As the secretory source of vitamins, peptides and hormones for neurons, the choroid plexus (CP) epithelium critically provides substances for brain homeostasis. This distributive process of cerebrospinal fluid (CSF) volume transmission reaches many cellular targets in the CNS. In ageing and ageing-related dementias, the CP-CSF system is less able to regulate brain interstitial fluid. CP primarily generates CSF bulk flow, and so its malfunctioning exacerbates Alzheimers disease (AD). Considerable attention has been devoted to the blood-brain barrier in AD, but more insight is needed on regulatory systems at the human blood-CSF barrier in order to improve epithelial function in severe disease. Using autopsied CP specimens from AD patients, we immunocytochemically examined expression of heat shock proteins (HSP90 and GRP94), fibroblast growth factor receptors (FGFr) and a fluid-regulatory protein (NaK2Cl cotransporter isoform 1 or NKCC1). CP upregulated HSP90, FGFr and NKCC1, even in end-stage AD. These CP adjustments involve growth factors and neuropeptides that help to buffer perturbations in CNS water balance and metabolism. They shed light on CP-CSF system responses to ventriculomegaly and the altered intracranial pressure that occurs in AD and normal pressure hydrocephalus. The ability of injured CP to express key regulatory proteins even at Braak stage V/VI, points to plasticity and function that may be boosted by drug treatment to expedite CSF dynamics. The enhanced expression of human CP 'homeostatic proteins' in AD dementia is discussed in relation to brain deficits and pharmacology. Accumulating evidence supports the idea that continually decreasing choroid plexus (CP) function in advanced ageing exacerbates Alzheimer's disease (AD). As part of a new paradigm to explain brain interstitium deterioration in age-related dementias, increasingly more attention is being paid to the role of compromised blood-CSF  and blooEfficient CSF turnover is essential for a healthy brain. It depends upon an exquisite balance between CSF formation and reabsorption . CompromIn serving the brain's metabolic needs by supplying 'biochemical goods', the CP uses CSF as a conduit for convecting substances . NumerouReduced formation of CSF and stagnated flow in ageing and AD  limits tStructure intimately relates to function in various epithelia. Therefore it is useful to analyze histopathological damage to CP at various stages of AD in order to clarify the onset of functional losses at the blood-CSF barrier. Ageing and AD cause similar degeneration in CP. Structural changes in AD though are usually greater than in non-demented control counterparts . Serot aSeveral features characterizing the CP in AD are highlighted in Table Due to the functional nexus of the CSF with brain interstitial fluid, the neuronal microenvironment in AD is impacted by markedly altered transport and permeability in CP. When neurodegenerative diseases, ischemia ,28 and e+] and immune molecule content of the CNS extracellular fluid [The CP is multifunctional, performing a wide range of homeostatic functions for the CNS . CSF homar fluid . AccordiMoreover to provide steady neuroprotection by regulating the extracellular milieu, the CP must protect itself against various stressor agents that build up during ageing and disease. There have been few investigations of the homeostatic systems within CP that stabilize choroidal functions in the face of ageing and AD. We have explored some candidate systems for compensatory responses by CP. Cytoplasmic heat shock proteins chaperone and perform housekeeping to maintain a healthy steady-state intraepithelial milieu ,32. GrowIt is difficult to functionally assess the CP in vivo ,33, partInformation about functional proteins in human CP is scarce. However protein expression was recently analyzed in autopsied lateral ventricle CP from normal adult brains and in those with confirmed AD ,37. The Ageing and AD dementia tax the CP and other CNS transport interfaces. In late life the deteriorating brain presents many potentially-destructive metabolites to the CSF for multi-site excretion into blood. The greater burden of macromolecule disposal in AD occurs when CP and arachnoid membrane, due to ageing debilities, are less able to transfer solutes. Nevertheless the CP seemingly attempts to maintain its 'epithelial soundness' when challenged to perform additional cleansing acts for the brain extracellular fluid (CSF).In healthly, young adults the CP epithelium sensitively acclimates to chemical and physical distortions in blood, CSF and parenchyma. Following cortical stabbing in rats, the CP upregulates TGFβ presumably to provide this CSF-borne growth factor for repairing the injury . A similAccordingly to assess pathophysiologic consequences of AD we investigated human CP's ability to upregulate certain functional proteins  in advanced states of AD dementia. With advancing knowledge about intricate cell physiology  it is relevant to evaluate components of cellular homeostasis. The term 'homeostatic proteins'refers to chaperones, receptors and transporters that stabilize the internal environment of the cell. CSF stability depends in large part upon CP epithelial homeostasis. Our group has analyzed several 'homeostatic proteins' involved in CP intracellular milieu stabilization:A wide spectrum of protection against neurodegeneration is provided by HSPs. The list of protective effects bestowed by HSP molecules is diverse and includes: accelerated degradation of misfolded proteins, maintenance of membrane lipid integrity, prevention of deleterious protein aggregation, and preclusion of damage to the translational apparatus ,42. HSPsTo evaluate human CP expression, we selected HSPs overexpressed in AD brains: GRP94 and HSP90. In contrast to previously found upregulation in brain, there was downregulated GRP94 in lateral ventricle CP. In aged controls there was abundant staining in the epithelial cytoplasm and stroma  ontogeny, ii) normal adult maintenance, and iii) neurodegeneration.FGF and FGFr are also fundamentally important in fostering neuron generation from stem cells in the subventricular zone (SVZ). This requires coordination between the CP-CSF and periventricular regions ,48. Pharnd but not 3rd gestational week [Receptor plasticity in aging and AD is better seen in light of information on FGF/FGFr expression dynamics in early life. In the fetus the formation of CP, neuronal stem cells and brain is promoted by CP growth factor secretion and CSF distribution ,49. InteFGF2 derived from CP is also conveyed by CSF bulk flow to the fetal germinal matrix where it acts on stem cell FGFr to promote neuronal maturation . By thisThe FGF/FGFr system also has a key role in adult CNS fluid homeostasis. CP helps the brain adapt to alterations in blood composition and flow. In otherwise healthy young adults, the imposition of dehydration or sudden ischemia upon the CNS elicits striking adaptive changes in CP epithelium. To endure insults by chemical or physical stressors , it is cDehydration seriously threatens CNS functions. Adjustments to dehydration in the healthy adult brain feature ion and water redistribution among fluid compartments. These compensatory responses to plasma hyperosmolality stabilize neuronal and interstitial volumes. Brain 'barriers' or transport interfaces are sites for the fluid homeostatic mechanisms. A working model for the restoration of fluid balance is offered: Dehydration or hyperosmolality upregulates the FGF2 and AVP peptides in CP ,52. FGF2Ischemia is another disorder with neuropathological consequences that are mitigated by growth factor upregulation or administration . BilaterFGF2 titers and FGFr receptor densities in degenerating CNS compartments provide insight on adaptive responses. FGF2 concentration is augmented in the AD brain . Moreove1-43 [FGF2 has an interesting relationship with amyloid. A worthwhile goal is to probe mechanisms of FGF2 interaction with amyloid in neuronal networks, extracellular matrix and CP-CSF. FGF2 co-localizes with several chemical forms of amyloid. Neuronal coexistence of FGF2 and amyloid precursor protein (APP) ,65 intim1-43  and atte1-43 . In the 1-43 ,70. ThisFGF2-mediated protective regulation of CP transport phenomena potentially affects the course of neurodegeneration. Considerable evidence points to CP's ability to remove Aβ from CSF -73. ThisThe diminished ability of CP to form fluid in advanced ageing and AD begs the question of how epithelial ion transport proteins are altered by distorted neurochemistry in senescence. In very old laboratory mammals the Na-K-ATPase activity of CP and the CSF generated by it are cut in half ,76. AnotThe cation-Cl superfamily of cotransporters includes NKCC1 and consists of 7 isoforms that actively transport Na and/or K electroneutrally with Cl . NKCC1 iThe NKCC1 helps cells and organs adjust to disrupted fluid balance. One thus expects homeostatic upregulation of this CP cotransporter in AD with its altered CSF dynamics. For delineating NKCC1 expression we used the T4 antibody to immunostain CP at various stages of AD dementia. Robust staining of the lateral ventricle plexus (even at Braak stage V/VI) occurred in the apical membrane and cytoplasm , then it might be feasible to prevent the early manifestations of AD (Braak stages I/II) from intensifying into the debilitating pathology of V/VI.One class of agents with considerable potential for AD therapeutics is the growth factor group. A useful paradigm for CSF growth factors and neuroprotection has evolved from experiments on transient forebrain ischemia in rats ,60. The Several other growth factors synthesized by CP and secreted into CSF should be explored in translational research dealing with interacting ischemia, hydrocephalus and AD ,103,104.In searching for agents to modify CP epithelial protein expression, the route of delivery of the active drug is of primary importance. Unlike the brain with its impermeable microvessels, the CP readily takes up water-soluble drugs from the plasma due to the highly-permeable choroidal capillaries. Consequently water-soluble agents freely diffuse to receptors or binding sites at the basolateral surface of the epithelium. Access of blood-borne hydrophilic compounds to the CSF-side of CP however is problematic because the tight junctions and basolateral membrane impede diffusing molecules as small as mannitol . MoleculTo circumvent the blood-CSF barrier, therapeutic agents are delivered into CSF by lateral ventricle catheters in experimental animals  or hydroA compelling aspect of CSF translational research is to identify agents that effectively regulate fluid formation by CP. Whereas the difficulty in congenital hydrocephalus is to downregulate CSF formation, the challenge in AD is to enhance CSF turnover perhaps by accelerating fluid production as well as outflow. Augmented flow of CSF enhances 'sink action' ,109. ThiAnother CP pathology meriting pharmacological attention is the massive fibrosis that progressively envelops the interstitium in old age. This interstitial fibrosis is even more extensive in AD . FibrosiThe CP has the main responsibility for CSF homeostasis. Therefore the functional status of the blood-CSF barrier is of great consequence to the CNS. Maintaining the CSF at a stable, specialized composition is of the utmost importance to neurons. CSF is prominent in regulating brain interstitial fluid with which it exchanges nutrients and waste products. Diseases markedly affect these molecular exchanges. Maintaining healthy bidirectional transport across the CP epithelium (CSF-blood) and ependyma (CSF-brain) is thus integral to a sound brain fluid environment.CSF macrocirculation through the ventriculo-subarachnoid system together with CSF microcirculation in the perivascular Virchow-Robin spaces ,111 perfAs the main generator of CSF, the CP has a pivotal role in helping the brain cope with the twin stressors of ageing and disease. More attention should be focused on the blood-CSF interface for pharmacologic opportunities to stave off CP dysfunction. Our findings on human CP expression of HSP90, FGFr and NKCC1 demonstrate that this epithelium in AD reacts to metabolic insults by upregulating certain proteins. This suggests that even diseased CP could respond to therapeutic agents, thus opening new vistas for treating CSF dysfunction in age-related dementias.Investigation of CP in AD is an area that is opening up. Translational research can now intensely focus on molecular factors that disable the CP to the point of reducing its ability to preserve brain integrity. Systematic CSF analyses using mass spectrometry and other cutting-edge biotechnology should generate neurochemical data specific for disease stages. New imaging approaches are essential to provide much needed functional data for CP, CSF and periventricular regions in AD patients. This should expedite the modeling of CP-CSF malfunctions and their resolution. Deeper insight into the pathophysiology of the blood-CSF transport interface will help to realize the development of novel therapeutic regimens for the AD family of diseases.AD, Alzheimer's disease; APP, amyloid precursor protein; Aβ, beta amyloid; AVP, arginine vasopressin; BBB, blood-brain barrier; CP, choroid plexus; FGF2, basic fibroblast growth factor 2; FGFr, receptor for fibroblast growth factor; GRP94, glucose regulatory protein 94; HSP90, heat shock protein 90; NGF, nerve growth factor; NKCC1, Na-K-2Cl cotransporter secretory isoform 1; NPH, normal pressure hydrocephalus; TGFβ, transforming growth factor beta; SVZ, subventricular zone; TFI, transient forebrain ischemia; VEGF, vascular endothelial growth factor; IGF-II, insulin-like growth factor II; HGF, hepatocyte growth factor;The author(s) declare that they have no competing interests.CJ had the primary responsibility of organizing and writing the review, and had NIH support (NS 27601) to do the NaK2Cl cotransporter experiments. PM carried out the NKCC1 immunostaining runs with the T4 antibody and provided interpretation of the regional stainings. RT conducted the experimental analyses of the heat shock proteins and generated figures. AS did the image processing analyses of the cotransporter expression and assisted with the literature analysis. JD contributed ideas for the altered CP-CSF dynamics in hydrocephalus and ventriculomegaly. GS has developed the model that CP-CSF malfunction exacerbates AD progression, and helped to revise the manuscript. ES was responsible for the FGFr experiments and interpreted the human CP data. All authors read and approved the final manuscript.


There is wide variation in the quality of care provided by primary care practices to individuals with chronic illnesses. Individual doctor attitudes and interest have been demonstrated to influence patient outcomes in some instances. Given the trend towards larger practices and part-time working, continuity of care is likely to fall and thus practice-based rather than individual general practitioner attributes and attitudes are likely to become increasingly important. The aim in this paper was to examine the extent to which individual general practitioner (G.P.) attitudes to the care of people with epilepsy cluster within practices and predict patient-rated quality of care.The sample consisted of 1255 people with active epilepsy (a recent seizure or on anti-convulsant medication for epilepsy) and 199 GPs from 82 general practices. Measures of GP attitudes (a 17-item GP attitudes questionnaire) and patient-rated quality of epilepsy care were obtained. 1210 individuals completed initial questionnaires and 975 patients filled in final questionnaires one year later. Responses were achieved from 64 practices  and 115 GPs .2 main factors were found to underlie GP attitudes to the care of people with epilepsy and these demonstrated clustering within practices "epilepsy viewed as a primary care responsibility"  0.40), and "medication skills". GP-rated scores on "epilepsy care being a primary care responsibility" were a significant predictor of patient-rated quality of GP care (p = 0.031). Other contributory factors were seizure frequency (p = 0.044), and patient-rated "shared decision making" (p = 0.022).Specific general practitioner attitudes to the care of people with epilepsy cluster within practices and are significantly associated with patient-rated quality of epilepsy care. It is important to take these findings into consideration when planning primary care interventions to ensure people with epilepsy receive the benefits of available medical and surgical expertise. There is wide variation in the quality of care provided by primary care practices to individuals with chronic illnesses . IndividThere is a marked trend to larger partnerships in primary care practices and more flexible working practices. It is likely, therefore, that continuity of care will continue to fall, and that patient experience of care of a particular condition will be based on contact with more than one general practitioner . Thus prIn the next few years there is likely to be considerable reorganisation of the way in which epilepsy care within primary care is delivered, with GPs taking on a more active role in providing care. Information on how individual general practitioners view and value their role in providing epilepsy care is considered as important . HoweverIn this paper, data from a completed community-based study on people with epilepsy are used to examine the following questions:1. To what extent do individual general practitioner attitudes to the care of people with epilepsy cluster within practices?2. Do general practitioner attitudes predict how people with epilepsy rate the quality of the general practitioner care of their epilepsy?General practitioners and adults with epilepsy taking part in an intervention study in Greater Manchester provided information for this study. The results of the intervention study  have been reported . No grouEthical approval was obtained in the 4 areas of Greater Manchester from where patients were approached. The patients who consented to participate in the study had their medical records examined to extract data on recording of clinical information about epilepsy and other markers of quality of care. Patients were also sent questionnaires for self-completion. These included both a generic quality of life measure- the EUROQOL 5D and a diGeneral practitioners completed a 17-item GP epilepsy attitudes questionnaire at the end of the study. Responses to items such as "I feel comfortable changing the type of anti epileptic drugs in my patients" were scored using a Likert scale. The attitudes scale was developed and validated for a previous study and results reported in an earlier paper .Mean practice scores for each item on the GP attitudes questionnaire were computed and significant factors underlying the grouping identified . Clustering of responses on the attitudes questionnaire were examined using the intra-class correlation coefficient both for individual items as well as for a mean score of responses to items loading on each of the main factors. Finally linear regression analysis with the patient-rated quality of care provided by the practice as the dependent variable and GP attitudes and other patient derived measures  as independent variables was carried out (using aggregated GP attitude and patient scores). Intervention group was adjusted for. P values of 0.05 and 95% confidence intervals were used to assess significance.1255 patients consented to participate in the study and 975 patients filled in final questionnaires. 199 GPs from 82 practices consented to participate in the study. Responses were obtained from 115 GPs  from 64 practices . 29 practices had a single respondent. These practices were excluded from the analysis of attitude clustering. In this study 54% of individuals were seizure-free in the previous year ("controlled" seizures) and 46% had reported a seizure in the previous year ("uncontrolled" seizures).Factor analysis with varimax rotation was undertaken on aggregate GP responses. Four factors had an eigenvalue of above 1.2. Three of these factors were selected after the scree test. Both the Kaiser-Meyer-Olkin measure of Sampling adequacy test (0.778) and Bartletts test for sphericity  suggested that factor analysis was appropriate for this data set. Using guidelines for identifying significant factor loadings based on sample size from Hair et al.[Responses to the 11 questions that were included in the first three factors were further examined. The aim was to detect if significant clustering of responses to these items occurred within practices. The average cluster size was 2.74.The results of the factor analysis and clustering of attitudes for the two main factors are listed in Table patient seizure frequency and patient-rated "shared decision making" and GP-rated score on "epilepsy care being a primary care responsibility" (Factor 1). Recording of clinical information about epilepsy was not a significant predictor of patient-rated quality of GP care.Data from 60 practices where both patient and general practitioner data were available were used in the linear regression analysis. As the data were obtained at the end of an intervention study, the intervention group was also included as an independent variable. The results of this analysis are given in Table Some further bivariate analyses were also undertaken. Recording of clinical information about epilepsy by GPs was not significantly associated with the GP-rated score on epilepsy care being a primary care responsibility but was associated with seizure frequency.In this study two main factors ("epilepsy viewed as a primary care responsibility" and "medication skills") were found to underlie GP attitudes to the care of people with epilepsy. Responses to questions constituting these factors demonstrated a high and significant level of clustering within practices. The main factor that accounted for the largest proportion of variance, general practitioner-rated "epilepsy viewed as a primary care responsibility", significantly predicted patient-rated quality of care. Patient-rated shared decision-making and seizure frequency were other significant predictors of patient-rated quality of GP epilepsy care. Recording of clinical information by GPs about epilepsy was not associated with GP attitudes to epilepsy care but was related to patient seizure frequency.In this study general practitioner attitudes to the care of people with epilepsy were found to cluster within practices to a considerable extent. This has not previously been shown in the U.K. A recent Dutch study  showed tThere is relatively little information of the relationship of GP attitudes to patient ratings of the quality of GP epilepsy care. Existing evidence suggests that GPs with a special interest in a particular condition improves outcomes . The resThe results of a multilevel analysis examining patient and doctor predictors of patient satisfaction from the Netherlands  suggestePatient ratings of the quality of care do vary according to whether individuals have controlled or uncontrolled seizures. Individuals with controlled seizures rate the quality of care provided higher than individuals with uncontrolled seizures. However why the ratings of care provided are higher is not clear as individuals with controlled and uncontrolled epilepsy differ from each other in other characteristics that may influence quality ratings apart from seizure frequency .At practice level GP attitudes are not related to mean practice patient seizure frequency. Although it is likely that individual GP attitudes will be influenced by whether an individual patient has controlled or uncontrolled seizures it is not possible to empirically demonstrate this relationship, as nearly all general practitioners will see a mix of individuals with "controlled" and "uncontrolled" seizures in a given year. Their attitudes to the care of people with epilepsy will be influenced by this spectrum of epilepsy severity .In terms of limitations of the results, some practices did not consent to take part in this intervention study and not all GPs who participated completed questionnaires. However there were no significant differences between practices that participated and did not participate in terms of size, average deprivation or training status . MoreoveSpecific general practitioner attitudes to the care of people with epilepsy are significantly associated with patient-rated quality of epilepsy care and cluster within practices. It is important to take these findings into consideration when planning interventions and services. General practitioners need to have good knowledge and skills in the management of epilepsy and should be aware of and utilise current guidelines for good clinical epilepsy care -19 to fuThe author(s) declare that they have no competing interests.AT designed and ran the study, undertook the analysis and wrote the manuscript. MR was involved in the design and running of the study and edited the paper.The pre-publication history for this paper can be accessed here:


Post-translational modification by Small Ubiquitin-like Modifiers (SUMO) has been implicated in protein targeting, in the maintenance of genomic integrity and in transcriptional control. But the specific molecular effects of SUMO modification on many target proteins remain to be elucidated. Recent findings point at the importance of SUMO-mediated histone NAD-dependent deacetylase (HDAC) recruitment in transcriptional regulation.Rad60, Esc2 and mouse NIP45 . Different proteins of the novel family are known to interact directly with histone NAD-dependent deacetylases (HDACs), structural maintenance of chromosomes (SMC) proteins, and transcription factors. In particular, the highly non-trivial designation of the first of the two successive SUMO-domains in non-plant RENi provides a rationale for previously published functionally impaired mutant variants.We describe the RENi family of SUMO-like domain proteins (SDP) with the unique feature of typically containing two carboxy-terminal SUMO-like domains. Using sequence analytic evidence, we collect family members from animals, fungi and plants, most prominent being yeast Till now, SUMO-like proteins have been studied exclusively in the context of their covalent conjugation to target proteins. Here, we present the exciting possibility that SUMO domain proteins, similarly to ubiquitin modifiers, have also evolved in a second line – namely as multi-domain proteins that are non-covalently attached to their target proteins. We suggest that the SUMO stable fusion proteins of the RENi family, which we introduce in this work, might mimic SUMO and share its interaction motifs . This presumption is supported by parallels in the spectrum of modified or bound proteins e.g. transcription factors and chromatin-associated proteins and in the recruitment of HDAC-activity. Among ubiquitin-related proteins, containing at least one domain with a ubiquitin-like fold, one can distinguish ubiquitin-like modifiers (UBLs) and ubiquitin domain proteins (UDPs) .UBLs can be covalently attached to target proteins analogously to ubiquitin. Unlike ubiquitin, UBLs mostly do not directly target proteins for degradation , althougIn contrast to UBLs, UDPs are not conjugated to other proteins and lack the C-terminal double glycine motif characteristic for ubiquitin and ubiquitin-like modifiers. They are a heterogeneous class of usually multi-domain proteins, which are unrelated outside of their ubiquitin-like domain . In seveThe biological relevance of non-conjugatable multi-domain proteins having a domain with clear relationship to UBLs like SUMO, rather than ubiquitin, is yet unknown. Here, we present a detailed sequence analysis of a family of SUMO-like domain proteins (SDPs) containing one or two SUMO-like domains. Members of the proposed RENi family act as factors in transcriptional regulation, chromatin silencing and genomic stability.During the study of the predicted nuclear subset of the Drosophila proteome, we encountered the unknown 424 amino acids long protein CG4449 NP_651134). Initial analysis of its sequence complexity shows that the disordered N-terminal half of the protein is followed by a likely globular segment . Indeed, a compositionally biased, polar low-complexity region (LCR) spans almost the entire N-terminal 220 amino acids (AA) as reported by CAST  [1134. IniThe C-terminal half of CG4449 turns out to contain an internally repeated segment identifiable with RADAR  region .In conclusion, we found that the Drosophila protein CG4449 NP_651134) has a tripartite architecture: with a N-terminal LCR followed by two globular domains with a SUMO-like fold (termed SD1 and SD2). Whereas SD2's similarity to single domain SUMO-like sequences can be easily detected with BLAST tools, the identification of SD1 is non-trivial , H. sapiens , M. musculus  and C. elegans . All these proteins contain a LCR at the N-terminus followed by two SUMO-like domains, the first of which has mostly diverged away beyond recognition thresholds using traditional sequence-profile searches  including the two SUMO-like domains, collects a family of animal proteins with the same tripartite organization in D. melanogaster CG4449 (220–424) and the corresponding sequences derived from A. gambie, X. laevis, C. elegans, C. briggsae, M. musculus and H. sapiens  A. thaliana , O. sativa , S. pombe , S. cerevisiae , Y. lipolytica , C. glabrata  and in other recently published fungi proteomes [S. cerevisiae and S. pombe homologs correspond to the studied Rad60 and Esc2 proteins, respectively [A multiple sequence alignment of the globular C-terminal half of s Figure  was usedroteomes . The S. ectively ,15; the These HMM-search results suggest the most likely plant and lower eukaryote orthologues to the animal NIP45-like proteins. For establishing the orthology relationship, these initial results need to be confirmed by reciprocal searches independently performed for fungal and plant proteins. Further below, we present this evidence for the homology between the C-terminal part of the proteins found in the various taxonomic groups.C. glabrata representative (CAG57776) and retrieving the best and significant hits in the proteomes of S. cerevisiae, S. pombe, K. lactis, C. albicans, Y. lipolytica, D. hansenii, A. nidulans [M. musculus , A. thaliana , C. elegans .The set of fungal RENi proteins can be autonomously collected using a BLASTP search started with the nidulans . The domnidulans ). A HMM A. thaliana  against the TIGR Gene indices of barley, maize, rice, potato and soybean [A. thaliana in Y. lipolytica  and H. sapiens proteomes  [Potential plant RENi homologs, derived in a full-length TBLASTN search with  soybean , show a  soybean ) and a ct 8e-05) .We propose to name the collected group of protein sequences the RENi-family after its most studied members Rad60, Esc2 and NIP45. All representatives have a similar sequence architecture involving a N-terminal low complexity region with many polar and (positively) charged residues and a C-terminal globular part with one (plant proteins) or two  SUMO-like domains.The use of a model representing the complete globular region of RENi proteins was essential for the successful collection of the family. A global HMM spanning the SD1 and SD2 domains tests for homology in the whole globular part and, correspondingly, directly collects the RENi family. In contrast, when using the C-terminal half of various RENi family members as query sequence in PSI-BLAST , the seaWhile RENi proteins of the fungal, metazoan and mycetozoan taxa contain two C-terminal SUMO-like domains SD1 and SD2), only the second one can be clearly defined in plant representatives   and rad663E  and A graph representation of the pair-wise similarity relationship for SD1 and SD2 sequences to other known ubiquitin-like domains  is involved in chromatin silencing via the recruitment or stabilization of the Sir (silent information regulators) complex [ complex ,28. It i complex  and, thu complex  and act  complex  as well  complex ,32. With complex  but also complex .NIP45, the one studied RENi in metazoa, has been implied in gene regulation, where it needs its DNA-binding partner NFATp for this activity . StrikinThere is little experimental data on the importance of the predicted SUMO-like domains in RENi proteins. Nevertheless, all listed functions of RENi proteins conform with the known role of SUMO in transcriptional regulation and the control of genome integrity . In the In this report, we use sequence-analytical methods to infer the homology relationships between RENi family members and determine their tripartite (bipartite for plant homologs) domain architecture. A N-terminal polar low-complexity segment and two consecutive SUMO-like domains in the C-terminal half characterize the functionally described fungal and metazoan RENi proteins. While the more C-terminal SD2 is easily detectable, it is the particularly divergent SD1 that was shown in fungi to be essential for the assayed molecular functions. Due to the likely limited sequential-  requirements, this SUMO-like domain is difficult to detect and has been missed in previous analyses of individual family members. The identification of the more N-terminal SUMO-like domain SD1 helps rationalizing experimental findings for mutant fungal RENi family members.RPS-BLAST  and FFASThe sequence analytic work was executed by MN. All authors  contributed to evaluating the results and making the discoveries reported here. MN prepared all the figures and, together with FE, the manuscript text. All authors read and approved the final manuscript.


Nova-1 and Nova-2 genes. Anti-Ri antibodies are typically detected in the serum and cerebrospinal fluids of patients with neurological disorders such as opsoclonus/myoclonus and cerebellar ataxia and in association with gynecologic and breast malignancies.The IgG autoantibody ANNA-2 (anti-Ri) is a type 2 antineuronal antibody that has been found to bind to highly conserved and widely distributed adult brain proteins encoded by the This report describes an unusual example of a 33-year-old female patient who developed short-term memory deficits over a 3-month period. An extensive neurological work-up, including a panel of paraneoplastic markers was negative with the exception of a high titer serum Anti-Ri . A large left ovarian mass was palpated, surgically resected and eventually diagnosed as a mature cystic teratoma. Post-operatively, memory deficits had disappeared within 1 month and serum Anti-Ri titers had decreased significantly to 1:256. An extensive diagnostic work-up for other malignancies was negative.Although, Anti-Ri antibodies are typically associated with malignancies, this case illustrates the potential association between benign tumors and this autoantibody. Paraneoplastic syndromes (PNS) are signs or symptoms attributable to tissue damage at sites that are remote from a primary malignancy or their metastases. PNS involving virtually every level of the neuro-muscular system have been described -3. TypicA wide variety of anti-neuronal antibodies have been associated with the many PNS-associated neurologic disorders . These aA 33-year-old nulligravid female with no significant past medical history presented to her physician with complaints of a short-term memory loss of approximately three months' duration. This included an inability to remember details about 24-hour old events. There had been no major socio-economic or personal changes in her life over this period. A detailed neurologic examination was notable only for her presenting complaint. Routine laboratory work-up, including a lumbar puncture were all within normal limits. A physical examination revealed a large right adnexal mass, which upon ultrasonographic assessment showed internal features suggestive of a malignancy. A panel of serum paraneoplastic autoantibodies was then requested, including anti-Hu, anti-Yo, anti-Ri, anti-Tr and anti-Ma1/2. All were normal with the exception of IgG anti-Ri, measured at 1:15,360 by an indirect immunofluorescence method. An extensive diagnostic work-up failed to reveal any malignancies. The patient subsequently underwent a right salpingo-oophorectomy, and her adnexal mass was diagnosed as a benign mature cystic teratoma of the ovary. Almost immediately following her surgery, the patient expressed a subjective improvement in her symptoms. Within a month, the serum anti-Ri had decreased to 1:256, and a detailed neurologic examination revealed resolution of her symptoms. She has not experienced any relapse in her symptoms in the 1 year since her surgery.Nova-1 and Nova-2 genes and which may play a role in neuronal maturation and homeostasis [In 1988 and 1991, Budde-Steffen at al  and Luqueostasis -9. As theostasis . Indeed,eostasis . The speeostasis . What theostasis , the vasIs it possible that the presence of the autoantibody and the ovarian tumor are completely fortuitous?. Given the temporal relationship between the resolution of her symptoms and the sharp decrease in her anti-Ri titer following her surgery, we believe this is unlikely. However, the precise mechanistic basis for this association as well as potential influence of outside factors remains to be elucidated. It should also be noted that high titers of Anti-Ri have been identified in patients with a history of ovarian cancer but without any evidence of a PNS, suggesting caution in assessing the specificity of this auto-antibody for PNS.In summary, we describe in this report an association of a common anti-neuronal antibody (anti-Ri) in a patient with a benign neoplasm, mature cystic teratoma of the ovary, and whose neurologic symptoms, short-term memory deficits, was apparently associated with her tumor. Although, Anti-Ri antibodies are typically associated with malignancies, this case illustrates the potential association between benign tumors and this autoantibody.The authors declare that they have no competing interests.Dr Fadare wrote the initial version of the manuscript.Dr Hart managed the patient, provided clinical information, and revised the manuscript.


However, some but not all studies in mice and humans (though none in rats) have found that CLA promotes insulin resistance. The molecular mechanisms responsible for these effects are unclear, and there are conflicting reports on the effects of CLA on peroxisomal proliferator-activated receptor-γ (PPARγ) activation and expression. We have conducted three experiments with CLA in obese mice over three weeks, and one over eleven weeks. We have also investigated the effects of CLA isomers in PPARγ and PPARα reporter gene assays.Studies in rodents and some studies in humans have shown that conjugated linoleic acid (CLA), especially its trans-10, cis-12 isomer in the diet of female genetically obese (oblep/oblep) mice for up to eleven weeks reduced body weight gain and white fat pad weight. After two weeks, in contrast to beneficial effects obtained with the PPARγ agonist rosiglitazone, CLA or CLA enriched with its trans-10, cis-12 isomer raised fasting blood glucose and plasma insulin concentrations, and exacerbated glucose tolerance. After 10 weeks, however, CLA had beneficial effects on glucose and insulin concentrations. At this time, CLA had no effect on the plasma TNFα concentration, but it markedly reduced the plasma adiponectin concentration. CLA and CLA enriched with either isomer raised the plasma triglyceride concentration during the first three weeks, but not subsequently. CLA enriched with its trans-10, cis-12 isomer, but not with its cis-9, trans-11 isomer, stimulated PPARγ-mediated reporter gene activity; both isomers stimulated PPARα-mediated reporter gene activity.Inclusion of CLA or CLA enriched with its oblep/ob lepmice. Activation of both PPARγ and PPARα may contribute to the improvement in insulin sensitivity. In the short term, however, another mechanism, activated primarily by trans-10, cis-12-CLA, which probably leads to reduced adipocyte number and consequently reduced plasma adiponectin concentration, may decrease insulin sensitivity.CLA initially decreased but subsequently increased insulin sensitivity in  The major components of CLA, are the cis-9, trans-11  and the trans-10, cis-12  isomers, both of which have biological activities. The t10, c12-isomer is the one primarily responsible for the effects of CLA on weight gain and insulin sensitivity. The CLA used in the present study contains these isomers in roughly equal proportions.The term conjugated linoleic acid (CLA) refers to a mixture of positional and geometric isomers of linoleic acid  mice, despite causing weight loss [falep/fa lepand Zucker diabetic fatty falep/fa leprats [In previous studies, CLA or t10, c12-CLA has exacerbated glucose tolerance and raised plasma insulin levels in normal and genetically obese α, since TNFα is associated with apoptosis of adipocytes but causes insulin resistance. TNFα mRNA levels were markedly increased in isolated adipocytes from normal mice that had been fed on CLA for as little as four days . Surprisdblep/db lepmice with CLA for eleven weeks improved glucose tolerance and reduced plasma insulin concentration during the glucose tolerance test, indicating improved insulin sensitivity. Treatment for five weeks also improved glucose tolerance but plasma insulin was raised [dblep/db lepmice because β-cell failure as well as insulin resistance affects glucose homeostasis, and glycosuria can cause weight loss. Therefore, to investigate the effect of CLA on glucose homeostasis further, we have conducted four experiments in oblep/ob lepmice. We have included rosiglitazone as a comparator in one experiment and compared the effects of t10, c12- and c9, t11-enriched CLA with CLA in another experiment. The first three experiments were over 22 days, whereas the fourth was extended to eleven weeks and included measurements of plasma TNFα and adiponectin. An intriguing finding is that whereas CLA initially exacerbated glucose tolerance and raised the plasma insulin level, after ten weeks it began to improve glucose tolerance and lower the plasma insulin levels.Although most studies in mice have found that CLA exacerbates glucose tolerance and raises the plasma insulin concentration, in one study treatment of s raised . It is sWe also describe activation of PPARγ by t10, c12 but not by c9, t11-CLA, whereas PPARα was activated by both isomers.oblep/oblep) mice reduced their body weight, or weight gain compared to mice fed on a diet containing a similar amount of sunflower oil  concentration in experiment 3 Table , and CLAAfter ten weeks CLA had no effect on the plasma TNFα concentration. The plasma adiponectin concentration, by contrast, was markedly decreased Table .CLA enriched with t10, c12-CLA (50 and 100 μM), but not with c9, t11-CLA, stimulated PPARγ-mediated reporter gene activity Figure . In contdblep/db lepmice [A number of studies have found that CLA reduces weight gain and fat accretion in both rats and mice. Those that have failed to show such effects are generally those that have used low levels of CLA or CLA that contained low concentrations of t10, c12-CLA . Studiespdb mice , show expdb mice -10. In hpdb mice , but doupdb mice -13, and pdb mice ,12,14. Tpdb mice .oblep/oblep) mice as our model of insulin resistance. There is only one previous report of the effect of CLA in oblep/ob lepmice [oblep/ob lepmice with CLA. These benefits were achieved faster with the higher  than the lower dose (10 g/kg diet) of CLA.Our study raises the possibility that an initial exacerbation of glucose tolerance, apparently due to insulin resistance, might, after prolonged treatment with a high dose of CLA, be followed by improved insulin sensitivity and glucose tolerance. We used genetically obese  and maintained at 23 ± 1°C with lights on from 07:00 to 19:00 h. They were housed in plastic cages with bedding and fed 'rat and mouse standard diet' . Six days before the start of the studies they were allocated to treatment groups (6 mice per group), such that each group had a similar mean bodyweight. The CLA and other treatments were mixed with powdered diet and given ad libitum. The mice were killed by cervical dislocation in experiments 1 to 3; in experiment 4 they were anaesthetised with sodium pentobarbitone  and exsanguinated through an aortic catheter. All procedures were conducted in accordance with our Home Office, UK project licence under the Animals (Scientific Procedures) Act and as agreed by the University of Buckingham Ethical Review Board.Female C57Bl/6 CLA (in acid form), including isomer-enriched CLA, and sunflower oil were provided by Loders Croklaan, Wormerveer, The Netherlands. The CLA used in experiments 1, 2 and 3 was Clarinol™ A-60, containing 30% c9, t11-CLA and 31% t10, c12-CLA and 25% oleic acid. The CLA used in experiment 4 was Clarinol™ A-80, containing 40% c9, t11-CLA and 40% t10, c12-CLA. Rosiglitazone was synthesised by Dextra Laboratories, Reading, Berks, UK.ad libitum, and after 22 days when they had been fasted for 16 hours, immediately before termination of the experiment.At the start of treatment the mice weighed 34.5 ± 4.3 g (mean ± S.D.). The treatment groups were high oleic (83.5%) sunflower oil ; rosiglitazone (10 mg/kg diet) plus high oleic sunflower oil (15 g/kg diet); CLA (Clarinol™ A-60: 15 g/kg diet). An oral glucose tolerance test was conducted after 14 days as described below. 30 min prior to giving glucose, when the mice had been fasted for 4.5 hours, blood (100 μl) was taken for the measurement of plasma insulin and triglycerides. Plasma insulin and triglycerides were also measured after 21 days when the mice were feeding At the start of the treatment the mice weighed 44.0 ± 3.2 g (mean ± S.D.). A group fed on powdered chow alone was included. The doses of CLA (Clarinol™ A-60) and sunflower oil in the other two groups were increased to 25 g/kg diet. The oral glucose tolerance test and other measurements were carried out as described for experiment 1, except that the glucose tolerance test was one day earlier (i.e. after 13 days).At the start of treatment the mice weighed 34.1 ± 2.1 g (mean ± S.D.). The treatment groups were high oleic sunflower oil (25 g/kg diet); CLA (Clarinol ™ A-60: 25 g/kg diet); CLA with the CLA component of Clarinol enriched to 90% with t10, c12-CLA (25 g/kg diet); similarly, CLA enriched to 90% with c9, t11-CLA (25 g/kg diet). The oral glucose tolerance test and other measurements were carried out as described for experiment 2. In addition, the plasma non-esterified fatty acid concentration was measured in the blood taken prior to the glucose tolerance test, and the liver, pancreas, parametrial white adipose tissue depot and interscapular brown adipose tissue depot were weighed at termination.At the start of treatment the mice weighed 31.6 ± 3.8 g (mean ± S.D.). The treatment groups were high oleate (as glyceride) sunflower oil (25 g/kg diet); CLA (Clarinol A80: 10 g/kg diet) plus high oleic sunflower oil (15g/kg diet); CLA (Clarinol A80: 25 g/kg diet). Oral glucose tolerance tests preceded by blood sampling for the measurement of triglycerides, insulin and non-esterified fatty acids were taken after 14, 35 and 70 days of treatment. After 77 days of treatment the animals were anaesthetised in the fed state and blood was taken from the thoracic aorta for the measurement of plasma TNFα and adiponectin. Tissues were weighed as in experiment 3. Body length was measured from the tip of the nose to the anus.In each experiment oral glucose tolerance was measured after 13 or 14 days. In experiment 4 it was also measured after five and ten weeks. The mice were fasted for five hours before being dosed with glucose . Blood samples (20 μl) were taken from the tip of the tail after applying a local anaesthetic (lignocaine) and immediately before and 30, 60, 90, 120 and 180 min after dosing the glucose. They were mixed with haemolysis reagent and blood glucose was measured in duplicate using the Sigma Enzymatic (Trinder) colorimetric method and a SpectraMax 250 . Areas under the glucose tolerance curve (0–180 min) and other manipulations and analysis of the data were carried out using Prism software, version 3.0 .Blood was collected into EDTA-coated microcuvettes  for the measurement of plasma insulin, non-esterified fatty acids and triglycerides. Plasma was stored at -80°C. 5 μl plasma samples were assayed using kits for triglyceride (Sigma enzymatic colorimetric method), non-esterified fatty acids  and insulin .For the measurement of plasma TNFα  and adiponectin , blood was taken into tubes containing 200 units of heparin and plasma was stored at -80°C.MluI and BglII and ligated into pNF-κ B-luc that had been cleaved with the same restriction endonucleases. Ligated DNA was transformed into competent JM109, E. coli cells (Promega). pcDNA3/PPARα was prepared by removing the human PPARα cDNA insert from pUC18/hPPARα as a NruI/BamHI fragment and ligating it into EcoRV/BamHI cleaved pcDNA3.1(-) (Invitrogen). Ligated DNA was transformed into competent JM109, E. coli cells (Promega).pRSV/hRXRα and pcDNA3/hPPARγ1 were both obtained from Professor VKK Chatterjee . PPPRE3TK-luc was prepared by replacing the NF-kB enhancer element of pNF-κB-luc (Clontech) with a cassette of 3 PPAR Response Elements (PPREs). A double-stranded PPRE cassette was prepared using the 'Klenow fill-in' technique. An 113 bp oligonucleotide (PPRE3) 5'- GCATTCACGCGTCAAATATAGGCCATAGGTCATTCTCGAGCAAATATAGGCCATAGGTCATTCTCGAGCAAATATAGGCCATAGGTCAGATTCGATCAATATAGGCCATAGGTCACTCGAGGCAACAGATCTTACGCATG -3' containing a triplet of PPREs and appropriate restriction endonuclease sites was used as a template for synthesis of a second DNA strand. This was primed by PPRE3R, 5'-CATGCGTAAGATCTGTTGCC-3', which is complementary to the 3' region of PPRE3. 20 μl annealed PPRE3 and PPRE3R, 1.5 μl 2 mM dNTPs, 1 × Klenow Buffer and 5 units DNA Polymerase I (Klenow fragment), were incubated for 1 hour at 37°C and then 10 minutes at 75°C, purified by ethanol precipitation and resuspended in sterile water. The double-stranded PPRE cassette was digested with 2. Transient transfections were performed using LipofectAMINE as directed by the manufacturers (GibcoBRL). For the PPARγ assay Cos-7 cells were plated in 24-well plates at a density of 0.375 × 105 cells/well. Cells were transfected in serum-free medium (DMEM containing 2 mM L-glutamine) with pPPRE3TK-luc, pRLTK, pRSV/hRXRα and pcDNA3/hPPARγ 1 at concentrations of 0.4, 0.03, 0.02 and 0.02 μg/well, respectively. Five hours after transfection cells were fed with 250 μl/well of serum-free medium containing various concentrations (0–100 μM) of CLA . Cell lysates were prepared after 46 hours using 100 μl 1 × passive lysis buffer (Promega) per well. Firefly and renilla luciferase activities were measured using a Dual Luciferase Assay kit (Promega), as described by the manufacturers. Measurements were performed on an MLX microtitre plate luminometer (Dynex). For the PPARα assay Cos-7 cells were plated in 24-well plates at a density of 0.5 × 105 cells/well. Cells were transfected in serum-free medium with pPPRE3TK-luc, pRLTK, pRSV/hRXRα and pcDNA3/hPPARα at concentrations of 0.4, 0.04, 0.03 and 0.03 μg/well, respectively. Five hours after transfection DMEM supplemented with 2 mM L-glutamine and 20% SBCS  was added to the cells. Following 18 hours incubation at 37°C/5% CO2 the medium was removed and replaced with medium (DMEM supplemented with 2 mM L-glutamine and 10% SBCS) containing various concentrations (0-100 μM) of CLA enriched to 85% with the c9, t11 isomer or 81% with the t10, c12 isomer prepared in 0.1% DMSO. After 24 hours of treatment cell lysates were prepared and luciferase activity measured as described above.Cos-7 cells (ECACC No. 87021302) were routinely cultured in DMEM containing 10% FCS, 2 mM L-Glutamine, 100 iu/ml penicillin and 100 μg/ml streptomycin at 37°C/5% COData were analysed by one-way analysis of variance followed by LSD test with the sunflower oil treatment as the control. Means are of 6 values with SEM.in vivo experiments and analysed materials from these. AM conducted the in vitro experiments. MS and MC conducted the initial analysis and interpretation of the data. JA reanalysed some of the data and wrote the manuscript with input from the other authors, and in particular from AE and IM with respect to interpretation and perspectives.MC, LB, IM and MS devised the experiments. EW, MS and CS, supported by JO and SW conducted the


Prochlorococcus sp. are marine bacteria with very small genomes. The mechanisms by which these reduced genomes have evolved appears, however, to be distinct from those that have led to small genome size in intracellular bacteria. Prochlorococcus species, the smallest and most abundant photosynthetic organism in the ocean, have recently been published. Comparative genome analyses reveal that genome shrinkage has occurred within this genus, associated with a sharp reduction in G+C content. As all examples of genome reduction characterized so far have been restricted to endosymbionts or pathogens, with a host-dependent lifestyle, the observed genome reduction in Prochlorococcus is the first documented example of such a process in a free-living organism.Three complete genomes of P. marinus SS120 that is even more pronounced in P. marinus MED4. This acceleration has affected every functional category of protein-coding genes. In contrast, the 16S rRNA gene seems to have evolved clock-like in this genus. We observed that MED4 and SS120 have lost several DNA-repair genes, the absence of which could be related to the mutational bias and the acceleration of amino-acid substitution.Our results clearly indicate that genome reduction has been accompanied by an increased rate of protein evolution in Prochlorococcus have to be selectively neutral, as the large size of populations imposes low genetic drift and strong purifying selection. We assume that the major driving force behind genome reduction within the Prochlorococcus radiation has been a selective process favoring the adaptation of this organism to its environment. A scenario is proposed for genome evolution in this genus.We have examined the evolutionary mechanisms involved in this process, which are different from those known from host-dependent organisms. Indeed, most substitutions that have occurred in  Mycoplasma genitalium [Buchnera [Nanoarchaeum equitans [Buchnera genome is devoted to biosynthesis of amino acids which are essential to its host [The size of bacterial genomes is primarily the result of two counteracting processes: the acquisition of new genes by gene duplication or by horizontal gene transfer; and the deletion of non-essential genes. Genomic flux created by these gains and losses of genetic information can substantially alter gene content. This process drives divergence of bacterial species and eventually adaptation to new ecological niches . In somenitalium  or phytonitalium  and symbBuchnera -8 or theequitans . In the equitans  and 35 kequitans , respectits host .Prochlorococcus [5 cells/ml) in all nutrient-poor areas of the world's oceans between 40°N and 40°S and is probably the most abundant photosynthetic organism on Earth [b to a ratio; the second inhabits the upper layer of the ocean and has a low divinyl-chlorophyll b to a ratio [Prochlorococcus marinus MED4 [P. marinus SS120 [Prochlorococcus species MIT9313 [So far, all characterized examples of genome reduction have been associated with a change from a free-living to a host-dependent lifestyle . It is trococcus -15. The on Earth ,17. It hon Earth . The fir a ratio . The gennus MED4 , and of us SS120  and Proc MIT9313 , have reProchlorococcus sp. MIT9313 branches at the base of the Prochlorococcus radiation, close to the Synechococcus group [Prochlorococcus HL clade, encompassing the MED4 strain, appears to be the most recently evolved Prochlorococcus group, consistent with the fact that this clade is much less diversified than are the LL clades.Phylogenetic trees based on 16S rRNA sequences  or 16S-2us group . In contSynechococcus sp. WH8102 genome [Despite the close relatedness of these strains, their genomes vary widely in terms of size, G+C content and the number of protein-coding genes Table . While t2 genome , MED4 ha2 genome . HoweverSynechococcus and Prochlorococcus but also between MIT9313 and the two other Prochlorococcus strains  and more amino acids encoded by A+T-rich codons .The downsizing of MED4 and SS120 genomes during evolution is associated with a genome-wide adenine (A) and thymine (T) enrichment Table . Second,-12) for BLAST comparisons. In contrast, our analysis is based on identification of reciprocal best hits without the use of any particular threshold (apart from the default BLAST threshold) and consequently allows the detection of orthologous relationships whatever the gene lengths or the level of similarity. Still, our ortholog identification process is rather strict and the set of orthologs identified in this study probably corresponds to a lower estimate of the actual number of orthologs shared by the four genomes. This set of genes represents a substantial percentage of the total pool of all protein-coding genes in P. marinus MED4 (73.2%) and SS120 (69.2%) and about half of the gene set in Prochlorococcus sp. MIT9313 (56.2%) and Synechococcus sp. WH8102 (51.1%). These percentages are consistent with the differences in the respective number of genes within these genomes  and probably constitute an estimate of the core of genes conserved in all marine picocyanobacteria. This is sensibly more than the pool of around 1,000 orthologs identified by W.R. Hess . The difr genome .Prochlorococcus genomes, we have investigated whether it also affects the rate of protein sequence evolution in these genomes. We used the 1,306 orthologs common to the four genomes to estimate the amino-acid substitution rate in each genome. Branch lengths calculated for a given tree topology  indicate that protein sequences evolved at significantly different rates (P < 0.001) between MED4, SS120 and MIT9313. Therefore the hypothesis of a constant evolutionary rate between these strains can be rejected for protein-coding genes. The calculation of branch-length ratios reveals that the amino-acid substitution rate is 2.04-fold higher inMED4 than in SS120 (dA/dB) and 3.81-fold higher in MED4 than in MIT9313 ((dA+dX)/dC). This rate is also 2.31-fold higher for SS120 than for MIT9313 ((dB+dX)/dC). Computation of branch lengths for each functional category shows that the increased rate of amino-acid replacement in protein sequences concerns every category  to the rate of synonymous substitutions (dS) is commonly used to measure the relative rate of purifying selection acting at the protein level. We determined dS and dN for each gene pair of every group of orthologs and their values were averaged for each genome. Surprisingly, we observed saturation at synonymous sites for all genome pairs (dS > 2) and the calculation of the dN/dS ratio was thus impossible. Still, the average dN was higher between MED4 and SS120 (0.36) than between SS120 and MIT9313 (0.32). The lowest dN was observed between MIT9313 and WH8102 (0.24), a finding which is consistent with the relative acceleration of amino-acid substitutions in MED4 and in SS120.The ratio of the rate of nonsynonymous substitutions ), as well as several other marine Synechococcus spp. , it is reasonable to assume that the common ancestor of all Prochlorococcus species also had a genome size around 2.4 Mbp. Under this hypothesis, the genome reduction which has occurred in MED4 would correspond to around 31%. By comparison, the extent of genome reduction in the insect endosymbiont Buchnera, as compared to a reconstructed ancestral genome, is around 77% [P. marinus SS120 - and a fortiori the MED4 genome - is considered to be near minimal for a free-living oxyphototrophic organism [The process of genome reduction which has occurred within the ound 77% . The genorganism  and dataProchlorococcus lineages and host-dependent organisms have undergone genome reduction associated with accelerated substitution rates, these phenomena must have arisen from very different causes as the resulting gene repertoires of the two types of organisms differ tremendously. Indeed, the genome evolution of endosymbionts and obligatory pathogens is driven by two main processes which have mutually reinforcing effects on genome size and evolutionary rates. Being confined inside their host, these bacteria have tiny population sizes and are regularly bottlenecked at each host generation or at each new host infection. Consequently, they experience a strong genetic drift [If both ic drift  involvinic drift ,29 as weic drift . This geic drift ,31-33. Fic drift -6,8,9,32Prochlorococcus, the very large size of field populations [P. marinus SS120 only two such genes are missing . One noteworthy exception is the presence in Prochlorococcus, but not Synechococcus, of flavodoxin and ferritin, two proteins that possibly give Prochlorococcus a better resistance to iron stress. Apart from that, Synechococcus appears more like a generalist, in particular with regard to nitrogen or phosphorus uptake and assimilation [a priori be more suited to sustain competition. Hence, we assume that the key to the success of Prochlorococcus resides less in the development of a specific complex or pathway to cope better with unfavorable conditions than in the simplification of its genome and cell organization, which can allow this organism to make substantial economies in energy and material for cell maintenance.If it is neither the relaxation of purifying selection nor an increase in genetic drift that has been the main factor causing hococcus ,34, stromilation , and shoper se is a potential source of substantial economies for the cell, as it reduces the amount of nitrogen and phosphorus, two particularly limiting elements in the upper part of the ocean, which are necessary, for instance, in DNA synthesis. Another advantage is that it allows a concomitant reduction in cell volume. It has been previously suggested  that, fProchlorococcus genomes. Using a rate of 16S rRNA divergence of 1% per 50 million years [Prochlorococcus and Synechococcus. The ancestral Prochlorococcus cells must have developed in the LL niche, a niche probably left free by other picocyanobacteria. Given the considerable difference in genome size between the LL strains MIT9313 and SS120, it appears that genome reduction itself must have started in one  lineage(s) within the LL niche some time after Prochlorococcus differentiation from its common ancestor with marine Synechococcus species. Why the selection has affected only one (or some?) and not all Prochlorococcus lineages remains unclear. Examination of the gene repertoire of P. marinus SS120 [ada gene, which may be responsible for the shift in base composition, but also possibly several others, not necessarily involved in GC to AT mutation repair  and were secondarily lost in the LL-adapted lineages.Later during evolution  one LL population which probably already had a significantly reduced cell and genome size must have progressively adapted to the HL niche and eventually recolonized the upper layer. How this change in ecological niche was possible is still hard to define. Comparison of the gene set that differs between the LL-adapted SS120 and the HL-adapted MED4 shows that very few genes might be sufficient to shift from one to the other niche, including a multiplication of li genes  and the Prochlorococcus has similar features to that in host-dependent prokaryotes: genome reduction, bias toward a low G+C content, acceleration in the evolution rate of protein-coding genes, and loss of DNA-repair genes. In contrast to the latter organisms, however, in Prochlorococcus this evolution does not appear to be the result of genetic drift or relaxed selection being exerted on some gene categories. Indeed, purifying selection is very efficient in Prochlorococcus, as rRNA genes have evolved at a similar rate in all genomes. Despite the decrease in G+C content and an accelerated rate of evolution of protein-coding genes, purifying selection must also act on these genes and avoid potentially deleterious mutations. We hypothesize that a reduction in genome size  can constitute a selective advantage for life in the open ocean, both at depths where photon energy is low and in surface waters where nutrients are scarce.Genome evolution in the free-living genus Prochlorococcus has led to populations highly specialized to narrow ecological niches, at the expense of versatility and competitiveness in changing conditions. Indeed, not only is the distribution of the Prochlorococcus genus limited to low latitudes  but theProchlorococcus marinus MED4, P. marinus SS120, Prochlorococcus sp. MIT9313 and Synechococcus sp. WH8102  were downloaded from the Genome division of the NCBI Entrez system. A few additional genes which were modeled in at least one genome and were present in the other genomes but not modeled  were included in our dataset .The complete genome sequences and annotations of Genome sequences translated in their six reading frames were aligned with the Promer program of the MUMmer 3.0 system .Codon usage was computed for every open reading frame (ORF) of each genome with the EMBOSS program cusp. Amino-acid usage was derived from the results produced by cusp.We used a sequence-similarity based approach which is similar to the procedure used for the cluster of orthologous groups (COGs ). For eaProchlorococcus genomes (hypothesis of the molecular clock). The same analysis was applied to orthologs of each functional category.Protein sequences from each of the groups of four orthologous genes were aligned using ClustalW  with defdS) and non-synonymous (dN) substitution rates were obtained from the Yn00 program of the PAML 3.13 package [Nucleotide sequences of each group of orthologs were aligned with Protal2dna according to alignments of their corresponding amino-acid sequences . Pairwis package .The following additional data are available with the online version of this article. Additional data file The orthologous genes classified by functional categoryClick here for additional data fileA a fasta file of orthologous genes which were modeled in at least one genome and present but not modeled in the other genomesClick here for additional data file


Haemophilus influenzae. The list has grown considerably since then: add over 160 bacterial species (and counting), most major model organisms, and an ever-growing list of mammals—including, of course, humans. With 99% of our genome now fully sequenced, the Human Genome Project's next major goal is to identify all the functional elements contained in our 2.85 billion nucleotides. Such an effort is hardly trivial: producing the sequence of a mammalian-size genome can run from $10 to $50 million, the estimated price tag of the Cow Genome Project.It's hard to believe it was just ten years ago that scientists reported the first complete genome sequence of an organism, the bacterial pathogen In an ideal world, any organism would be fair game for sequencing, but in the real world, sequencing resources are scarce. Comparing genome sequences turns out to be a great way to identify regions that have important functions, but comparative genomics studies would be far more efficient if scientists could figure out in advance which genomes would reveal the most information about a particular question. Taking up that challenge, computational biologist Sean Eddy reports a statistical model that predicts how many genomes, and at what evolutionary distance, are needed for effective comparative genomic analyses. In addition to confirming some working principles of comparative genomics, the model also reveals a surprisingly simple guideline for future studies.Comparative genomics works by aligning sequences of different organisms to identify patterns that operate over both large and small distances. Aligning mouse chromosomes with human chromosomes, for example, shows that 99% of our protein-coding genes align with homologous sequences in mice. Underlying such analyses is the principle that DNA sequences that are highly conserved are likely to be functionally important. A common assumption is that adding more comparative genomes to the alignment helps distinguish functionally significant from irrelevant conserved sequences.How do you go about creating an abstract model that captures what Eddy calls the “essential flavor of comparative genomic analysis”? His model puts aside the specific characteristics of individual organisms, genomic features, and analysis programs in favor of identifying higher-level patterns and scaling relationships, specifically between the number of genomes, evolutionary distance, and feature size (features include genetic elements like exons and transcription factors).The model shows that the number of genomes required to identify conserved regions—that is, regions evolving under selection—scales inversely with the size of the feature being sought. Thus, to look for conserved sequences half as long, you need twice as many genomes, assuming a constant evolutionary distance and statistical power. For example, to identify a conserved human feature the size of a coding exon (about 50 nucleotides), it is sufficient to compare just the human and mouse genomes. But to identify conserved single nucleotides, you would need 55 comparative genomes at “mouse-like” evolutionary distances (roughly 75 million years).Things get a little trickier when varying evolutionary distance. We can see a substitution only at a given point in time: we can't tell how many times a site has changed, for example, or whether it changed at some point and then changed back. But at short evolutionary distances—where it's safer to assume no sites have changed more than once—the evolutionary distance is roughly the same as the fraction of sites identified as changed, and evolutionary distance and the number of genomes needed scale inversely. Therefore, the closer the evolutionary distance, the more genomes needed: one would need seven times as many comparative genomes using human/baboon distances, for example, compared to human/mouse distances. So when it comes to using primate sequences to study the human genome, our most distant relatives (such as lemurs) offer far more comparative analysis power than our next of kin (chimps and bonobos).While this model confirms the intuitive assumption that identifying smaller features requires more genomes, it reveals an inverse scaling relationship far more direct, and precise, than previously imagined. With the next phase of the Human Genome Project under way, Eddy's model offers valuable guidelines for identifying which genomes and how many might best meet this ambitious goal.


Gdf5 gene (a bone morphogenetic protein [BMP] family member) to develop new mouse lines that can be used to either activate or inactivate genes specifically in developing joints. Expression of Cre recombinase from Gdf5 bacterial artificial chromosome clones leads to specific activation or inactivation of floxed target genes in developing joints, including early joint interzones, adult articular cartilage, and the joint capsule. We have used this system to test the role of BMP receptor signaling in joint development. Mice with null mutations in Bmpr1a are known to die early in embryogenesis with multiple defects. However, combining a floxed Bmpr1a allele with the Gdf5-Cre driver bypasses this embryonic lethality, and leads to birth and postnatal development of mice missing the Bmpr1a gene in articular regions. Most joints in the body form normally in the absence of Bmpr1a receptor function. However, articular cartilage within the joints gradually wears away in receptor-deficient mice after birth in a process resembling human osteoarthritis. Gdf5-Cre mice provide a general system that can be used to test the role of genes in articular regions. BMP receptor signaling is required not only for early development and creation of multiple tissues, but also for ongoing maintenance of articular cartilage after birth. Genetic variation in the strength of BMP receptor signaling may be an important risk factor in human osteoarthritis, and treatments that mimic or augment BMP receptor signaling should be investigated as a possible therapeutic strategy for maintaining the health of joint linings.Articular cartilage plays an essential role in health and mobility, but is frequently damaged or lost in millions of people that develop arthritis. The molecular mechanisms that create and maintain this thin layer of cartilage that covers the surface of bones in joint regions are poorly understood, in part because tools to manipulate gene expression specifically in this tissue have not been available. Here we use regulatory information from the mouse  Through genetic manipulation, these authors have reduced signaling by bone morphogenetic factors in joint regions, and created a valuable model for the study of arthritis Thin layers of articular cartilage line the bones of synovial joints and provide a smooth, wear-resistant structure that reduces friction and absorbs impact forces . Loss orJoint formation begins during embryogenesis, when stripes of high cell density called interzones form across developing skeletal precursors . ProgramWnt14 is expressed in stripes at the sites where joints will form, and it is capable of inducing expression of other joint markers when misexpressed at new locations in the limb  containing the Gdf5 locus was modified by homologous recombination in bacteria to insert a cassette encoding Cre-internal ribosome entry site (IRES)-human placental alkaline phosphatase (hPLAP) into the translation start site of Gdf5  line used for all subsequent breeding experiments was seen to precede LACZ expression during successive development of joints in the digits  mice to analyze the pattern of Cre-mediated lacZ recombination throughout development. Joints in developing limbs begin forming in a proximal-distal pattern such that the shoulder joint forms prior to the elbow joint. In addition, three major stages of early joint development have been defined by histology as (1) interzone formation, (2) three-layer interzone formation, and (3) cavitation  mice were crossed with vitation . Consistvitation B and 1C,vitation . Sectionvitation D and 1E elopment . Adult ehemistry .lacZ expression. Starting at E13.5, LACZ activity is detected in an anterior and posterior domain of the limb bud . Previous studies have shown that the recombined floxPBmpr1a allele mimics a null allele of the Bmpr1a locus when transmitted through the germline . BMP signaling is known to be required for growth of the external ear of mice . Finally, mutant animals showed obvious skeletal changes in whole-mount skeletal preparations. At some sites in the ankles, joints seemed to be missing entirely, with fusion of bones that would normally be separate. For example, the second distal tarsal was fused to the central tarsal bone in every conditional knockout animal examined (18 of 18), a phenotype not observed in controls (zero of 18)  B and 3C.o of 18) B–3E  is down-regulated in the interzone region .In most joints of ions see C, no chaions see D, no difCol2a1 and Aggrecan (Agg), the genes encoding the major structural proteins of cartilage matrix , a gene expressed specifically in the periarticular and perichondral regions of developing joints . Less severe reductions were also seen in articular cells of tarsals and metatarsals in the hindfeet (unpublished data). By 2 wk of age, Col2a1 expression was reduced in most cells of the articular region  and Collagen 10 (Col10a1) (Collagen 1 (Col1a1) expression, increase proliferation, and result in cells with flattened, fibroblast-like morphology . While recombined LACZ marker expression was detected in most articular cartilage cells, it was also observed in scattered subarticular chondrocytes, growth plate chondrocytes, and osteoblasts . Together, these data suggest that BMPR1A activity is required in postnatal joint articular cartilage to maintain expression of many genes encoding structural components of cartilage matrix.By 1 wk after birth, obvious differences began to be detected in the articular regions of mutant animals. The expression of r region L and 5Q,r region K and 5P,Col10a1) M and 5R Col10a1) . Inhibitrphology . Howevereoblasts O and 5T Sox9 is required for normal cartilage differentiation, for expression of cartilage extracellular matrix (ECM) genes including Agg, and is a direct transcriptional regulator of the key cartilage matrix gene Col2a1 , toward the regions where surface articular cartilage was severely eroded or missing .Agg and Col10 expression were all reduced in mutant articular regions of the forefeet and hindfeet by 7 wk of age, and the beginning signs of cartilage loss were observed (unpublished data). By 9 mo of age, many regions of articular cartilage were completely missing or extremely fibrillated, leaving regions of exposed bone on the surface . The overall shape of mutant knee skeletal elements appeared similar to controls, although the fibrocartilaginous meniscus that resides between the femur and tibia appeared much less dense in mutants at E16.5. Some cartilage formed in the meniscus region, but the size of these elements was greatly reduced and contained abundant cells with fibrous, noncartilaginous appearance (unpublished data). This reduction of the meniscus can also be seen in sections from 7-wk- and 9-mo-old animals . By postnatal day 7, Safranin O staining and Bmpr1a is required cell-autonomously in articular cartilage .At 7 wk of age the normally domed tibial epiphysis was flattened and depressed in the knees of mutant animals, markedly reducing the distance between the growth plate and articular surface F and 7I.floxPGdf5-Cre/Bmpr1a mutant animals showed a highly significantly reduced ability to grasp and remain suspended on a slender rod . Mutant mice also showed a clear decrease in the maximum range of mobility of two different joints in the digits, as assayed by passive manipulation . The structural, histological, marker gene expression, and functional changes in mutant mice demonstrate that BMPR1A is required for normal postnatal maintenance of articular cartilage.The histological signs of joint arthritis were accompanied by functional impairments in both grasping ability and range of motion in mutant animals. Gdf5-Cre recombination system bypasses the early embryonic lethality of Bmpr1a mutations, and provides important new information about the role of this receptor in limb and skeletal development.Previous studies suggest that BMP signaling is involved in a large number of developmental events. Many of these events occur early in embryogenesis, and complete inactivation of BMP receptors causes death by E9.5 . The GdfBmpr1a with Gdf5-driven Cre include webbing between digits, lack of joint formation at specific locations in the ankle, and failure to maintain articular cartilage after birth, resulting in severe arthritis. Previous studies have shown that manipulation of BMP signaling alters interdigital apoptosis during development of the limb, but no experiment has identified a specific member of the BMP signaling pathway that is required for this process  and the R26R LACZ marker (expressed following Gdf5-Cre recombination) suggests that recombination-stimulated changes in gene expression may be delayed for a 0.5–1 d in the digit region  type II receptor also disrupts normal articular cartilage maintenance  followed by IRES-hPLAP  directly behind the ATG start site of Gdf5. In the process, 583 bp of the first exon of Gdf5 was removed and no functional GDF5 protein is predicted to be produced. The 5′ homology arm was subcloned from a PCR product tailed with XhoI and Bsp120I restriction sites that contains 781 bp of 5′ genomic Gdf5 sequence ending at the ATG translation start site . Cre was subcloned from a 1.1-kb Bsp120I/EcoRI fragment of pML78. IRES hPLAP was subcloned from a 2.1-kb PCR product tailed with EcoRI and SpeI sites that contains the hPLAP translation stop site . The 3′ homology arm was subcloned from a 0.8-kb PCR product amplified from a 0.9-kb XhoI Gdf5 genomic subclone containing part of the first exon and downstream intron. The forward primer contains the 3′ end of the first exon and is tailed with a SpeI site; the reverse primer is from the T7 promoter of the vector containing the 0.9-kb subclone and flanks the intronic XhoI site . The targeting construct was built and verified in pBSSK , then digested with XhoI and subcloned into pSV1, the vector used for homologous recombination (A mouse 129x1/SvJ BAC library (Invitrogen) was screened to identify a 140-kb BAC from the  E. coli  to placebination . SoutherloxP site present in the BAC vector, pBeloBAC11, was removed to prevent the addition of undesired Cre target sites into the genome. To do this, BAC DNA was prepared by CsCl separation, digested with NotI to free the insert from the vector, and size-fractionated over a sucrose gradient. Aliquots of fractions were run on a pulse-field gel and Southern blotted using vector-specific DNA as a probe. Fractions containing unsheared insert and almost no detectable vector DNA were dialyzed in microinjection buffer (10 mM Tris [pH 7.4] with 0.15 mM EDTA [pH 8.0]) using Centriprep-30 concentrators . This purified insert DNA was adjusted to 1 ng/μl and injected into the pronucleus of fertilized eggs from FVB/N mice by the Stanford Transgenic Facility. Transgenic founder mice were identified by PCR using Cre-specific primers 5′-GCCTGCATTACCGGTCGATGCAACGA-3′ and 5′-GTGGCAGATGGCGCGGCAACACCATT-3′, which amplify a 725-bp product, and were assessed for absence of BAC vector using vector-specific primers 5′-CGGAGTCTGATGCGGTTGCGATG-3′ and 5′-AGTGCTGTTCCCTGGTGCTTCCTC-3′, which amplify a 465-bp product. Three lines of Gdf5-Cre mice were established and maintained on the FVB background. Matings with R26R Cre-inducible LACZ reporter mice . Cryosections of tissue were assayed by TUNEL using the In Situ Cell Death Detection Kit, Fluorescein . Following TUNEL, slides were washed in PBS, blocked with PBS + 0.05% Tween-20 + 5% goat serum, washed again, and incubated with a 1:200 dilution of a rabbit anti-phospho-histone-H3 antibody called Mitosis Marker  to identify cells in mitosis. Cy3-labeled anti-rabbit secondary antibody was used to detect the antibody. Cell nuclei were labeled with DAPI, and slides were mounted in Vectamount  and visualized at 100× magnification. The area of selected anatomical sites were measured, and the number of TUNEL-labeled nuclear fragments and the number of Cy3-labeled nuclei were counted from three 10-μm sections spanning 50 μm, from three control and three mutant animals. The number of labeled cells in the metacarpal-phalangeal and metatarsal-phalangeal joints was counted in a 290 μm × 365 μm rectangle placed around the center of the joint. The posterior region of the fifth digit was defined by drawing a line from the tip of the digit down 2.15 mm and across to the lateral edge of the tissue. For this analysis, the Tissue from animals ranging from stages E14.5 to P14 was prepared for analysis by fixing in 4% paraformaldehyde (PFA) in PBS for 45 min to 4 h depending on the stage; washing three times in PBS, once in PBS + 15% sucrose for 1 h, and once in PBS + 30% sucrose for 2 h to overnight depending on the stage; and then freezing in OCT. Tissue from animals aged 7 wk to 9 mo was processed similarly to earlier stages except that it was decalcified in 0.5 M EDTA (pH 7.4) for 4 d prior to incubating in sucrose. All solutions were prechilled and used at 4 °C with agitation, and skin from tissues of P0 or older mice was lacerated or removed prior to processing.Tissue was then cryosectioned at 12 μm and processed. Staining of sections with Safranin O, Fast Green, and Harris' hematoxylin was carried out using standard histological procedures. Detection of LACZ activity with X-Gal was performed as described  and was Bmpr1a .RNA in situ hybridization was performed as described , with thBmpr1a , Col2a1   in PBS (pH 5) at 37 °C for 30 min to 2 h depending on the stage. Slides were then washed in PBS, treated with 0.3% hydrogen peroxide in 100% methanol for 30 min, washed, blocked with PBS + 0.05% Tween20 + 5% goat or fetal bovine serum, washed again, and incubated with primary antibodies in PBS + 0.05% Tween 20 + 1% goat or fetal bovine serum overnight at 4 °C. Biotin-labeled secondary antibodies (Vector Labs) were tagged with HRP using the Vectastain Elite ABC kit (Vector Labs) followed by detection with DAB (Vector Labs). Primary antibodies and dilutions used were: goat anti-mouse MMP13, 1:100 ; rabbit anti-human SOX9, 1:500 ; rabbit http://www.ncbi.nih.gov/Genbank/) accession numbers for the genes discussed in this paper are Gdf5 (AC084323) and Bmpr1a (NM_009758).GenBank (


The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two  HCMV is a member of the betaherpesvirus family ,48. OtheSor OriL) and a DNA cleavage/packaging signal [cis-acting functions can be relatively small ranging from ca. 90–150 base pairs (bp) for the ori and ca. 250–300 bp for the a sequence. The functional HCMV oriLyt is much more complex than either of the HSV oris; the HCMV oriLyt consists of multiple direct and inverted repeats and extends over at least 1500 bp [a sequence varies in size from ca. 550 bp to 762 bp, however, the conserved pac-1 and pac-2 cis-elements which determine the sites for cleavage of replicated viral DNA are present [Defective HSV viruses created by high multiplicity serial passage of virus stocks have been described on numerous occasions and have been characterized in detail at the molecular level ,43,52,67g signal ,57,60-62 1500 bp ,37. HCMV 1500 bp . The HCM present ,58,64,65et al. 2003 [In contrast to HSV, HCMV does not efficiently produce defective virus genomes, this difference may be related to the distinct biology of the two viruses . Howeveral. 2003 , describHCMV infects cells of the hematopoietic lineage ,39,55,68varRIT , were propagated in human fibroblasts (HF) cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum . Recombinant HCMV, RC2.7EGFP, expressing enhanced green fluorescent protein (EGFP) , under the control of the major early 2.7 promoter, was constructed by cotransfection of plasmid pEAG2.7EGFP with a set of overlapping cosmid clones derived from HCMV Toledo .HCMV Toledo , and HCMV Townea sequence, was obtained from Ed Mocarski (Stanford University). pEAG2.7EGFP was derived by cloning the EGFP gene from plasmid pEGFP-N2  between the EagI and SmaI site of the β2.7 gene taken from Toledo . HCMV amplicon plasmid Tn9-8 was derived by inserting the 6 kpb DraI fragment of TownevarRIT  , and cloned into the HCMV amplicon Tn9-8 at the PstI site in both orientations. The gpt gene in Tn9-8-gpt was derived by cloning a PCR fragment from Escherichia coli DH5α using the primer pairs 5'CTGCAGCTAGTCTAGACTGGGACACTTCACATGAGC3'and 5'CTGCAGCTATGTATCTAGAGCCAGGCGTTGAAAAGATTA3'.Plasmid pON205 , contains the Towne strain ) Figure , spannin et al., . The pla4 precipitation of approximately 4 μg of Tn9-8 amplicon DNA. The Tn9-8 DNA was transfected into approximately 1 × 106 passage 16 human fibroblast (HF) cells. At 24 hours post transfection, the cells were infected with CMV Towne at a multiplicity of infection (MOI) of 5 plaque forming units (PFU) per cell. Fresh medium was added to cells four days after infection and cells were harvested at 6 to 7 days post infection as described previously . Virus stocks are prepared by three freeze-thaw cycles. Serial passages of amplicon-containing viral stocks on fresh HF cells were superinfected with CMV Towne as a helper virus at a MOI of 1.Plasmid DNA was transfected by CaPOViral DNAs were digested with restriction enzyme, electrophoresed in 0.8% agarose gels, transferred to Hybond-N+ nylon membranes (Amersham Corp.), , and immobilized with a UV Crosslinker 1000 . DNA on the membrane was probed with fluorescein-labeled pUC9 DNA using conditions previously described .5 CD34+ cells were used for each infection with TN9-8GF5 amplicon containing stocks, RC2.7EGFP virus, CMV Towne virus, or uninfected cell control. The cells mixed with virus were centrifuged at 500 × g for 10 mins at room temperature and were then placed in 37°C water bath for one hour. Following this the cells were cultured in 6-well cell culture plates (Costar) for 18–72 hours. At the end of the incubation the cells were harvested for CD34 staining.The isolation of cord blood CD34+ stem cells was carried out by All Cells Inc.  using CD34 Progenitor Cell Isolation Kit . The positive selection of the CD34+ cells was carried out using hapten-conjugated antibody to CD34+ followed by anti-hapten antibody coupled to MACS Microbeads. The magnetically labeled cells are enriched on positive selection columns in the magnetic field. The purity of the CD34+ population was >95% as analyzed by flow cytometry. The purified CD34+ cells were suspended in Iscove's modified Dulbecco's Minimal Essential Medium containing 5% fetal bovine serum. 2 × 10Amplicon containing viral stocks prepared from passage 1 were used to infect HF or human CD34+ cells maintained in 12 well culture plates. At 24 hour intervals post infection, the wells were observed for EGFP expression with a Nikon TE2000 microscope under UV illumination. Immunostaining for CD34+ cells was done using Phycoerythrin (PE)-conjugated anti-CD34 antibody . Infected or control cells were incubated with 20 μl of PE-labeled anti-CD34 antibody for 45 minutes at room temperature and subsequently were washed twice with PBS containing 0.1% BSA. The cells were directly analyzed for EGFP and CD34+ staining on a FACSCalibur instrument , at 18 and 36 hours post infection.a sequences was constructed. Theoretically, due to the large size of the HCMV genome, an amplicon derived from this virus should be able to carry the large DNA inserts and be capable of efficient introduction into hematopoietic cells by infection.In order to exploit the natural tropism of HCMV for cells of the hematopoietic lineage, in a nonlytic manner, an HCMV amplicon i.e. a plasmid containing the HCMV oriLyt and EcoRI E fragment of Towne that was not present in the Toledo strain [EcoRI E region spans in part the complex oriLyt region [BamHI sites in Figure During analysis of cosmid clones of HCMV strain Towne, sequence heterogeneity was observed in the o strain . The Ecot region ,2,24,37.t region . This secis-acting functions required for the propagation of the defective virus genomes in the presence of helper virus  [EcoRI site of pON205. The resulting amplicon was designated Tn9-8    cells, and subsequently infected with HCMV Towne strain at an MOI of 5 to provide helper virus replication functions. Seven days later, infected cells were harvested, sonicated, and viral stocks were prepared for passage to fresh HF cells. Fresh HF cells were infected with the progeny of the transfection/infection and incubated for 7 days. The DNA from these infected cells was harvested (designated passage 1), restricted with  blotted . Southera Figure , lane 2.r Figure . This regpt), were serially passaged in HF cells. Defective viruses could be detected at passage 3 when probed with plasmid pUC9; however, the copy number appeared to diminish upon serial passage (not shown). Selection with mycophenolic acid on Tn9-8-gpt amplicons did not enhance recovery.Packaged defective viral genomes derived from Tn9-8 or a derivative containing a selectable marker (Tn9-8-gpt), digested with Pst I and Hind III, respectively, in order to analyze monomeric repeat units. The Hind III-digested DNA was circularized by ligation and used to transform E. coli bacteria to analyze structure and to demonstrate shuttle vector capability between eucaryotic and bacterial hosts. A number of plasmids prepared from the rescue attempt had a restriction enzyme pattern indistinguishable from the input . Digestion with NaeI produced a fragment of the predicted size of a unit length a sequence (762 bp), and this product hybridized with an a sequence specific probe (PstI-SgrAI fragment from Tn9-8),  promoter was used as test reporter gene. Two resulting amplicon plasmids designated Tn9-8GF5 and Tn9-8GF7 both expressed EGFP following transfection of HF cells in the absence of helper virus, as expected (not shown). Packaged amplicons were generated by introduction of Tn9-8GF5 into cells and infecting with HCMV 24 hours later at an MOI of 5. Transfection-derived viral stocks were passaged onto fresh HF cells supplemented with Towne helper virus at an MOI of 1. Viral stocks prepared from passage 1 were used to infect HF cells and grown on 12-well tissue culture plates. A limited number (ca. 0. 1%) of brightly fluorescing cells could be seen by microscopic examination at 24, 72 and 96 hours post-infection  promoter prepared from passage 0 and passage 1 were used to infect CD34+ cells derived from cord blood. Starting at 24 h after infection, CD34+ cells were examined for EGFP expression by fluorescent microscopy. EGFP expression was observed in TN9-8GF5 amplicon-infected CD34+ cells starting at 24 h post-infection. The cells remained positive for EGFP expression for more than 96 hrs, at which point the cells were terminated  population infected with either TN9-8GF5 amplicon containing viral stocks  or with the CMV-EGFP virus, RC2.7EGFP (3.6%)  Figure , &7b or ) Figure  &7c. The) Figure , and 7e.We have shown that a replication-defective virus vector system that is derived from HCMV is capable of delivering and expressing foreign genes in infected primary cells including progenitor stem cells such as human CD34+ cells. Further improvement and optimization of the system offers the potential to deliver gene-based therapies to multipotent cells.Foremost among the advantages of the vector system we have described is the potential ability to efficiently infect and deliver genetic information to hematopoietic stem cells CD34+) and other dividing and non-dividing cell types which may support HCMV infection + and oth,39,55,68in vivo and in vitro [ex vivo culture conditions, which preserve the developmental properties of the stem cells [in vitro with HCMV [in vitro culture of cord blood derived CD34+ cells. In a separate study, cord blood derived CD34+ cells cultured with IL-3 in vitro showed a progressive decline of the CD34+ population and more differentiated cells originating in the CD34(-) population [in vitro culture has been observed. EGFP expression was also seen in the CD34(-) population (Fig The infectivity of CD34+ cells from seropositive and seronegative subjects with HCMV has been tested both in vitro . Furtherin vitro . The HCMin vitro . The appem cells ,22. Umbiem cells . In our ith HCMV , reportepulation . In our tion Fig , &7c. Itcis-acting ori and packaging sequences, and have no structural gene sequences. However, amplicon containing viral stocks are a mixture with HCMV replication competent helper virus. HCMV induced cell-differentiating effect, if any, might be minimized using a helper virus-free amplicon system. In this regard, it should be possible to test a number of strategies to prepare helper virus-free stocks [Further studies of HCMV infection in CD34 cells will help in defining whether CD34+ infected cells undergo cell differentiation by increased expression of other markers such as CD33, CD38, HLA-DR or cytokines. It is also relevant to note here that HCMV virus carries homolog sequences for HLA-related and cytokine-related molecules and infection can induce cellular cytokines ,46,50,66e stocks ,63. ThesThe authors of this publication are supported financially by salary and shares of MedImmune Inc., during the completion of this work. A patent application has been filed with the United States Patent Office relating to the content of this manuscript. The authors have assigned all rights of ownership to MedImmune Inc. The authors declare that they have no other competing interest.a' sequence analysis. GWK provided critical intellectual input. RRS provided the original idea and experimental design as well as cloning of the amplicon, generation of amplicon stocks and Southern analysis.KM carried out the expression analysis in human fibroblasts and CD34 cells. MNP & GMD generated amplicon stocks and MNP did the '