Proventricular dilatation disease (PDD) is a fatal disorder threatening domesticated and wild psittacine birds worldwide. It is characterized by lymphoplasmacytic infiltration of the ganglia of the central and peripheral nervous system, leading to central nervous system disorders as well as disordered enteric motility and associated wasting. For almost 40 years, a viral etiology for PDD has been suspected, but to date no candidate etiologic agent has been reproducibly linked to the disease.Analysis of 2 PDD case-control series collected independently on different continents using a pan-viral microarray revealed a bornavirus hybridization signature in 62.5% of the PDD cases (5/8) and none of the controls (0/8). Ultra high throughput sequencing was utilized to recover the complete viral genome sequence from one of the virus-positive PDD cases. This revealed a bornavirus-like genome organization for this agent with a high degree of sequence divergence from all prior bornavirus isolates. We propose the name avian bornavirus (ABV) for this agent. Further specific ABV PCR analysis of an additional set of independently collected PDD cases and controls yielded a significant difference in ABV detection rate among PDD cases  compared to controls  . Partial sequence analysis of a total of 16 ABV isolates we have now recovered from these and an additional set of cases reveals at least 5 distinct ABV genetic subgroups.These studies clearly demonstrate the existence of an avian reservoir of remarkably diverse bornaviruses and provide a compelling candidate in the search for an etiologic agent of PDD. Cyanopsitta spixii), have been severely impacted by PDD.Proventricular dilatation disease (PDD) is considered by many to be the greatest threat to aviculture of psittacine birds (parrots). This disease has been documented in multiple continents in over 50 different species of psittacines as well as captive and free-ranging species in at least 5 other orders of birds -5. Most,PDD is an inflammatory disease of birds, first described in the 1970s as Macaw Wasting Disease during an outbreak among macaws (reviewed in ). PDD prThe cause of PDD is unknown, but several studies have raised the possibility that PDD may be caused by a viral pathogen. Evidence for an infectious etiology stems from the initial outbreaks of Macaw Wasting Disease, and other subsequent outbreaks of PDD ,10. RepoTo address this question, we have turned to a comprehensive, high throughput strategy to test for the presence of known or novel viruses in PDD affected birds. We employed the Virus chip, a DNA microarray containing representation of all viral taxonomy to interrogate 2 PDD case/control series independently collected on two different continents for the presence of viral pathogens. We report here the detection of a novel bornavirus signature in 62.5% of the PDD cases and none of the controls. These bornavirus-positive samples were confirmed by virus-specific PCR testing, and the complete genome sequence has been recovered by ultra-high throughput sequencing combined with conventional PCR-based cloning.Bornaviridae family and represents the first full-length bornavirus genome cloned directly from avian tissue. Subsequent PCR screening for similar ABVs confirmed a detection rate of approximately 70% among PDD cases and none among the controls. Sequence analysis of a single complete genome and all of the additional partial sequences that we have recovered directly from the PDD case specimens suggests that the viruses detected in cases of PDD form a new, genetically diverse clade of the Bornaviridae.Bornaviruses are a family of negative strand RNA viruses whose prototype member is Borna Disease Virus (BDV), an agent of encephalitis whose natural reservoir is primarily horses and sheep . AlthougTo identify a possible viral cause of PDD, we applied the Virus chip, a DNA microarray containing 70 mer oligonucleotide probes representing all known viral sequences conserved at multiple nodes of the viral taxonomic tree ,18 to idBornaviridae signature was detectable in 62.5% of the cases and none of the controls  and random hexamer primers, and then PCR-amplified using a modified random amplification strategy compatible with the Solexa sequencing platform . An initial set of 1.4 million 33 mer reads was obtained from this template material. Filtering on read quality, insert presence, and sequence complexity reduced this data set to 600,000 unique reads. Additional ELAND and iterative BLAST analyses  from PDD cases and controls were screened for ABV sequences in a blinded fashion . For this analysis, we targeted three regions of the genome: 1) the L gene region of the genome that we used for PCR confirmation of the microarray results, Figure . In contBecause we applied stringent inclusion criteria for the above-described association analysis study, a number of ABV (+) and ABV (-) samples were excluded. From these materials, six additional ABV isolates were detected – 5 derived from cases considered clinically suspicious and a sixth isolate derived from a confirmed PDD case for which only GI content and liver specimens were available. Additional PCR screening of a set of 12 PDD control crop biopsy specimens provided to us unblinded again yielded solely ABV PCR (-) and GAPDH (+) results. These samples were excluded from the association analysis because we knew their clinical status prior to screening. We note that inclusion of these samples in statistical analyses would not diminish the association of ABV with known or suspected PDD.Bornaviridae. Here, our description of results is restricted to comparison with representative members of the 4 major isolates of BDV, but virtually identical results were obtained when all available BDV sequences were analyzed.Recovery of partial sequence from additional isolates of ABV ) from 3 distinct regions of the ABV genome provided the opportunity to further investigate the genetic diversity within this new branch of the th subgroup among the ABVs  that are likely to play a significant role in this disease. Through microarray analysis and follow-up PCR, we detected ABV sequences in 62.5% of the PDD cases in a set of specimens from two carefully collected PDD case/control series originating from two different continents. We confirmed that these assays faithfully reflect the presence of full-length bornavirus in ABV PCR positive specimens through cloning of the complete ABV vRNA sequence directly from RNA extracted from one of these ABV PCR positive PDD case specimens. We next found evidence for a significant association between the presence of ABV and clinically confirmed PDD in follow-up blinded PCR screening of a set of additional PDD cases and controls, with ABV was detected in 71% of PDD cases and none of the controls .Almost all prior sightings of bornaviruses in nature have been among mammals, and the mammalian isolates have been remarkably homogeneous at the sequence level  PDD cases have well-described neurological symptoms such as ataxia, proproceptive defects and motor abnormalities; and (ii) the central GI tract pathology in the disorder results from inflammation and destruction of the myenteric ganglia that control peristaltic activity. However, despite our success in ABV detection in PDD, we did not observe ABV in every PDD case analyzed. There are several possible explanations for this result. First, we do not know the tissue distribution (tropism) of ABV infection, or how viral copy number may vary at different sites as a function of the stage of the disease. By weighting our sample collection towards clinically overt PDD, we may have biased specimen accrual towards advanced disease. At this stage, where destruction of myenteric ganglial elements is often extensive, loss of infected cells may have contributed to detection difficulties . There are many precedents for such temporal variation in clinical virology – for example, in chronic hepatitis B viral loads typically decline by several orders of magnitude over the long natural history of the infection . It is aFinally, there could actually be multiple etiologic agents in PDD, with ABV infection accounting for only ~70% of the cases. Certainly both human and veterinary medicine are replete with examples of multiple agents that can trigger the same clinical syndrome – for example, at least 5 genetically unrelated viruses (hepatitis viruses A-E) are associated with acute hepatitis, and at least 3 of these can be implicated in chronic liver injury; similarly, several agents  are implicated in bronchiolitis. To investigate this possibility, further high-throughput sequencing analysis of PDD cases that were negative for bornaviruses by PCR screening is currently underway.Bornaviridae family now opens the door to such investigations.Although ABV is clearly a leading candidate etiologic agent in PDD, formally establishing a causal role for ABV in PDD will require further experimentation. Such experiments could include (i) attempts to satisfy Koch's postulates via cultivation of ABV, followed by experimental transmission of infection and disease in inoculees, (ii) examination of seroprevalence rates in flocks with high and low PDD incidences, (iii) documentation of seroconversion accompanying development of PDD-like illnesses and (iv) examination of PDD cases by immunohistochemistry or in situ hybridization for evidence of colocalization of ABV infection at sites of histopathology. The recovery and characterization of a complete ABV genome and multiple isolates from this diverse new branch of the Bornaviridae family is not confined to a set of genetically homogeneous species as was previously thought, but actually encompasses a set of heretofore unanticipated genetically diverse viral species.By combining clinical veterinary medical investigation with comprehensive pan-viral microarray and high throughput sequence analyses, we have identified a highly diverged set of avian bornaviruses directly from tissues of PDD cases, but not controls. These results are significant for a number of reasons. First, they provide a compelling lead in the long-standing search for a viral etiology of PDD, and pave the way for further investigations to assess the link between ABV and PDD. Second, these results also unambiguously demonstrate the existence of an avian reservoir of bornaviruses, expanding our understanding of the bornavirus host range. Finally, these results also provide the first evidence that the Two independent sets of PDD case and control specimens collected from two distinct geographic locations were independently prepared for pan-viral microarray screening and subsequent PCR screening. Sampling collection and inclusion criteria for each set are described below. Detailed information on each sample, along with results from histology, microarray, and PCR assays are provided in Additional file ater  solution immediately upon harvest and frozen within 2 minutes of collection at -20°C to -80°C according to manufacturer's protocol, and held in this manner until shipped. A duplicate sample was fixed in 10% buffered formalin for routine histological examination with H & E stain. Time of frozen storage varied (2 weeks to 12 months) as samples were accumulated prior to shipping frozen. Clinically affected birds submitted as positives were euthanized under isoflurane anaesthesia and mixed tissues  were placed into RNAlater within 1 minute of death and frozen within 2 minutes of death. Duplicate samples were collected for histopathologic diagnosis of PDD.All specimens provided for initial screening were crop tissue biopsies obtained from live psittacine birds to be used as normal controls or multiple tissue samples collected from clinically diseased birds at the time of euthanasia. Specimens were collected from client-owned birds from approximately August 2006 to May 2008 (All samples collected by S. Clubb). All of these samples originated from the southeast region of Florida. Crop biopsy tissue was collected from live birds under isoflurane anaesthesia. Following routine surgical preparation and sterile technique, the skin was incised over the center of the crop. The crop tissue was exposed and a section of tissue removed taking care to include large visible blood vessels. Fresh crop biopsy tissue was trimmed into tissue slices < 5 mm thick and submersed in RNAlPDD-positive cases were required to meet the following criteria 1) Clinical history of characteristic wasting/malabsorption syndrome with dilation of the proventriculus and/or ventriculus and presence of undigested food in the stool and in most cases, a clinical history of ataxia or other CNS signs consistent with clinical PDD, and 2) histopathology confirming the presence of moderate to extensive lymphoplasmacytic ganglioneuritis affecting crop tissue and at least one of the following additional areas: proventriculus, ventriculus, brain, adrenal gland, or myocardium. PDD-negative controls were required to be from birds with no evidence of lymphoplasmacytic neurogangliitis on histopathology derived either from 1) normal birds with no clinical history of PDD or no known exposure to PDD or 2) birds which died of other causes. Crop biopsies from samples from living birds classified as suspicious cases were also submitted. Suspicious cases were defined histologically as having lymphocytes and plasma cells surrounding neurons but not infiltrating into the neurons. An additional specimen derived from a live bird raised with two necropsy-confirmed PDD birds in Virginia was also collected for analysis. Here, only cloacal swab and blood specimens were available and the lack of histopathological confirmation and crop tissue excluded this specimen from the ABV-PDD association analysis. However, we did perform ABV PCR on these clinically suspicious specimens and the resulting viral sequences isolated were included in the subsequent comparative sequence analyses.For RNA extractions, specimens were thawed in RNALater, sliced into 0.5 mm × 0.5 mm pieces, transferred to 2 ml of RNABee solution , homogenized with freeze thawing and scapel mincing, then extracted in the presence of chloroform according to manufacterer's instructions. Resulting RNA was next incubated with DNase  to remove any potential contaminating DNA present in the specimen.Tissue samples were obtained from psittacine birds submitted to the Division of Avian and Fish Diseases, Kimron Veterinary Institute (KVI) Bet Dagan, Israel, for diagnostic necropsy between July 2004 and March 2008. A few additional specimens were obtained through private veterinarians. Some tissues were kept for nearly 4 years frozen either at -20°C or -80°C prior to testing, while others were fresh tissues from recent cases. The types of banked frozen tissue varied from case to case, while for some of the older cases only gastrointestinal content had been banked. Clinical histories for these birds were available from the submission forms or through communication with the submitting veterinarians. The results of ancillary tests performed at the KVI were available through the KVI computerized records.Only cases for which appropriate histological sections were available for inspection were considered for this study. These had to include brain and at least two of the following tissues: crop, proventriculus, ventriculus. The tissue-types examined for each bird for which specimens were provided are listed in Data File S1. PDD-positive cases were required to have evidence of lymphoplasmacytic infiltration of myenteric nerves and/or ganglia within one or more of the upper GI tract tissues mentioned above. These were all derived from birds that had been suspected to have PDD based on their clinical case histories and/or necropsy findings. PDD-negative controls had no detectable lesions and no evidence of non-suppurative encephalitis. For most birds in the PDD-negative group, a cause of death (other than PDD) has been determined. Two birds that came from a known PDD outbreak, but showed only cerebral lymphoplasmacytic perivascular cuffing, were classified as 'suspicious'. These were excluded from the statistical analysis, as were all other birds for which a PDD status could not be clearly determined and classified as 'inconclusive' .3 of each tissue was macerated by two passages through a 2.5 ml sterile syringe and transferred into sterile test tubes containing 4 ml nuclease-free PBS. The content of the tubes was mixed by vortex for 30 sec, and the tubes were placed overnight at 4°C. RNA extraction was performed on the following day, using either the QIAamp® viral RNA kit  or the TRI Reagent® kit , following the manufacturers' instructions. The end product was either provided lyophilized  as a dry pellet, or re-suspended in 40 ul nuclease-free water.When possible, a sample of brain as well as a combined proventricular/ventricular sample was prepared for RNA extraction for each bird. If not available, other tissues and/or gastrointestinal content were used  that are 100% conserved across the 12 other fully sequenced bornavirus genome isolates in NCBI  were utilized for initial follow-up PCR and sequence confirmation of microarray screening results. Briefly, 1 ul of the randomly amplified nucleic acid prepared for microarray hybridization from all specimens was utilized as template for 35 cycles of PCR, under the following conditions: 94°C, 30 seconds; 50°C, 30 seconds; 72°C, 30 seconds. Resulting PCR products were gel purified, subcloned into the TOPO TA cloning vector pCR2.1  and sequenced with M13F and M13R primers.Initial PCR primers were generated based on two of the 70 mer microarray probes with hybridization signal in the Bornaviridae PCR assay, these ABV consensus primers were found to be > 100X more sensitive for ABV detection compared to BDV_LconsensusF and BDV_LconsensusR primers, and were thus utilized to re-screen the initial set of PDD case and control samples provided for microarray analysis  and all subsequently provided samples. Two additional PCR primers in the N (ABV_NconsensusF: 5'-CCHCATGAGGCTATWGATTGGATTAACG-3' and ABV_NconsensusR: 5'-GCMCGGTAGCCNGCCATTGTDGG-3') and M (ABV_MconsensusF: 5'-GGRCAAGGTAATYGTYCCTGGATGGCC-3' and ABV_PconsensusR: 5'-CCAACACCAATGTTCCGAAGMCG-3') that mapped to conserved sequences shared between the complete ABV genome sequence and the 14 other fully sequenced BDV genomes in the NCBI database were also employed for PCR screening of PDD cases and controls.Sequences recovered from BDV_LconsensusF and BDV_LconsensusR PCR products were aligned, and an additional set of ABV consensus primers biased towards the ABV sequences were identified: ABV_LconsensusF, 5'-CGCCTCGGAAGGTGGTCGG-3'  and ABV_LconsensusR, 5'-GGCAYCAYCKACTCTTRAYYGTRTCAGC-3' . Using identical PCR cycling conditions as described above for the microarray-based st strand synthesis step. The resulting aRNA was next used as input for a modified version of Genomic DNA sample preparation protocol for ultra high-throughput Solexa sequencing . 400 ng of the input aRNA was reverse-transcribed with reverse transcriptase  using a random nonamer tailed with 19 bp of the Solexa Long (5'-CACGACGCTCTTCCGATCTNNNNNNNNN-3') primer sequence . Following termination of reaction, first strand cDNA products were purified from the reaction with Qiagen MinElute spin column . To ensure stringent separation from primers, the MinElute eluate was then filtered through a Microcon YM30 centrifugal filter . The resulting eluate served as template for 2nd strand synthesis in a standard Sequenase 2.0  reaction primed with a random nonamer tailed with 22 bp (5'-GGCATACGA GCTCTTCCGATCTNNNNNNNNN-3') of the Solexa Short primer sequence . Double-stranded DNA products were separated from primers and very short products through a second Qiagen MinElute spin column run followed by a Microcon YM50 centrifugal filter. This eluate was used as template for 10 cycles of PCR amplification with the full length Solexa L and S primers using KlenTaq LA DNA polymerase mix . PCR product was purified from the reaction with a MinElute spin column. Following cluster generation, Solexa sequencing primer was annealed to the flow cell, and 36 cycles of single base pair extensions were performed with image capture using a 1G Genome Analyzer . The Solexa Pipeline software suite version 0.2.2.6  was utilized for base calling from these images. Using software default quality filters, cycles 4–36 were deemed high quality, resulting in a total of 1.4 million 33 mer reads for downstream sequence analyses.500 ng of total RNA derived from one of the PDD case specimens was linearly amplified via modification of the MesssageAmp aRNA kit . To ensure the amplification of both mRNA and vRNA present in the specimen, T7-tailed random nonamer was mixed in an equimolar ratio with the manufacturer-provided T7-oligo(dT) primer during the 1Efficient Local Alignment of Nucleotide Data) and BLAST analyses. To filter reads from our analysis potentially derived from psittacine host tissue, the working set of reads were aligned to a database of all Aves sequences from NCBI  using ELAND, which tolerates no more than 2 base mismatches, and discards both low quality reads and reads with low sequence complexity. Reads that did not align to the Aves database by ELAND analysis were next re-aligned to the Aves database for high stringency blastn analysis , followed by progressively lower stringencies , corresponding to reads containing only 22 nucleotide identities to sequences in the Aves database. To identify reads with some homology to Bornaviridae sequences in the resulting set of 322,790 host-filtered reads, we re-implemented the ELAND/iterative blastn analysis strategy (down to ≥ 15 nucleotides identity) using a database of all NCBI BDV sequences (n = 207) augmented by our previously recovered ABV sequences (n = 5). An additional iterative tblastx analysis was incorporated to capture distantly related reads that shared similarity to the known BDV sequences only at the level of predicted amino acid sequence (down to ≥ 6 amino acid identity).Reads sharing 100% identity to each other or the Solexa amplification primers were filtered, reducing our initial set of 1.4 million reads to a working set of 600,000 unique reads. In order to quickly assess the homology of this set of reads to different sequence databases, we employed an iterative strategy using ELAND , ABV2r, 5'-CCTGTGAATGTCTCGTTTCTG-3' (maps to residues 5754-5733 of vRNA), and ABV3r 5-TTCTTTCAGCAACCACTGACG-3' (maps to residues 2563-2543 of vRNA). Reverse transcription was carried out at 50°C for 1 hr with SuperScriptIII  according to manufacturer's instructions. Following RNase H treatment, PCR was performed on the resulting cDNA with Phusion polymerase  with the primers used for reverse transcription and the following primers: ABV1f: 5'-GGATCATTCCTTGATGATGTATTAGC-3', (maps to residues 5567-5589) ABV2f: 5'-CAAATGGAGAGCCTGATTGG-3' (maps to residues 2378-2397) ABV3f: 5'-AATCGGTAAGTCCAGAGTCAAGG-3' (maps to residues 155-177). All products were amplified for 35 cycles under the following conditions: 98°C, 3 minutes; 98°C, 10 seconds, 50°C, 30 seconds, 72°C 3 minutes. Resulting products were gel purified, and subcloned into the TOPO T/A cloning vector pCR2.1 after incubation with Taq polymerase and dATP for 10 minutes at 72°C. For each product, 4 independent transformants were prepared for standard dideoxy sequencing on an ABI3730 sequencer . Forward and reverse reads spanning each clone were generated using M13F and M13R and additional overlapping primers spaced at 600–800 bp intervals across the each of the clones.vRNA RT-PCR products containing uncapped vRNA termini were captured using the First Choice RLM RACE kit  with the following modifications to the standard protocol: 1) tobacco acid phosphotase treatment was omitted, 2) a phosphorylated RNA, RNAligate, 5'-p-GUUAUCACUUUCACCC-3'  was substituted for the 3' RNA ligation-mediated RACE primer provided in the kit and ligated to 3' ends as per manufacterer's 5' RACE protocol, and 3) in the 3' RACE reverse transcription reactions, two reverse transcription reactions were performed and carried forward in parallel: one with random decamers and one with a DNA oligo complementary to oJSmer utilized in the RNA ligation step . For 5' RACE, a single round of PCR was sufficient to generate a product using the vRNA specific primer ABV5RaceOuter, 5'-CAGTCGGTTCTTGGACTTGAAGTATCTAGG-3' (maps to residues 346-317 of vRNA) and manufacturer provided outer PCR primer. For 3' RACE, nested PCR was required to recover detectable PCR product of expected size using outer PCR primers oJSmerRC and the gene specific primer ABV3RaceOuter, 5'-CCCGTCTACTGTTCTTTCGCCG-3' (maps to residues 8479-8497 of vRNA), followed by inner PCR using Tailed_RNAligateRC, 5'-AAGCAGTGGTAACAACGCAGAGTACGGGTGAAAGTGATAAC-3' and the gene specific primer, ABV3RaceInner, 5'-GCAATCCAGGAATAAGCAAGCACAAA-3' (maps to residues 8595-8620 of vRNA). Both of the RACE PCR reactions were carried out with Platinum Taq polymerase  in 35 cycles of gradient PCR : 94°C, 30 seconds; 55–58°C, 30 seconds; 72°C, 30 seconds. Resulting PCR products were gel purified and subcloned into TOPO T/A cloning vector pCR 4.0. For the 5' RACE products, 7 independent transformants from 3 independently generated PCR products were subcloned and sequenced with M13F and M13R primers. For the 3' RACE products, 6 independent transformants from 4 independently generated PCR products were subcloned and sequenced with M13F and M13R primers. Terminal sequences reported here reflect the majority consensus sequence obtained from these reads.Genome sequence assemblies from both initial genome sequence recovery and vRNA genome sequence recovery were generated using Consed, version 16.0 software . All basBeyond the initial set of 16 specimens provided for microarray analysis, specimens from a total of 38 additional PDD cases, PDD controls, and PDD suspicious birds with varied clinical histories were provided to us blinded by our 2 collaborators . Specimens provided from Israel in the form of extracted RNA were similarly DNAse-treated and amplified prior to PCR screening.Gallu gallus GAPDH sequence: Gg_GAPDHf: 5'-AGTCATCCCTGAGCTSAAYGG*GAAGC-3' , * indicates the junction of GAPDH exon 8 and 9 spanned by this primer), Gg_GAPDHr 5'-ACCATCAAGTCCACAACACGG-3' , maps to GAPDH exon 12). After PCR results were tallied, clinical information on all specimens tested was unmasked. A complete accounting of ABV, GAPDH PCR results, specimen type and clinical status is provided in Additional file 1 ul of the randomly amplified material generated from these RNA samples was used as input template for ABV consensus PCRs as described above. In parallel, as an independent control for input specimen RNA integrity, PCR for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was performed on all specimens using designed based on Friedman-Einat et al  and GallTo reduce potential confounding due to differences in viral detection resulting from specimen tissue source, only specimens derived from upper GI tract tissue  that tested positive by GAPDH mRNA PCR were included in association analysis presented in Table The remaining 17 samples were excluded from the analysis because they were either 1) GAPDH-positive or GAPDH-negative samples derived from specimen other than upper GI tract tissue  or 2) derived from cases that were histologically or clinically 'suspicious', but unconfirmed PDD cases. Six additional ABV PCR positives were identified among this set of samples excluded from the statistical analyses: 1 derived from GI content from a confirmed PDD case, and 5 derived from a variety of tissues from the PDD suspicious cases.Multiple sequence alignments of complete genome sequences or partial sequences derived from PCR screening studies were generated with ClustalW  version ABV: Avian bornavirus; BDV: Borna diseae virus; PDD: Proventricular dilatation disease.Sequence information obtained here has been disclosed for patenting purposes. ALK, AG, SC, PS-C, KF, KS, CYC, AL, AG, SKB, DG, and JLD were all party to this disclosure in conjunction with UCSF Office of Technology Management.ALK participated in the conception, design, and coordination of the study, performed specimen extraction of specimens from Florida case/control study, array analyses for both sets of PDD case/control series, follow-up PCR screening and sequencing of samples and wrote the manuscript, AG orchestrated and collected the PDD case/control specimens from Israel and coordinated the clinical and histopathology analyses, and nucleic acid extraction for samples from Israel, and participated in revising the manuscript, SC orchestrated and collected the Florida PDD case/control specimens and oversaw the clinical and histopathologic analyses of these samples from Florida, and participated in revising the manuscript, PS-C carried out filtering and iterative BLAST analysis of ultra high throughput sequence data for ABV genome sequence recovery, participated in primer design and complete genome sequence recovery, and drafting the manuscript, KF participated in array analysis, developed pipeline for ultra high throughput sequence analysis, and participated in design of filtering and iterative BLAST analysis, KS performed modified library preparation for ultra high throughput sequencing and participated in revising the manuscript, CYC performed ultra high throughput sequencing and participated in revising the manuscript, AL, SM, and YF participated in clinical evaluation, specimen collection and extraction of samples from Israel, AG participated in extraction of specimens from Florida and follow-up microarray analysis and high throughput sequencing, CCW developed additional primers for PCR follow-up studies, SBK assisted in the selection of the PDD case/control specimens from Florida and participated in review of clinical and histological status of cases and controls included in the study, DG and JLD oversaw the overall conception and design of the project and supervised all phases of its execution and the drafting and revision of the manuscript.Alignment of bornavirus genomes 5' and 3' termini. Bornavirus genome organization overview diagrammed as in Figure AB258389], Bo/04w [GenBank:AB246670], H1766 [GenBank:AJ311523], Ref [GenBank:NC_001607], V [GenBank:U04608], V/FR [GenBank:AJ311521], CRNP5 [GenBank:AY114163], CRP3B [GenBank:AY114162], CRP3A [GenBank:AY114161], He/80/FR [GenBank:AJ311522], He/80 [GenBank:L27077], pBRT7-HrBDVc [GenBank:AY705791], No/98 [GenBank:AJ311524], ABV [GenBank:EU781967].Click here for fileAlignment of transcription initiation and termination sites in bornavirus genomes. Panel A, alignment of the 3 bornavirus transcription initiation sites (TIS) and 6 nucleotides of flanking sequences. Panel B, alignment of the 4 bornavirus transcription termination sites. Source genomes for alignments are shown at left. Black trianges highlight ABV sequences.Click here for fileAlignment of splice donor and splice acceptor sequences in bornavirus genomes. Panel A, alignment of splice donor 1 and splice acceptor 1 sequences; Panel B, alignment of splice donor 2 and splice acceptor 2 sequences; Panel C, alignment of splice acceptor 3 sequences. Source genomes for alignments are shown at left.Click here for filePhylogenetic relationships between sub-genomic loci of ABV and representative BDV genomes. Neighbor-joining trees generated for the indicated nucleotide sequences of ABV and a representative set of BDV genomes are shown for each ORF in the bornavirus genome. Accession numbers of representative BDV genomes are: Ref/V [GenBank:NC_001607], H1766 [GenBank:AJ311523], He/80 [GenBank:AY705791], No/98 [GenBank:AJ311524].Click here for fileSummary of clinical and molecular data for specimens provided in this study. Microsoft Excel file containing two spreadsheet (US specimens and Israel specimens) summarizing clinical and epidemiologic information available for each specimen, as well as the associated results from the described microarray/PCR/sequence experiments.Click here for file


Syndecan (dSdc) as a positional candidate gene affecting variation in fat storage between two Drosophila melanogaster strains. Here, we first used quantitative complementation tests with dSdc mutants to confirm that natural variation in this gene affects variability in Drosophila fat storage. Next, we examined the effects of a viable dSdc mutant on Drosophila whole-body energy metabolism and associated traits. We observed that young flies homozygous for the dSdc mutation had reduced fat storage and slept longer than homozygous wild-type flies. They also displayed significantly reduced metabolic rate, lower expression of spargel (the Drosophila homologue of PGC-1), and reduced mitochondrial respiration. Compared to control flies, dSdc mutants had lower expression of brain insulin-like peptides, were less fecund, more sensitive to starvation, and had reduced life span. Finally, we tested for association between single nucleotide polymorphisms (SNPs) in the human SDC4 gene and variation in body composition, metabolism, glucose homeostasis, and sleep traits in a cohort of healthy early pubertal children. We found that SNP rs4599 was significantly associated with resting energy expenditure (P = 0.001 after Bonferroni correction) and nominally associated with fasting glucose levels (P = 0.01) and sleep duration (P = 0.044). On average, children homozygous for the minor allele had lower levels of glucose, higher resting energy expenditure, and slept shorter than children homozygous for the common allele. We also observed that SNP rs1981429 was nominally associated with lean tissue mass (P = 0.035) and intra-abdominal fat (P = 0.049), and SNP rs2267871 with insulin sensitivity (P = 0.037). Collectively, our results in Drosophila and humans argue that syndecan family members play a key role in the regulation of body metabolism.Syndecans are a family of type-I transmembrane proteins that are involved in cell-matrix adhesion, migration, neuronal development, and inflammation. Previous quantitative genetic studies pinpointed Drosophila  Obesity is a condition characterized by an excess of adipose tissue that adversely affects human health D. melanogaster has emerged in recent years as a powerful model organism for studying the genetics of fat storage and obesity D. melanogaster, Oregon R (Ore) and 2bdSdc gene, which encodes a member of the syndecan gene family The role of genes in human obesity and related phenotypes is well established SDC1, SDC2, and SDC3, are expressed in a tissue-specific manner, whereas the fourth, SDC4, is expressed in a variety of cell types Syndecans are type-I transmembrane proteins that are present on the surface of all adherent cells dSdc was a candidate gene contributing to variation in TAG storage between Ore and 2b. To investigate this hypothesis, we performed quantitative complementation tests by crossing mutants of dSdc with flies of the Ore and 2b genotypes and found that allelic differences at dSdc produced differences in TAG storage between these two strains. We next examined the functional role played by dSdc in fat storage by testing whether flies homozygous for a hypomorphic mutation of dSdc (BGdSdc02774) and flies homozygous for the corresponding wild-type allele differed in whole-body energy metabolism and associated traits in Drosophila. Collectively, our results in Drosophila showed that dSdc plays an important role in the regulation of body composition and metabolism in young flies.Based on these observations, the results of our genetic mapping experiment, and the similarity in structural characteristics between Drosophila and mammalian syndecans, we reasoned that SDC4 gene includes 5 exons spanning 19.7 kb on chromosome 20q12 SDC4 gene and variation in traits associated with body composition, energy metabolism, and sleep duration in a cohort of 252 healthy early pubertal children. Although small in size, this human cohort was chosen for two main reasons. First, the phenotypic data available for each subject included robust measurements of body composition and glucose-insulin dynamics. Second, the cohort was characterized by genetic admixture, which allowed us to adjust for ancestry within ethnic groups and therefore limit false-positive results SDC4 genotypes as independent variables revealed an impact of SDC4 variation on resting energy expenditure (REE), insulin dynamics, and sleep duration in our cohort of young children. These results motivate future genetic studies in independent human populations to verify the effects of this gene.In humans, the dSdc and TAG storage, we first used complementation tests with two mutations of dSdc (10608Sdc and BG01305Sdc). 10608Sdc failed to complement the TAG phenotype of Ore and 2b QTL in males  [CS (B)] strain by the insertion of a p[GT1]-element in the second intron of dSdc  experiments using RNA isolated from three body parts . We found that the overall expression of dSdc is significantly reduced in the three body parts of BG02774Sdc flies  and body weight-adjusted protein (18%) and glycogen (40%) contents, while male BG02774Sdc did not differ from the control strain  genes. Several of the dilps (dilp1-5) are expressed at high levels in insulin producing cells (IPC) of the pars intercerebralis in the brain of both larvae BG02774Sdc had a defect in the production of DILPs, we measured mRNA levels of dilp2, dilp3, and dilp5, which are expressed in the IPC of adult flies dSdc mutants and controls. We also investigated the expression level of the gene that encodes the adipokinetic hormone (AKH), a putative glucagon homolog. Like pancreatic insulin and glucagon-producing cells in mammals, cross-regulatory interactions exist between DILP- and AKH-producing cells in Drosophila Akh were previously shown to be increased in IPC-deficient larvae and adults dilp2, dilp3 and dilp5, respectively, and a 38% increase in Akh expression in BG0277Sdc4 flies compared with controls  signaling controls organ growth and final body size controls . In maleof dilp2 .dilp1-5 were poorly fertile and had reduced metabolic activity. A significant reduction in egg-production was also observed by Gronke et al. dilp2-3,5 mutant females. Therefore, we tested the impact of the dSdc mutation on these phenotypes. First, we measured fecundity by counting the total number of eggs laid over a five day period. As expected, we found that BG02774dSdc females laid significantly less eggs (42% less) than control flies (2) production. Because no differences were observed between male and female flies in CO2 production, we pooled male and female data for statistical analysis. We observed that dSdc mutants displayed a 22% reduction in metabolic rate as compared to controls . Reduced insulin receptor/PI3K/Akt signaling in Drosophila induces nuclear accumulation of the dFoxO, which in turn activates the transcription of target genes, such as the translational regulator Thor, the Drosophila homolog of mammalian EIF4EBP1Thor mRNA levels in dSdc mutants and controls and found no differences between male and female flies; therefore we pooled the data for the analysis. Interestingly, in contrast to our prediction, we observed that Thor expression was significantly reduced in dSdc mutants (71%) compared to control flies  flies. Once again, we did not observe differences in transcript levels between male and females and therefore we pooled the data for the statistical analysis. We found that mRNA levels of spargel were reduced 52% in the mutant flies relative to controls (Peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1) family members play a pivotal role in the control of energy homeostasis in mammals controls .+-linked respiratory substrates (pyruvate plus proline) that deliver electrons into complex I of the mitochondrial electron transport chain. In the analysis averaged across sexes, we observed that the ADP-dependent state 3 respiration rate (a measure of oxidative phosphorylation capacity) was reduced by approximately 15% in BG02774Sdc compared with the controls  flies over the course of a 24 h day. As expected, sleep was increased by 32% during nighttime and by 76% during daytime in BGdSdc02774 female flies compared to controls  Project (http://www.hapmap.org): rs1981429 (T/G) and rs2267871 (T/A), which map in the intron 2, and rs4599 (C/T) in the 3′UTR region. All genotype groups were in Hardy-Weinberg equilibrium (P<0.0001) and rs4599 (P = 0.0002), which is consistent with genotypic data reported by the HapMap Project. Low to moderate pair-wise linkage disequilibrium estimates were observed among the SNPs in the three ethnic groups (We conducted a population-based association study on 252 European American (EA), African American (AA) and Hispanic American (HA) children. Anthropometric and metabolic characteristics of the study subjects are shown in ilibrium . The Minilibrium . There wc groups .P = 0.0038) and nominally associated with fasting glucose levels and sleep duration  to show that variation in this gene affects inter-individual variability in TAG storage between two strains of D. melanogaster. We then confirmed the effect of dSdc on TAG storage using a hypomorphic dSdc mutant (BG02774Sdc) and showed that flies homozygous for the mutation had significantly lower TAG storage than control flies under ad libitum feeding conditions. They also displayed significantly reduced metabolic rate and lower mitochondrial ADP-stimulated (state 3) respiration rate. Furthermore, female mutants had lower body weight, were leaner, had reduced glycogen levels, and were less fecund than controls. DILPs produced by IPCs in the brain of Drosophila have been shown to regulate body size, energy metabolism, and fecundity in several studies et al. dilp2-3,5 mutant flies had significantly reduced body weight, higher levels of lipids and glycogen, and were less fecund than controls. Another study found that flies homozygous for a deletion of dilps1-5 not only were smaller and less fertile but also displayed reduced metabolic activity and TAG storage than controls BG02774Sdc flies had significantly lower expression of dilp2-3,5 genes compared to controls. A significant reduction in dilp2 expression was also observed in males. Thus, these data suggest that some of the effects associated with reduced dSdc function may be mediated by the reduced levels of DILPs. Further studies however will be required to determine not only if reduced expression of DILPs is causal in these effects, but also to understand how dSdc regulates the expression levels of dilps in Drosophila. We also observed that female mutants had significantly higher expression levels of Akh, which encodes a hormone involved in mobilization of glycogen reserve from the fat body BG02774Sdc flies have lower levels of glycogen than controls.The syndecans are a family of cell-surface heparan sulphate proteoglycans that are expressed on the surface of all adherent cells ThorThor expression in BG02774Sdc flies. This result is quite surprising considering that previous work reported that Thor transcript levels are up-regulated in dilp2-3,5 mutants dSdc mutant flies have a defect in the mechanism(s) involved in dFoxO translocation and transcriptional regulation. This hypothesis is supported by the observation that, despite reduced dilp2 transcript levels, male dSdc mutant flies did not show a compensatory increase in dilp3 mRNA as a result of the reduced insulin signaling in the IPCs, an autocrine regulatory mechanism proposed by previous studies dilp3 transcript in the IPCs was significantly reduced in dFoxO null flies dilp3 acts as a positive regulator of dilp2 and dilp5 expression dilp2-3,5 observed in female BG02774Sdc flies. In this context, another unanticipated finding in our study is that reduced expression of dilps did not lead to extended life span and increased starvation resistance as observed in several other studies BG02774Sdc flies showed significantly reduced survival. The mechanism behind this finding is unknown at present. However, it has been previously reported that Thor null flies had an impaired response to nutrient deprivation and shorter lifespan than controls Thor transcript observed in the BG02774Sdc flies could be responsible for these phenotypes. Experiments are currently underway in our laboratory to test this hypothesis.Reduced insulin receptor/PI3-kinase/Akt signaling pathway results in nuclear accumulation of the transcription factor dFoxO and transcriptional activation of its targets, such as spargel gene expression is induced by insulin signaling via a pathway parallel to dFOXO spargel in BG0277Sdc4 mutant flies compared to controls. BG0277Sdc4 flies also showed a significant reduction in mitochondrial state-3 respiration rate, suggesting reducing mitochondrial oxidative phosphorylation capacity. In mammals, skeletal muscle metabolism influences whole-body metabolic rate dSdc might control Drosophila whole-body metabolic rate via the insulin signaling in skeletal muscle, which in turn regulates spargel expression and mitochondrial respiration.In mammals, PGC-1α and PGC-1β have been shown to play a pivotal role in the control of energy homeostasis. They regulate glucose and fat oxidation in muscle and fat tissue dSdc mutant flies slept longer than controls. This increased sleep length is due largely to increases in the average duration of sleep bouts rather than their number (data not shown). Previous microarray analyses of fly heads revealed three genes with predicted functions in lipid metabolism that increased expression during sleep P-element insertions in metabolic pathway genes impacted sleep duration and bout number BG02774Sdc mutants in combination with reduced TAGs may be indicative of a strategy to conserve energy, an idea long postulated as a possible function of sleep dSdc mutant flies.In the present study we also found that BG02774Sdc on several phenotypes. Sex-specific effects on TAG storage were also observed in our initial complementation tests with different mutant alleles of dSdc. Such sex-specific allelic effects are commonly observed in Drosophila dSdc can act to modulate cell signaling, alternative alleles could be highly sensitive to different levels of signaling molecules that naturally occur between the sexes. Along these same lines, in the comparison of the control CS (B) flies, the baseline expression of dSdc was much higher in control females compared with the control males. Thus, we would expect to observe greater phenotypic effects of the mutation in females compared with males and this is what we observed in most traits. Understanding the mechanism underlying these sex specific allelic effects is an important goal for future study.One of the interesting findings of this component of the study was the sex-specific nature of the effects of SDC4 gene is associated with REE, sleep duration, and insulin dynamics in children. These results may shed light on physiological pathways underlying disease-related phenotypes in humans, perhaps by mechanisms that involve REE and/or sleep duration. Low REE has previously been associated with obesity- and diabetes-related traits in adults SDC4 genetic associations with REE and lean mass highlight the inter-relationship between these two traits. Previous studies reported that syndecan-4 is essential in skeletal muscle development and regeneration The results of the human study parallel the results of the experiments in flies. Overall, we observed that genetic variation in the human SDC4 polymorphisms and sleep duration in early pubertal children. Previous reports have demonstrated that “sleep need” for this age group is approximately 9 hours per night Another point of potential interest is the relationship between the 2b and Ore were used to establish the recombinant inbred lines in which QTL affecting TAG were previously mapped BG01305Sdc and BG02774Sdc lines were established by the Berkeley Drosophila Gene Disruption (BDGD) Project via P-element insertion into the second intron of the dSdc gene in the 1118;Canton Sw (CS) strain 10608Sdc line is a P-element mutation that was established by the BDGD Project and maintained over a balancer  chromosome The unrelated isogenic laboratory lines To control for larval density, we allowed the parents of the experimental flies to mate for 3 hours to generate egg collections on apple juice/agar medium in laying plates. After 24 hours, we picked groups of 100 first-instar larvae from the surface of the medium and put into replicate vials. To minimize the influence of genetic variation in reproduction on energy metabolism, we performed all the metabolic assays on 3–5 day old virgin flies that were randomly collected from the replicate vials. We reared flies in vials containing 10 ml of standard cornmeal, agar, sugar, and yeast medium at a constant temperature of 25°C, 60–75% relative humidity, and a 12-hr light-dark cycle.Ore and 2b female flies to males of dSdc mutation stocks. These crosses produced four F1 genotypes: M/2b, Bal or CS/2b, M/Ore, and Bal or CS/Ore, where M denotes the Sdc mutation. We measured TAG storage in each genotypic class for each sex using the same experimental design described in y  =  µ + L + G + W + L×G + E, where L and G are the fixed cross-classified main effects of line  and genotype , W is the covariate body weight, and E is the within-vial variance. We inferred significant failure of the mutation to complement quantitative TAG phenotypes of Ore and 2b alleles if the main effect of the L×G interaction term was significant, the contrast [M/Ore – M/2b]) was significant, and the contrast [Bal or CS/Ore – Bal or CS/2b] was not significant We performed quantitative complementation tests by crossing virgin 2PO4 buffer. TAG content was measured for each homogenate spectrophotometrically using a commercially available kit (Sigma-Triglyceride Assay Kit) following the manufacturer's suggested protocol. Total protein levels were measured using a standard Lowry protein assay. Glycogen content was measured from the same homogenates using the protocol described in We measured body weight, TAG and total protein levels using the protocol described in We estimated female fecundity by standard procedures 2 production using a flow-through respirometry system  and a modification of the method described in 2 was measured for 10 minutes/chamber with a 30 second flush period between measurements. The amount of CO2 produced by each group of flies was calculated using C950 Data Acquisition software . Each group of flies was sampled at the same time of the day. The data analysis was performed using ANCOVA, with body weight used as covariate.We measured metabolic rate as COWe maintained adult virgins at 30 flies to a single-sex vial to ensure that each line was exposed to identical levels of social dSdc mutation on the lifespan of virgin flies, we measured survival using population cages. To obtain virgin offspring for each population cage, we standardized the parental density of each genotype (10 pairs) and allowed them to lay eggs for five days. Virgin offspring were collected over four successive days and added to a population cage at the end of this period. We set up three population cages of each sex and genotype  and removed and counted dead flies every other day. We used Cox regression To examine the effects of the We measured survival under starvation conditions by placing 10 flies per genotype/sex on 1.5% agarose medium and recording the number of flies alive at 8-h intervals until all were dead. Statistical significance was determined by one-way ANOVA.+-linked substrates (5 mM pyruvate + 5 mM proline) as implemented in DatLab Version 4.1.0.8. Statistical significance was determined by the two-tailed Student's t test.We placed live flies into 200 µl of ice-cold isolation buffer  supplemented with protease inhibitors  in a 1.5 ml Eppendorf tube. The samples were pounded gently 126 times over a 2 minute period, using a motorized micromortar, and filtered through a 5 µm nylon mesh. We then raised the volume to 400 µl by washing the nylon membrane with additional isolation buffer. After a cycle of low-speed centrifugation followed by a centrifugation of the filtered solution for 10 min at 3000 g, at 4°C, the pellet was re-suspended in 100 µl of isolation buffer. Protein concentrations in the mitochondrial fractions were determined using a Lowry assay. We performed mitochondrial respiration assays using freshly isolated mitochondria by measuring oxygen consumption in a two-chamber polarographic oxygen sensor . We measured state 2, state 3 and state 4 respiration rates using NADt test.We isolated total RNA using the TriPure RNA isolation kit (Roche). Isolated RNA was then used to make cDNA, using the First Strand Synthesis kit (Invitrogen). We performed RT-qPCR using a Syber Green Master mix and 50 ng total of cDNA per reaction and run in a Stratagene Mx3000P® qPCR machine. Statistical significance was determined by the two-tailed Student's A total of 252 children between the ages of 7 and 12  were evaluated for the human association study. Subjects were participants from an ongoing study conducted at the University of Alabama at Birmingham. Race was determined by self-reported African-American, Caucasian or Hispanic ancestry in both parents and grandparents.The study protocol was approved by the Institutional Review Board for human studies at the University of Alabama at Birmingham. A written informed consent was obtained from all study participants before enrolling in the study.Height and body weight in human subjects were measured in light indoor clothes and without shoes. Body composition  was measured by DXA using either a Lunar DPX-L densitometer  or a LUNAR Prodigy densitometer in the Department of Nutrition Sciences at UAB. Subjects were scanned in light clothing while lying flat on their backs with arms at their sides. Intra-abdominal adipose tissue was analyzed in the Department of Radiology by computed tomography scanning with a HiLight/Advantage Scanner .Following an overnight fast, blood samples were obtained to establish the basal levels of glucose and insulin, and a frequently sampled intravenous glucose tolerance test (FSIGTT) was performed as described elsewhere 2) uptake and VCO2 production were measured continuously for thirty minutes.REE was measured in the morning immediately after awakening during the overnight visit. A computerized, open-circuit, indirect calorimetry system with a ventilated canopy  was used. While lying supine on a bed, the head of the subject was enclosed in a plexiglass canopy. Subjects were instructed not to sleep and remain quiet and still, breathing normally. One-minute average intervals of oxygen (VOSleep patterns were assessed by a questionnaire that was administered to the parents of the subjects. Parents were inquired about the amount of hours their child spent sleeping at night. We used hours sleeping at night as continuous phenotype.SDC4 polymorphism by Pyrosequencing technology www.preventiongenetics.org). Approximately 100 ancestry informative markers (AIMs) were utilized for the study. Information regarding marker sequences, experimental details, and parental population allele frequencies has been submitted to dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) under the handle PSU-ANTH We determined the genotypes of each We assessed Hardy-Weinberg equilibrium, allelic frequencies, and D' linkage disequilibrium coefficients using Haploview v3.2


Understanding the mechanisms that coordinate cell proliferation, cell cycle arrest, and cell differentiation is essential to address the problem of how “normal” versus pathological developmental processes take place. In the bristle lineage of the adult fly, we have tested the capacity of post-mitotic cells to re-enter the cell cycle in response to the overexpression of cyclin E. We show that only terminal cells in which the identity is independent of Notch pathway undergo extra divisions after CycE overexpression. Our analysis shows that the responsiveness of cells to forced proliferation depends on both Prospero, a fate determinant, and on the level of Notch pathway activity. Our results demonstrate that the terminal quiescent state and differentiation are regulated by two parallel mechanisms acting simultaneously on fate acquisition and cell cycle progression. Despite substantial progress that has been made, we still know little about how single precursor cells undergo a limited number of cell divisions before arrest. Discovering the mechanisms by which terminal cells maintain cell division arrest is essential for understanding “normal” development, as well as the origin of pathological deregulations. Using the bristle cell lineage, a model system widely employed to analye cell identity acquisition, we observed that only two out of four terminal cells in this lineage are unable to re-enter the cell cycle and proliferate. Our study shows that in these cells, cell division arrest is maintained by the action of the transcription factor Prospero and the signalling pathway Notch. Since both of these factors also control cell identity in this lineage, this finding demonstrates that common elements acting simultaneously and in parallel regulate the terminal quiescent state and differentiation. This system provides a unique animal model in which to understand how the mechanisms involved in cell fate acquisition and those controlling cell division intermingle to produce cell lineages resulting in terminal cells in the right number and at the right place and time. C. elegans, cell divisions are not completely blocked after genetic ablation of cycE −/− mouse cells are resistant to oncogenic transformations suggesting that normal and oncogenic proliferation have different requirements for CycE Drosophila, the core mechanism of the cell cycle is not redundant and down-regulation of CycE arrests the cell cycle. Thus, CycE appears to be the most important G1 cyclin in all Drosophila cell divisions studied so far. In addition, it has been shown that ectopic expression of CycE after terminal mitosis induces re-entry into the S-phase resulting in additional cell cycles Drosophila CycE seems to be central to the generation of ectopic rounds of cell divisions after cell cycle deregulation.A high degree of coordination between cell proliferation, cell cycle arrest and cell differentiation is essential for proper development. Disruption of this coupling can lead to malformations and eventually cancer. Cell cycle progression relies primarily on the activity of cyclin-dependant kinases (Cdk) that are regulated by their association with factors like cyclins or cyclin kinase inhibitors (CKI) and by phoshorylation or dephosphorylation Drosophila bristle lineage, which produces the external mechanosensory organs called microchaetes, cell cycle progression and cell determination are intimately related In the p72neuralized -GAL4 line (neur) was used as a driver to simultaneously overexpress CycE and Histone2B::YFP (H2B::YFP), which highlights the DNA In order to analyse the mechanisms that maintain arrest in terminal cells of the bristle lineage, we forced proliferation by specific overexpression of CycE using the UAS/Gal4 system in vivo imaging to follow the formation of the bristle lineage in neur>H2B::YFP, PON::GFP . The second involved a pIIIb daughter cell identified as the future neuron by its position and its inheritance of Pon::GFP during pIIIb mitosis  see . This suin vivo time-lapse followed by immunodetection experiments (not shown). Taken together these data indicate that the shaft cell and neuron are the only two cells in the terminal bristle lineage to undergo an ectopic division when CycE is overexpressed  immunoreactivity) together with bristle lineage cells, neurons or socket cells. xpressed .To further analyse cell behaviour in response to CycE overexpression, we sought to determine the critical period during which cells were competent to undergo additional mitosis. To do so, CycE was expressed under the control of a heat-shock promoter at different moments in the cell lineage. Antibody staining was carried out twelve hours after heat shock to distinguish socket cells and neurons. in vivo recordings (see in vivo recording data showing that Pon::GFP seems to be distributed uniformly between both daughter cells (The data presented above indicate that the first extra mitosis is asymmetric and generates two different cells (a shaft and a socket cell) while the second one is symmetric giving rise to two identical cells (two neurons). In the normal bristle cell lineage all mitosis are asymmetric. The cell-fate determinants, Numb and Neuralized (Neu), co-segregate to one daughter cell, where both act to bias the Notch-mediated fate decision ings see . In contings see . This iser cells . These dneur>CycE). N pathway was inhibited by expressing Numb under the control of a thermo-inducible promoter in both pIIa daughter cells . When CycE was overexpressed together with mild induction of Numb, we observed that 28% of the clusters harboured four pIIa daughter cells with two socket cells and two shaft cells . Under these same conditions, in vivo analysis showed that both pIIa daughter cells divided (not shown). Such clusters were never observed when CycE was overexpressed alone (neur>CycE column 4). Thus, when N activity was reduced and CycE overexpressed, the future socket cell divided asymmetrically giving rise to a shaft and a socket cell. This suggests that, similarly to the shaft cell, the socket cell retains pIIa features shortly after birth. Taken together, these data show that full activation of the N pathway is necessary to prevent the future socket cell from entering mitosis upon CycE overexpression.The extra division of the future shaft cell was not correlated to different levels of CycE overexpression . Moreoveintra) intra was induced at 20 h APF by a 30 min pulse at 34°C. Under these conditions, only 45% of clusters were transformed . We took advantage of this mild penetrance to analyse the effect of CycE overexpression together with weak Notch dependant transformation. We observed that the number of clusters containing three pIIa daughter cells (two of which were socket cells) was reduced from 91% under CycE overexpression alone to 38% under conditions of N and CycE overexpression . This reduction was associated with an increase in the number of clusters having a normal set of pIIa daughter cells . This indicates that the extra divisions induced by CycE overexpression were blocked by mild activation of the N pathway. These data show that in the pIIa sub-lineage, activation of the N pathway is involved in maintaining the future socket cell in a quiescent state. As a consequence, CycE overexpression induced cell division exclusively in the future shaft cell. A similar anti-mitotic action of the N pathway has been reported in Drosophila follicle cells. In these cells, lack of N activity has been shown to induce extra mitoses at the expense of endocycles To further analyse the action of N on the proliferative capacity of terminal cells, we performed reciprocal experiments and activated the N pathway in the shaft cells. This pathway was activated after overexpression of the intracellular domain of the N receptor (Nneur>CycE). Under these conditions, the overexpression of Numb was not sufficient to modify cell identity, since both an ELAV and a Pros positive cell (neuron and sheath cell respectively) were present in all clusters. Surprisingly, even though the conditions of Numb overexpression were similar to previous experiments, we observed no change in the number of clusters with duplicated sheath cells when CycE was overexpressed (0% vs 4% in neur>CycE (n = 126) and in hs-numb, neur>CycE (n = 118) respectively). Similarly, no change was observed in the number of clusters with duplicated neurons (19% vs 22% in neur>CycE (n = 126) and in hs-numb, neur>CycE (n = 118) respectively).Similar to the situation in the pIIa sub-lineage, only the N-on cell of the pIIIb sub-lineage, namely the sheath cell, did not undergo extra mitosis upon CycE overexpression. To test whether activation of the N pathway was necessary to prevent ectopic division of the sheath cell, we reduced the activation of this pathway by overexpressing Numb  together with CycE  17/+pros heterozygous background. Results depicted in 17/+pros, neur>CycE (n = 129) compared to 1% in neur>CycE alone (n = 71). Interestingly, we also observed an increase in the percentage of clusters containing multiple neurons (86% n = 129). These results suggest that with CycE overexpression, Pros affects the proliferative properties of the sheath cell and the neuron even if in this later Pros is transiently expressed. Similar results were obtained by overexpressing CycE using a hs-CycE construct at the time of pIIIb division (22 h APF) in a 17/+pros background (data not shown). To determine the origin of the duplicated cells, we combined time lapse imaging to follow the pattern of cell division and immunostaining to identify the fate of pIIIb daughter cells. Within the 25 lineages followed, three types of extra divisions were observed: (i) in 64% of the cases, only one pIIIb daughter cell underwent an ectopic mitosis, giving rise to two neurons; this is similar to what was observed upon overexpression of CycE alone  being a CycE inhibitor.The fact that the response of pIIIb daughter cells to CycE overexpression was invariant after Numb overexpression suggests that factors other than N can prevent mitosis in these cells. One candidate that may impede extra mitoses is the cell determinant Pros, as loss of function of cE alone ; (ii) inivisions . The absTaken together, these data indicate that Pros has a dual function in the bristle lineage. In addition to its involvement in neuron and sheath fate determination neur>CycE) while downregulating N activity (hs-numb) in a 17/+pros background. Induction of Numb expression (30 min heat shock at 38°C) was performed under visual control on living pupae followed by immunostaining to identify sheath cells and neurons. Only clusters heat shocked within one hour following the pIIIb division were analysed. We observed a significant increase in the proportion of sheath cells that underwent an extra division  versus 19% in 17/+pros, neur>CycE (n = 72) and 4% in hs-numb, neur>CycE (n = 104). These data indicate that decreasing N pathway activity in a 17/+pros mutant background favours the division of the future sheath cell upon CycE overexpression.The data presented above indicate that activation of the N pathway prevents mitosis of the socket cell upon CycE overexpression. A similar role is played by Pros in the sheath cell. Since the N pathway is active in the sheath cell, N and Pros could act redundantly to prevent extra divisions in this cell. To analyse this possibility, we overexpressed CycE , we observed a supplementary pIIIb daughter cell identified by its small nucleus  supplementary cell divisions were observed only in those terminal cells in which the Notch pathway is not endogenously activated, (2) supplementary cell divisions were observed in sheath cells and neurons in a 17/+pros background; (3) activation of the N pathway blocked the ectopic division of the shaft cell; (4) socket cells underwent an extra division after reduction in N pathway activity and (5) additional sheath cells underwent extra divisions when both N and Pros activity was reduced.In this study we have shown that in the bristle lineage terminal cells, the shaft cell and the neuron, but not the socket and sheath cells, undergo supplementary cell divisions after CycE overexpression. These supplementary cell divisions were not due to a cell-specific differential expression by per se was observed (as in pros null background or pros heterozygous pupae) or in which only a small proportion of cells were transformed (as after mild overexpression of Numb or Nintra). As such, the observed effects were considered to be the result of N and Pros acting directly on the maintenance of the state of cell cycle arrest rather than a modulation of cell fate acquisition. Furthermore, we never observed changes in bristle cell identities after CycE overexpression. This is in contrast to Drosophila neuroblasts, in which CycE seems to control cell identity independently of its role in the cell cycle In this study, the proliferative capacity of terminal cells was analysed when cell divisions were forced by CycE overexpression under different Notch and Prospero backgrounds. The analysis was performed under conditions in which either no cell transformation When N-activity was impaired and cell divisions were forced, both pIIa daughter cells divided like their mother, pIIa, each producing a shaft and a socket cell. These observations reveal that, just after birth, pIIa daughter cells are not yet committed to their final fate and retain pIIa characteristics. These results suggest that cell fate in pIIa daughter cells is acquired in a sequential manner. Initially, both pIIa daughter cells appear to be equivalent. Then, the N-pathway is rapidly activated in one daughter cell due to a N-signal originating from its sister cell cycE expression induced extra mitosis even in fully differentiated neurons that were identified by 22C10/Futsch immunodetection Our results show that 15 minutes after birth, pIIa daughter cells are committed to their normal fate and do not respond to CycE overexpression. This suggests that extra divisions observed in these cells result from a delay in the cell cycle exit rather than a re-entry into the cell cycle. In contrast, cycE expression were induced only in a rbf1 mutant background dap background (AA unpublished results). Thus, Dap is involved in neuronal cell arrest. Since Dap is also expressed in shaft cells The fact that CycE induced extra divisions in terminal cells suggests that these cells are arrested in G1. Two main mechanisms trigger G1-arrest: repression of Cyclin/Cdk2 activity by CKI and repression of E2F activity by RB proteins . Thus, the mechanisms by which N and Prospero maintain cell cycle arrest in terminal cells of the bristle organ remain to be elucidated.N-signalling is pleiotropic and either promotes or represses cell cycle progression depending on the cellular context In conclusion, our results demonstrate that fate determination factors such as Notch and Prospero participate in maintaining a quiescent state in terminal cells. Under normal conditions, several mechanisms act in concert to ensure cell cycle arrest. Dap appears as a plausible candidate to ensure this role in N-off cells like the neuron and the shaft cell. N cooperates to maintain a quiescent state in sheath and socket cells and, finally, Pros acts only on the sheath cell. Consequently, the terminal quiescent state and cell differentiation do not seem to be regulated by mutually exclusive mechanisms. We favour the notion that these phenomena are regulated by parallel mechanisms involving factors having dual actions on fate acquisition and cell cycle progression.neur-GAL4 driver was used to specifically express H2B::YFP CycE construct, one on the second and one on the third chromosome neur>CycE and neur>CycE, 17/+pros pupae, overexpression was performed using a strain bearing GAL80ts (gift from D. Coen). Fly crosses, embryonic and larval development were carried at 18°C, and white pupae were transferred to 30°C to allow the expression of GAL4. Overexpression induced by heat-shocks were performed using the hs-CycE (Bloomington); hs-numbNintra17prosts-1Ny, w,UbxFLP; FRT82B ubi-nls::GFP (gift of J. Knoblich) to generate pros-null somatic clones.The neur>CycE, UAS-H2B::YFP, UAS-PON::GFP pupae was carried out as described previously Live imaging of the bristle lineage in Pupal nota were dissected between 17 h and 35 h APF and processed as previously described Figure S1neur-Gal4. Immunodetection of CycE in neur>CycE pupae at 23 h (A), 32 h (B) and 48 h APF (C) and at 23 h APF in (D) hs-numb, neur>CycE, (E) hs-Nintra, neur>CycE and (F) 17/+pros, neur>CycE pupae. Sensory cells are revealed by GFP  and sheath cells by Prospero (blue) immunoreactivity. CycE immunoreactivity is in red and in black (inverse colour) in A'–F'.  Pupae were heat shocked at 20 h APF and dissected three hours later. Note that CycE accumulation fades away in the neurons at 48 h APF (arrowheads in C).CycE expression driven by (8.94 MB TIF)Click here for additional data file.Figure S2neur>CycE, H2B::YFP pupa. (A) Representative frames from a time-lapse observation of a neur>H2B::YFP; Pon::GFP pupae (control). (B) Representative frames from a time-lapse observation of a neur>CycE; H2B::YFP; PON::GFP pupae. Arrows indicate extra divisions. Abbreviations: g, glial cell; n, neuron; s, sheath cell; so, socket cell; sh, shaft cell. Time (h/min) APF is indicated in each panel. Anterior is upwards and the view is dorsal.Time-lapse observation of a (3.91 MB TIF)Click here for additional data file.Figure S317pros clonal analysis. (A–C) Clones were detected by the lack of GFP staining (green), their limits are shown with a white dotted line. Sensory organs are identified with anti-Cut  antibodies. (A) Four cells are present in all sensory organs (red) in 17pros somatic clones. Nota from pupae at 28 hr APF. (B) CycE immunoreactivity (red) was more intense in sensory organs inside the 17pros clone (arrows) than outside the clone (arrowhead). Nota from pupae at 22 hr APF. (C) Dap expression (red) was unchanged in sensory organs in- or outside of 17pros clones. Arrowheads show the pIIIb cells. Nota from pupae at 21 hrs APF.(7.67 MB TIF)Click here for additional data file.Video S1In vivo observation of a neur>H2B::YFP, PON::GFP pupa (control). H2B-YFP is in green, PON-GFP is in red. Anterior is upwards and the view is dorsal.(4.66 MB MOV)Click here for additional data file.Video S2In vivo observation of a neur>CycE, H2B::YFP, PON::GFP pupa. H2B-YFP is in green, PON-GFP is in red. Arrows indicate extra divisions. Anterior is upwards and the view is dorsal.(35.59 MB MOV)Click here for additional data file.Video S3In vivo observation of a neur>CycE, H2B::YFP; 17+/−pros pupa. Arrows indicate extra divisions and asterisk the glial cell death. The view is dorsal.(8.90 MB MOV)Click here for additional data file.


Escherichia coli populations by simple synthetic suicide circuits containing quorum components and a lysis gene. Contrary to design expectations, oscillations required neither the quorum sensing genes (luxR and luxI) nor known regulatory elements in the luxIP promoter. Instead, oscillations were likely due to density-dependent plasmid amplification that established a population-level negative feedback. A mathematical model based on this mechanism captures the key characteristics of oscillations, and model predictions regarding perturbations to plasmid amplification were experimentally validated. Our results underscore the importance of plasmid copy number and potential impact of “hidden interactions” on the behavior of engineered gene circuits - a major challenge for standardizing biological parts. As synthetic biology grows as a discipline, increasing value may be derived from tools that enable the assessment of parts in their final context.Synthetic biology seeks to enable programmed control of cellular behavior though engineered biological systems. These systems typically consist of synthetic circuits that function inside, and interact with, complex host cells possessing pre-existing metabolic and regulatory networks. Nevertheless, while designing systems, a simple well-defined interface between the synthetic gene circuit and the host is frequently assumed. We describe the generation of robust but unexpected oscillations in the densities of bacterium  In principle, a perfect understanding of these factors enables the development of models that accurately predict behavior for a proposed design. In reality, the scale and scope of cellular physiology, coupled with an imperfect understanding of the system and host components, make the construction of such models quite challenging. For this reason, simplified models that assume a well-defined interface between the circuit and host, while ignoring the background processes of host metabolism, are generally employed. If the predictions generated by the simple models deviate from the experimental implementation it indicates that the model insufficiently encompassed the critical components and interactions of the system. In this manner, the validity of common simplifying assumptions can partially addressed and refined during the design process.Synthetic biology In this work we present a genetic circuit (ePop) that causes oscillations in bacterial population density over time. The oscillations arise through the unanticipated interplay of growth conditions, the host cell, and one of the circuit's “background processes” – plasmid replication. Specifically, a density-dependent rise in plasmid copy number leads to an increase in gene dosage and concomitant increase in the expression of a plasmid-borne toxin gene. Toxin expression causes cell lysis and decreases the population density, allowing growth recovery, and generating oscillations as multiple cycles proceed. Although this mechanism was not intended during original circuit design, a refined mathematical model that incorporates plasmid copy number control captures the circuit dynamics, and predictions based on the model are validated by experimental results. Conditional plasmid amplification as a control mechanism for population density has not previously been described and evokes its potential for other applications. Furthermore, these results emphasize that copy number deserves increased attention when designing plasmid-based synthetic gene circuits.One synthetic biology focus has been exhaustive documentation and standardization of individual biological parts E) from phage φX174 luxI promoter (luxIP) from Vibrio fischeri, recently reclassified as Aliivibrio fischeriE protein is an inhibitor of MraY, an enzyme that catalyzes the production of the first lipid intermediate in E. coli cell wall synthesis. Cells deficient in MraY activity lyse during septation, a process that requires newly synthesized cell wall luxR gene (luxR*) and an intact luxI gene under control of an isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter (lac/ara-1P). The luxR and luxI genes originate from A. fischeri and constitute a quorum sensing pair. LuxI produces a quorum signal, an acyl- homoserine lactone (AHL), from cellular precursors. LuxR is a transcription factor that responds to AHL and activates the luxIP promoter. In ePop, luxR* contains a frame-shift mutation that introduces an early stop codon and completely abolishes the luxR DNA-binding domain, as determined by homology to TraR The gene circuit (ePop) contains two modules . The firWhen transformed into MC4100z1 cells, ePop generated multiple cycles of population oscillations during long-term batch cultures . These oV. fischeri, anaerobic growth conditions might have contributed to oscillations. Aerobically grown cells underwent one or two rounds of lysis, but did not oscillate as well. These results indicate that oxygen availability contributed to but was insufficient to account for the observed oscillations.To further characterize the oscillations, we investigated effects of culturing conditions specific to our growth assay, including media, temperature, and oxygenation. Complex media (Luria Broth) might have contributed to oscillations as shifts in preferred nutrient sources during growth could impact E protein expression. However, cells grown in defined M9 minimal media supplemented with glycerol exhibited oscillations similar to those grown in LB supplemented with glycerol . CulturiE gene must be triggered at elevated cell density. However, the luxR truncation and the low impact of IPTG induction suggested that the luxR and luxI genes were not responsible for density sensing. To test their role, we constructed two new versions of the ePop circuit. By design, both have the same luxI-EP module as ePop. ePop-lite contains functional luxR but no luxI  to oscillate.For oscillations to occur, the lysis phenotype conferred by the  no luxI ; ePop-minor luxI . Both eP by ePop  or ePop-op  and represses the right operon (luxICDABE). In ePop, the original CRP site is present and the E gene is located at the position of right operon. E expression and lysis was therefore predicted to be repressed by cAMP and stimulated glucose (which reduces cAMP concentrations). Paradoxically, glucose supplementation abolished oscillations and allowed elevated culture densities (lux operon.It has been shown that cyclic adenosine monophosphate (cAMP) and cAMP receptor protein (CRP) play a significant role in ensities . Neverthlux box, cAMP site, or both maintained oscillations. Only those that deleted the core promoter site and the ribosome binding site abolished oscillations. These results demonstrate that transcriptional activation from upstream promoter elements, such as the lux box or CRP site, were not required for oscillations.To address whether glucose and cAMP operated at the promoter level, as well as whether specific promoter regions required for oscillations could be isolated, a promoter deletion series was constructed . ePop coV. fischeri as well as E. coli transformed with the lux operon from V. fischeriIn the apparent absence of transcriptional regulation, we surmised that changes in gene dosage at higher cell density could cause oscillations. Modulation of DNA levels has been implicated in effects of iron deprivation on luminescence in for reviews seeRegulation of replication in ColE1-type plasmids has been well characterized, relA+ and relA strains  and is somewhat enhanced for plasmid lacking the Rom/Rop protein Relevant to the ePop dynamics, studies have identified several environmental factors that increase ColE1 copy number. Chloramphenicol causes the arrest of protein synthesis and chromosomal replication while stimulating replication from the ColE1 origin resulting in drastic plasmid amplification Details on a mechanistic link between environmental factors and ColE1 replication have recently emerged and suggest that uncharged tRNAs directly catalyze the degradation of RNA I thereby dis-inhibiting replication The complexity and number of inputs inherent to the both the stringent response and ColE1-type plasmid regulation  is set to zero, lysis does not occur and the model reduces to a simplified treatment ColE1 copy number control. Increasing δ1 in this context minimizes the plasmid amplification that occurs as stationary phase is reached and mirrors the impact of glucose on the accumulation of the ePop backbone pNewTet.E  buffered with 100mM MOPS (pH = 7.0) was used for cell growth. 3oxoC6 homoserine lactone (AHL) was synthesized by the Duke Small Molecule Synthesis Facility. DPD was produced using pfs and luxS genes cloned from MG1655, expressed in BL21 (DE3) cells using pET-21 and purified using an immobilized metal affinity chromatography column . The enzymatic reaction was performed as previously described MC4100Z1 cells were the gift of M Elowitz. BW25113 and MG1655 were obtained from the Cells were grown in 48 well Tissue Culture Plates  inside a Perkin-Elmer VICTOR3 plate reader heated to 37°C unless otherwise noted. Wells containing 500µL fresh media and the appropriate chemicals were inoculated with 50µL of a starter culture . 250µL mineral oil was layered above each well to prevent evaporation over the course of the experiment. Care was taken to ensure starter cultures did not reach densities high enough to induce lysis. Prior to each measurement, plates were shaken for 5 sec in an orbital pattern. OD at 600nm was measured every 15 min. Data presented are the raw OD values that result from 500uL culture in the plates used. They have not been modified to account for path length.R) was constructed using the lux box region (140 bp upstream of luxI in V. fischeri) from pluxGFPuv E gene coding sequence from φX174 (NEB). Each region was PCR-amplified and then joined together in an overlap PCR reaction. The ‘lux box-E gene’ fragment was inserted into the AatII site of host vector pLuxRI2 mini was constructed by inserting the lux box-E gene fragment (AatII digest of ePop) into pLuxR2 lite was constructed by digesting ePop with HindIII  and re-ligating the larger fragment from an agarose gel purification. Promoter deletion mutants were generated using divergent primers flanking the deletion of interest. Each primer introduced a terminal NheI site. Digestion and intramolecular ligation generated plasmids where the region targeted for deletion was replaced by a 6 base pair NheI “scar”. pNewTet.E was generated by introducing a 40bp oligo with NdeI and NheI sites into the AatII site of pProTet.E (Clontech).ePop . 750µL of starter culture was added to 2L of LB or LB+glucose  in 6L non-baffled flasks and grown at 37C and 180rpm. Chloramphenicol was added to the media to a final concentration of 30.6 µg/mL or 106µg/mL. An equivalent OD of cells was removed at various time points using a previously determined calibration curve to account of the non-linearity of OD measurements. Cultures were centrifuged for 10min at 5000g and re-suspended in 2mL Tris-buffered Saline. 240µL of the resuspended culture was added to 2mL LB and OD was recorded. Resuspended OD values were all within 20% of one another, enabling linear normalization. The remaining re-suspended culture was used to perform triplicate 500µL minipreps using the Zyppy miniprep kit (Zymo Research). Miniprep yield was measured on a ND-1000 UV spectrophotometer (Nanodrop).Figure S1(A) or presence (B) of 1mM IPTG and OD (600nm) was measured (every hour in this case) in a plate reader. 1mM IPTG did not drastically change the nature of the oscillations but did affect the synchronization across different colonies and starter cultures (different colored traces) (C) Colony forming unit (CFU) experiments were performed every three hours and show that viable cell density correlated with OD. (D) MC4100z1 cells that do not carry any plasmids (red) do not show the oscillations exhibited by MC4100z1 cells that carry the ePop plasmid (green) demonstrating that strain and culturing conditions were insufficient to produce oscillations.OD monitoring of MC4100z1 cells carrying the ePop circuit. Cells were grown in the absence (0.48 MB TIF)Click here for additional data file.Figure S2(A) Cells grown in buffered LB supplemented with glycerol (top) showed similar oscillations to cells grown in M9 minimal media supplemented with glycerol (bottom). Different colored traces represent individual colonies. Oscillations are therefore not due to some unknown component in complex media or shifts in preferred media source. (B) Cells grown at 33C (black) exhibit an elevated lysis density and longer period than those grown at 37C (red). (C) Cultures treaded with DPD exhibited one of two phenotypes in response. Cells either oscillated with a similar period but recovered more quickly from an initial round of lysis (top) or had oscillation period significantly increased by DPD (bottom). The differences between the two types of colonies, presumably of genetic origin, have not been determined. DPD and AI-2 did not appear to be the feedback signal X, however, because neither prevented growth or caused increased lysis.Effect of changing culturing conditions. (0.57 MB TIF)Click here for additional data file.Movie S1Solid phase growth monitoring of BW25113 cells carrying the ePop plasmid. Microscope slides were prepared by allowing warm LB agarose (1%) containing the appropriate antibiotics and inducers  to be drawn under a suspended cover slip. After cooling, the coverslip was removed and the agarose pad cut to size with a clean razor blade. 1 µL low density starter culture  was placed on the agarose and a fresh coverslip was applied. Slides were sealed with mineral oil and nailpolish to prevent evaporation. Cells were placed on a Leica inverted microscope in an environmentally controlled enclosure set to 37°C. After 1–2 hours, a region for monitoring was selected on the basis of exhibiting cell division. Images were captured using a Hammamatsu 1384 ORCA-ERA camera at 4 frames per minute overnight using autofocus feature to maintain the focal plane.(4.17 MB MP4)Click here for additional data file.


Saccharomyces cerevisiae, sister chromatids separate gradually from centromeres to telomeres, accompanied by regional chromosome stretching and subsequent recoiling. The stretching results from residual cohesion between sister chromatids, which prevents their immediate separation. This residual cohesion is at least partly dependent on cohesins that have escaped removal by separase at anaphase onset. Meanwhile, recoiling of a stretched chromosome region requires condensins and generates forces to remove residual cohesion. We provide evidence that condensins promote chromosome recoiling directly in vivo, which is distinct from their known function in resolving sister chromatids. Our work identifies residual sister chromatid cohesion during early anaphase and reveals condensins' roles in chromosome recoiling, which eliminates residual cohesion to complete sister chromatid separation.Sister chromatid separation is initiated at anaphase onset by the activation of separase, which removes cohesins from chromosomes. However, it remains elusive how sister chromatid separation is completed along the entire chromosome length. Here we found that, during early anaphase in  ► Sister chromatid cohesion partially persists after anaphase onset in budding yeast ► Residual cohesion is partly dependent on cohesins and causes chromosome stretching ► Condensins directly promote recoiling of stretched chromosomes during early anaphase ► Chromosome recoiling generates forces to remove residual sister chromatid cohesion Cohesion between sister chromatids is established during S phase and maintained until anaphase. A multisubunit protein complex cohesin plays a central role in establishment and maintenance of sister chromatid cohesion . Upon anPrior to chromosome segregation triggered by removal of sister chromatid cohesion, sister chromatid DNAs must be disengaged from each other. This is facilitated by topoisomerase II, which removes catenation between sister chromatids that remains after completion of DNA replication . DecatenIt is thought that sister chromatid resolution is achieved by cooperation between topoisomerase II and condensins . Indeed,Meanwhile, several reconstitution studies have revealed that condensins can promote supercoiling of DNA, in vitro e.g., . HoweverSaccharomyces cerevisiae may prove a useful system as its chromosomes show little condensation upon the transition from interphase to mitosis  fused with green fluorescent protein (GFP) and thus forming microscopic fluorescent “dots,” in the vicinity of CEN15, at the HIS3 locus, and close to the telomere  on the right arm of chromosome XV , the CEN-HIS distance was enlarged momentarily  at its maximum during anaphase compared with this resting length  on the right arm of chromosome XV with tet operators   C.The greater stretching (averaged for 10 kb) of shorter chromosome arm intervals indicates that at any given time during anaphase, stretching occurs along a relatively small chromosome arm region, rather than uniformly over a long region. We infer that, as a centromere is pulled toward a spindle pole immediately after anaphase onset, the region around the centromere is stretched C and subHIS locus (marked with GFP), after anaphase onset  but before any spindle pulling force was applied to the HIS locus. To monitor this period, the ADE2 locus  was marked with lac operators, bound by Lac repressors (LacI) fused with cyan fluorescent protein  and degraded through auxin-induced poly-ubiquitylation  or by a global mechanism uniformly working all along chromosome arms? Our results in smc2-8 and ycg1-2 ; subsequently the CEN-HIS region stretched, similarly to wild-type. However, in these mutants, there was a significant delay in the completion of HIS dot segregation  happened with a significantly lower velocity than in wild-type cells , which was unable to grow  and reached a similar conclusion  mutant, in which chromosome segregation defects result from the inability to resolve catenation between sister chromatids  see FigA. We stu4 mutant  , TEL segregation speed should be similar in wild-type and condensin mutants as sister chromatids has been separated all along their arms by this point. On the other hand, if condensins are directly involved in recoiling chromosome arms, TEL segregation speed of dots should be lower in condensin mutants.To address further the primary role of condensins in chromosome segregation, we next evaluated the segregation speed of the TEL dot segregation (shortening of the CEN-TEL distance) to the bud was significantly lower in the smc2-8 mutant (and the few ycg1-2 cells that showed TEL segregation) than in wild-type cells (smc2-aid mutant (see TEL segregation speed in condensin mutants was caused by remaining sister catenation within the short region (18 kb) between the TEL dot and the end of the chromosome; if such catenation were involved and subsequently dissolved, the TEL segregation speed would have been enhanced later, but it was almost constant as it proceeded (We found that the speed of the tant see Bv. It isroceeded Bi. ColleHIS locus in the top2-4 mutant (see CEN3 (eCEN3), under the control of a galactose-inducible promoter . The spectrum in YCG1+ was well fitted by a power-law , similar to the top2-4 mutant but distinct from condensin mutants , i.e., not only Scc1 degradation but also entry to anaphase was necessary for the TEL dot segregation. Thus reduced cohesion partially rescues chromosome segregation when condensins are defective. A corollary is that condensin-dependent chromosome recoiling facilitates elimination of residual cohesion to complete separation of sister chromatids along their arms  and methods for yeast culture were as described previously . Unless