterogeneity represented a single protean disorder or an underlying genetic heterogeneity. This problem has been further complicated by the extremely small gene pool of Basenjis in the American breeding population and the multiple inbreeding loops apparent in pedigree analysis.In the Basenji breed, the adult onset of PRA was observed, with initial visual loss in dim light (night blindness), which gradually progressed to total blindness. Initial visual loss affects the peripheral visual field, but unless the dog is used for high visual performance tasks such as agility work, the reduction in the visual field (tunnel vision) may not be apparent.Despite tunnel vision and night blindness, many Basenjis affected with PRA retain adequate forward daylight vision for many years, sometimes for their entire natural life. This phenotype highly resembles progressive rod-cone degeneration  enables genome-wide association mapping in populations even when the extended pedigree information normally required for linkage analysis is either incomplete or missing entirely. Furthermore, the development of genome-wide single nucleotide polymorphism (SNP) chip arrays has made such mapping widely available for humans and several other species, including the dog. In humans, mapping has been particularly successful in isolated populations -20. In tSAG that cosegregated with the disease phenotype and caused the disease. This creates an opportunity to study the molecular basis of SAG-associated retinal diseases, and a model for evaluating potential therapeutic approaches for Oguchi disease and SAG-associated RP in humans.In this study, we report a genome-wide association study (GWAS) to map a specific form of PRA in the Basenji breed of dog, using six affected dogs and three control dogs. Together with homozygosity analysis and consideration of the mode of inheritance, we identified a non-stop mutation in tentatively affected with PRA, 18 nonaffected controls beyond the assumed age of risk (at least 6 years of age), and 37 with no assigned phenotype either because they were younger than the assumed age of risk or due to lack of information. Blood was stored at 4 °C or -20 °C until DNA extraction. DNA was similarly extracted from blood samples of 110 purebred dogs from 22 different breeds not known to segregate this disease (Appendix 1).All procedures involving animals were conducted following the guidelines of the Institute for Laboratory Animal Research , the U.S. Public Health Service , and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Blood samples were collected by cephalic or jugular venepuncture into vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) anticoagulant from 80 purebred Basenji dogs: 19 diagnosed clinically as affected with PRA, six diagnosed clinically as Ascertainment of disease phenotype was based entirely on indirect ophthalmoscopic examination by board-certified veterinary ophthalmologists. In the minority of cases, the ophthalmoscopic diagnosis was confirmed with clinical electroretinography. The cases studied in the present report include only dogs examined and diagnosed by either or both of two of the authors , although in several cases the initial diagnostic examination was previously performed by other veterinarians.Of the 19 purebred Basenji dogs diagnosed clinically as affected with PRA, six were selected as cases for association analysis, based on confidence in the disease ascertainment (dogs affected with stage II PRA), consistency of the disease phenotype among the selected dogs, pedigree information supporting autosomal recessive inheritance identical by descent (IBD), and choosing the least closely related set of dogs affected with PRA available (four dogs had no parents or grandparents in common).Among 43 Basenji dogs not expressing PRA symptoms, three dogs not affected with PRA were selected for the control group based on having no parents or grandparents in common with each other, being clinically diagnosed as nonaffected with PRA at an age beyond the apparent age of risk for onset of the disease , being free of other ocular abnormalities that might obscure or confuse the presence of PRA, and being close relatives of the affected dogs chosen as cases. The unaffected dog at age 6, although borderline for the age of onset, was a sibling of an affected dog, and was chosen to accomplish the fourth criterion.PLINK) [Samples were genotyped using the Illumina Canine SNP chip (HD Canine SNP Chip), which comprises 173,662 SNP loci, following the manufacturer's standard protocol. Genotypes were called using GenomeStudio . Genotype calls were converted into Plink-format files, and association was tested using the association command without pedigree or sex information (PLINK) . The genHomozygous block analysis was performed on affected dogs only (n=6) using Plink, under the following criteria: sliding window criteria: 1,000 Kb, 150 SNPs, five missing calls, one heterozygous call, 0.05 threshold; homozygous segment criteria: length 1,000 Kb, 150 SNPs, 50 density (Kb/SNP). The output was then filtered for those chromosomes where all six cases showed a minimum of one homozygous segment anywhere in the chromosome. The segments then were aligned within each chromosome, to identify those where all six dogs shared the same homozygous block. For all such regions, genotype calls were retrieved to determine whether all homozygous blocks were homozygous for the same haplotype. If so, then the “affected haplotype” was compared to haplotypes observed in the control group.SAG), which has been associated with Oguchi disease, as well as retinitis pigmentosa [KCNJ13), which has been associated with snowflake vitreoretinal degeneration and Leber congenital amaurosis [Within a homozygous block on chromosome 25 (CFA25), two genes were considered candidates: S-antigen, arrestin . KCNJ13 gene screening was conducted on six dogs: two affected Basenji dogs, three unaffected Basenjis, and one normal Boxer dog. Nine primer pairs were designed to amplify the three exons of the gene (Appendix 2B). PCR products of both genes were sequenced, and sequences aligned using Sequencher 4.2.2 Software .SAG non-stop mutation was genotyped on the complete set of Basenji dogs available (N=80) with PCR using primer pairs flanking the mutation , followed by sequencing. An allele-specific extension test was then designed to screen 110 dogs from 22 breeds (Appendix 1). One primer pair, containing a forward primer specific to the wild-type allele, amplifies a 259 bp fragment, and a separate primer pair, containing a forward primer specific to the mutant allele, amplifies a 531 bp product (Appendix 2C). Both forward primers contain a mismatch in the 3′ penultimate base.The identified . Basenjis affected with PRA typically exhibited evidence of stage I PRA at about 5 years of age. These dogs when reexamined ophthalmoscopically at a later age typically showed progression of disease by thinning (attenuation) of the retinal vasculature, which usually became ophthalmoscopically evident by 6 or 7 years of age. This phenomenon, taken as evidence of reduced blood flow through the retinal vasculature, is referred to as stage II or mid-stage PRA. In all of these respects, Basenjis affected by PRA closely resemble dogs of other breeds affected by the prcd form of PRA.PRA as a clinical disease in the Basenji was, in broad terms, typical adult onset canine retinal degeneration. The first ophthalmoscopically observed evidence of disease was irregular hypo- and hyperreflectivity of the tapetal fundus. This phenomenon, taken as evidence of retinal thinning, is referred to as stage I or early stage PRAClinical diagnosis of PRA in the Basenji was complicated by several factors, however. These included a common prevalence of conus in dogs affected with PRA and nonaffected dogs. Conus refers to a roughly triangular region immediately superior to the optic nerve head in which the tapetum is yellow (and may be hyperreflective) compared to the surrounding green tapetal region. This phenomenon is seen, though less frequently, in normal dogs of other breeds, but also is seen in dogs of other breeds as one of the earliest signs of retinal thinning. In the fundus of Basenjis affected with PRA, this area of conus is sometimes observed to expand and become hyperreflective. Second, the optic nerve head is typically less myelinated and thus less extensive over the fundus than is typical of other breeds of dog. Also seen repeatedly in Basenjis affected with PRA and nonaffected Basenjis was a strange mottled golden-yellow-brown discoloration of the tapetal fundus that obscured the tapetal reflection in a patchy manner, and gave the fundus a beaten bronze appearance. This fundus change, termed bronzing, could either mask the earliest signs of retinal degeneration in some Basenjis affected with PRA or produce a mottled variation in tapetal reflectivity that to some extent mimicked the earliest signs of retinal degeneration in Basenjis that eventually proved to be not affected with PRA.Finally, the fundus appearance in Basenjis affected with PRA was often patchy, or non-uniform, within a given eye, between the two eyes of a given affected dog, and among affected dogs. To some extent, this fundus appearance is observed in other forms of canine PRA, but was particularly evident in the eyes of Basenjis affected with PRA .The breeding population of Basenjis in the United States of America  descends from a small number of founders, and has a complicated pedigree structure with multiple inbreeding loops and overlapping generations. In combination with the late age of onset, and the heterogeneous clinical nature of the disease, this made pedigree analysis more uncertain than usual in terms of establishing the likely mode of inheritance. A subset of six affected dogs were thus chosen for the genome-wide association study with criteria (see the Methods) intended to maximize the likelihood that all six dogs were affected with a single autosomal recessive disease inherited from a common ancestor, and thus identical by descent .10(P) value of 4.65 was obtained for 12 SNPs: one SNP on CFA4, seven on CFA13, and four SNPs on CFA25  spanning these seven SNPs in affected dogs showed five homozygous blocks containing one or two of these seven significant SNPs, with distances ranging from 45 Kb to 929 Kb . AlthougThe four SNPs on CFA25 were all within one 2.09 Mb homozygosity block , in which all affected dogs were homozygous for one allele for each SNP, and the three unaffected dogs were homozygous for the complementary allele. One SNP at position 48,147,815 appeared to break this homozygosity block into two blocks of 1.25 Mb and 0.83 Mb, but this was considered unreliable as all genotype calls for this SNP were either heterozygous or no call (Appendix 3).Genotype calls from the affected dogs only (n=6) were analyzed for homozygosity blocks greater than 1.0 Mb, excluding sex chromosomes. Five hundred and thirty-nine such blocks were identified. After sorting by chromosome and aligning homozygous regions within each chromosome, five loci were identified where all six dogs shared a homozygous segment : CFA 6, KCNJ13 and SAG. Primer pairs were designed to amplify all exons of both genes, including flanking sequences from the 5′ untranslated region (UTR), introns, and 3′ UTR , and used to generate amplicons from DNA samples representing six dogs: two affected Basenji dogs, three non-affected Basenjis, and one normal Boxer dog.From association and homozygosity analyses, the association peak on chromosome 25 was considered the best potential candidate region to investigate further. Within the 2.09 Mb homozygosity block encompassing all four SNPs with the maximum p value, two genes were evaluated as positional candidates: KCNJ13, previously associated with snowflake vitreoretinal degeneration and Leber congenital amaurosis in humans [KCNJ13 was not further investigated as a candidate.n humans ,31, was SAG, as identified in the Canfam2 genome sequence at chr25:47,814,602–47,845,820, comprised 16 exons, 15 of which are coding. Eighteen amplicons were generated encompassing all exons and their flanking 5′ and 3′ UTRs and intronic sequence using primers flanking each exon (Appendix 2A), and sequenced. Ten exonic variations were identified (Appendix 4B). Two (Nos.1 and 2) were in the 5′ UTR in exons 1 and 2, respectively; five (Nos. 3–7) were in exonic coding sequence but all represented synonymous third-base codon changes and two (No. 9 and 10) were in the 3′ UTR.SAG variations, all Basenjis  were homozygous for one allele that differed from the allele in the boxer and the reference sequence. The two exceptions were variations 8 and 9, which segregated informatively. Since SNP number 9 was located in the 3′ UTR of the gene, it seemed unlikely to represent a causative mutation.Furthermore, for eight of these ten Variation number 8 (Appendix 4B), however, was a much more significant tyrosine to cysteine transition mutation at position CFA25:47,845,680 .The OMIA) currently lists 614 traits and disorders in dogs, of which 238 are Mendelian. For 164 of those, the causative mutation is known. More pertinently, 24 mutations have currently been reported in 18 genes as causatively associated with retinal diseases in at least 58 dog breeds. Approximately 12 genes result in a PRA phenotype while the rest affect other retinal cells or structures [The online Mendelian inheritance in the animal database , and from pedigree analysis demonstrated probable IBD recessive inheritance; and controls were selected that were free of retinal disease at an age likely to be beyond the apparent age of risk (at least 6 years of age) and were appropriately related to the dogs selected as cases. This reduced the number of “high confidence dogs” selected to just six affected dogs and three controls.GWAS initially identified three potentially significant loci associated with the disease phenotype, on CFA4, CFA13, and CFA25. Haplotype analysis, assuming recessive inheritance, excluded the CFA4 locus. Although homozygosity blocks were observed on CFA13 and CFA25 in affected dogs, the LD expected from the Basenji breed structure strongly favored CFA25 over CFA13. The success of association mapping combined with homozygosity analysis to map the disease to a single locus using fewer than ten dogs confirms that the minimum number of dogs needed for GWAS is breed dependent, and can be low in breeds where LD blocks are large. However, the extensive LD (in this case 2.09 Mb) presents a potentially frustrating tradeoff. If either no obviously appealing candidate genes had been present within the homozygosity block, or many were, a much larger number of affected dogs would have been needed to reduce the LD, the number of potential causative genes, and the genomic region to be sequenced and further evaluated.MERTK [THAP1 [C8orf37 [A parallel approach using homozygosity analysis to directly identify candidate regions was also undertaken. This method has successfully mapped autosomal recessive diseases in humans, especially in consanguineous families. Examples include mapping of a severe autosomal recessive RP to subsequently identify a causative mutation in MERTK ; mappingK [THAP1 ; and hom[C8orf37 . BecauseSAG mutation in all six dogs affected with PRA in the study set. Non-stop mutations are point mutations that convert the stop codon to code for an amino acid, resulting in a deduced elongation of the C-terminus of the protein since the translation continues into the  3′ UTR. In one human study, such non-stop mutations represented about 0.2% of the codon-changing mutations (119 examples in 87 genes) [SAG sequence, the first alternative in-frame stop codon is 72 bases after the mutated stop codon and 37 bp upstream from the polyadenylation signal, suggesting that the mutated protein, if translated, would be extended by a further 25 amino acids  . These mno acids . This ape et al.  and no oexpasy) indicates that the additional 25 amino acids would change the isoelectric point to 6.78 (from 6.0 in the wild-type) and would add four positively charged residues, for a total of 56 in the mutant versus 52 in the wild-type. Although the estimated half-life, predicted localization, and instability index would not change, it is not certain that the mutant protein would fold correctly. If it does, then the mutant protein's ability to bind appropriately to its G-protein-coupled receptor, rhodopsin, would likely be impaired, and this would likely lead to the retinal degeneration phenotype. SAG (arrestin) is critically involved in quenching the photoactivated phosphorylated rhodopsin apoprotein (P-Rh*), a process that involves conformational changes in rhodopsin and SAG [SAG protein were produced, an elongation of its C-tail by 25 amino acids would likely impair its ability to be anchored in its N-domain and to respond accurately, appropriately and in a timely manner to changes in its receptor.Computational protein analysis of the deduced protein  and more than ten non-refseq genes. Because the present studies were undertaken in a patient population for which only phenotypic data and DNA were available, ongoing and future studies in purpose-bred dogs will provide tissues, previously and currently unavailable, for functional, molecular, transcriptomic, proteomic, and cytologic studies to address critical questions that arise from the present investigations, particularly the effect of the non-stop mutation on the pathology of the disease, and the involvement of other genes within the LD interval on the course of the disease. Basenji dogs affected with PRA can thus serve as a novel large animal model of SAG-associated retinal degeneration, offering investigational opportunities to better understand the primary disease mechanism, explore causes for the wide variation of disease as observed in humans and Basenji dogs, investigate the effect of the non-stop mutation on residual function of the protein, test the potential role of light-exposure on the phenotype, and evaluate potential therapeutic approaches.Within the 2.09 Mb LD-homozygous interval identified as the disease locus, four refseq genes are located (