In vitro and in vivo effects of kisspeptin antagonists p234, p271, p354, and p356 on GPR54 activation.

Kisspeptins (KPs) and their receptor (GPR54 or KiSS1R) play a key-role in regulation of the hypothalamic-pituitary-gonadal axis and are therefore interesting targets for therapeutic interventions in the field of reproductive endocrinology. As dogs show a rapid and robust LH response after the administration of KP10, they can serve as a good animal model for research concerning KP signaling. The aims of the present study were to test the antagonistic properties of KP analogs p234, p271, p354, and p356 in vitro, by determining the intracellular Ca2+ response of CHEM1 cells that stably express human GPR54, and to study the in vivo effects of these peptides on basal plasma LH concentration and the KP10-induced LH response in female dogs. Exposure of the CHEM1 cells to KP-10 resulted in a clear Ca2+ response. P234, p271, p354, and p356 did not prevent or lower the KP10-induced Ca2+ response. Moreover, the in vivo studies in the dogs showed that none of these supposed antagonists lowered the basal plasma LH concentration and none of the peptides lowered the KP10-induced LH response. In conclusion, p234, p271, p354, and p356 had no antagonistic effects in vitro nor any effect on basal and kisspeptin-stimulated plasma LH concentration in female dogs.

The effects of kisspeptin agonist canine KP-10 and kisspeptin antagonist p271 on plasma LH concentrations during different stages of the estrous cycle and anestrus in the bitch.

Kisspeptin (KP) plays a key role in the regulation of the hypothalamic-pituitary-gonadal axis via the release of GnRH. As normal KP signaling is essential for reproductive function, it could be an interesting new target for therapeutic interventions, e.g., nonsurgical contraception in dogs. The aims of the present study were to investigate the effect of KP-10 administration on plasma LH concentration in different stages of the reproductive cycle and to investigate the suitability of p271 as KP antagonist in the bitch. Two groups of six adult Beagle bitches were used. In one group, plasma LH concentration was determined before (40 and 0 minutes) and 10, 20, 40, and 60 minutes after the intravenous administration of 0.5-µg/kg body weight (BW) canine KP-10. In the other group, the bitches received a continuous intravenous infusion with p271 (50 µg/kg BW/h) for 3 hours, and 0.5-µg/kg BW canine KP-10 was administered intravenously 2 hours after the start of the p271 infusion. Their plasma LH concentration was determined before (-40 and 0 minutes) and 30, 60, 90, 120, 130, 140, 160, and 180 minutes after the start of the p271 infusion. In both groups, the experiments were performed during the follicular phase, the first and second half of the luteal phase, and during anestrus. Canine KP-10 induced an increase of plasma LH concentration during all estrous cycle stages and anestrus. There was no difference in LH response between the two groups. The lowest LH response was seen during the follicular phase and the highest response during anestrus. The area under the curve (AUC) for LH and LH increment in the follicular phase were lower than those in anestrus. The AUC LH and LH increment in the first half of the luteal phase were lower than those in the second half of the luteal phase and anestrus. The AUC LH and LH increment in the second half of the luteal phase were not different from those in anestrus. Continuous administration of the antagonist p271 did not alter basal plasma LH concentration and could not prevent or lower the LH response to KP-10 in any of the cycle stages and anestrus. It can be concluded that the LH response to KP-10 is dependent on estrous cycle stage and that peripheral administrated p271 cannot be used as KP antagonist in the dog. This provides new insight in reproductive endocrinology of the bitch, which is important when KP signaling is considered for therapeutic interventions, such as for estrus induction or nonsurgical contraception in the bitch.

Dietary management of labrador retrievers with subclinical hepatic copper accumulation.

BACKGROUND: Genetic and environmental factors, including dietary copper intake, contribute to the pathogenesis of copper-associated hepatitis in Labrador retrievers. Clinical disease is preceded by a subclinical phase in which copper accumulates in the liver. OBJECTIVE: To investigate the effect of a low-copper, high-zinc diet on hepatic copper concentration in Labrador retrievers with increased hepatic copper concentrations. ANIMALS: Twenty-eight clinically healthy, client-owned Labrador retrievers with a mean hepatic copper concentration of 919 ± 477 mg/kg dry weight liver (dwl) that were related to dogs previously diagnosed with clinical copper-associated hepatitis. METHODS: Clinical trial in which dogs were fed a diet containing 1.3 ± 0.3 mg copper/Mcal and 64.3 ± 5.9 mg zinc/Mcal. Hepatic copper concentrations were determined in liver biopsy samples approximately every 6 months. Logistic regression was performed to investigate effects of sex, age, initial hepatic copper concentration and pedigree on the ability to normalize hepatic copper concentrations. RESULTS: In responders (15/28 dogs), hepatic copper concentrations decreased from a mean of 710 ± 216 mg/kg dwl copper to 343 ± 70 mg/kg dwl hepatic copper after a median of 7.1 months (range, 5.5-21.4 months). Dogs from a severely affected pedigree were at increased risk for inability to have their hepatic copper concentrations normalized with dietary treatment. CONCLUSIONS AND CLINICAL IMPORTANCE: Feeding a low-copper, high-zinc diet resulted in a decrease in hepatic copper concentrations in a subset of clinically normal Labrador retrievers with previous hepatic copper accumulation. A positive response to diet may be influenced by genetic background. Determination of clinical benefit requires further study.

Prevalence and co-occurrence of hip dysplasia and elbow dysplasia in Dutch pure-bred dogs.

Hip as well as elbow dysplasia (HD, ED) are developmental disorders leading to malformation of their respective joints. For a long time both disorders have been scored and targeted for improvement using selective breeding in several Dutch dog populations. In this paper all scores for both HD and ED, given to pure bred dogs in the Netherlands from 2002 to 2010, were analyzed. Heritabilities and correlations between HD and ED were calculated for the 4 most frequently scored breeds. Heritabilities ranged from 0.0 to 0.37 for HD related traits (FCI-score, osteoarthritis, congruity, shape and laxity (Norberg angle); FCI: Fédération Cynologique Internationale) and from 0.0 to 0.39 for ED related traits (IEWG score, osteoarthritis, sclerosis and indentation; IEWG: International Elbow Working Group). HD related traits showed high genetic and residual correlations among each other but were only to a minor extent correlated with ED related traits, which also showed high correlations among each other. Genetic correlations were higher than residual correlations. Phenotypic and genetic trends since 2001 for the four most scored breeds were slightly positive but decreasing over time, indicating that selection over the past decade has not been effective.

Urinary excretion of copper, zinc and iron with and without D-penicillamine administration in relation to hepatic copper concentration in dogs.

Hereditary copper-associated hepatitis in dogs resembles Wilson's disease, a copper storage disease in humans. Values for urinary copper excretion are well established in the diagnostic protocol of Wilson's disease, whereas in dogs these have not been evaluated. The objectives of this study were to characterize both basal and D-penicillamine induced urinary copper, zinc and iron excretion in dogs in relation to hepatic copper concentration. Beagles, Beagle-Bedlington terrier cross-breeds homozygous for the COMMD1 gene mutation that causes copper toxicosis, and Labrador retrievers with normal or increased hepatic copper concentrations were investigated. The hepatic copper phenotype was determined by histological evaluation of liver biopsies and measurement of the hepatic copper concentration by instrumental neutron activation analysis. Urinary excretion of copper, iron and zinc was measured via inductively coupled plasma optical emission spectrometry under basal conditions and after oral administration of a single dose (20mg/kg bodyweight) of the chelator D-penicillamine. There was a rapid increase in urinary excretion of copper and zinc, but not iron after D-penicillamine administration. This increase was not different between dogs with high or normal hepatic copper concentrations. D-penicillamine-induced urinary copper excretion and the copper/creatinine ratio did not correlate with hepatic copper concentrations in the dogs studied, although basal urinary copper/zinc ratios did correlate with hepatic copper concentrations in Labrador retrievers. The latter parameter may be useful in diagnostic and follow-up protocols for copper-associated hepatitis in Labrador retrievers.

Association of dietary copper and zinc levels with hepatic copper and zinc concentration in Labrador Retrievers.

BACKGROUND: Copper-associated hepatitis is an inherited disease in the Labrador Retriever. Apart from genetic factors, dietary intake of copper and zinc are suspected to play a role in the pathogenesis. OBJECTIVES: To investigate whether dietary copper and zinc levels of commercially available dry diets are associated with hepatic copper and zinc concentrations in Labrador Retrievers. ANIMALS: Fifty-five Labrador Retrievers that were fed a single brand and type of commercial dry food for at least 1 year. Of these, 44 dogs were family members of Labrador Retrievers with copper-associated hepatitis. METHODS: Liver biopsies, blood samples, and diet samples were obtained. Liver specimens were scored histologically and copper and zinc concentrations were quantified. Dietary concentrations of copper and zinc were measured. The association between dietary intake of copper and zinc and hepatic copper and zinc concentrations was investigated by linear regression analysis. RESULTS: High dietary copper and low dietary zinc levels were significantly associated with high hepatic copper levels. No association between dietary intake and hepatic zinc was present. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary copper and zinc at current levels in commercially available dry dog food can influence hepatic copper and can be a risk factor for the development of copper-associated hepatitis in Labrador Retrievers with a genetic susceptibility to copper.

Phenotypic and genetic evaluation of elbow dysplasia in Dutch Labrador Retrievers, Golden Retrievers, and Bernese Mountain dogs.

Canine elbow dysplasia encompasses four developmental diseases: ununited anconeal process, osteochondrosis of the medial part of the humeral condyle, fragmented medial coronoid process (FCP), and incongruity of the elbow joint. Four radiographic views per joint were used to evaluate 2693 Labrador Retrievers (LRs), 1213 Golden Retrievers (GRs), and 974 Bernese Mountain Dogs (BMDs) for the presence of elbow dysplasia between 2002 and 2009 in the Netherlands. The views were also graded for signs of osteoarthritis and sclerosis. FCP was diagnosed most frequently in LRs, GRs and BMDs, with an incidence of 6%, 5%, and 15%, and a heritability of 0.17, 0.24, and 0.06, respectively. Heritabilities were estimated using a sire model and all available ancestors. Sclerosis at the base of the medial coronoid process was the radiographic sign most strongly correlated with FCP (r=0.95, 0.92, and 0.95 in LRs, GRs and BMDs, respectively). The sex of the dog was significantly correlated with the presence of osteoarthritis in LRs, but not in GRs and BMDs. Male LRs were 1.7-fold more frequently, but not more severely, affected by osteoarthritis than female dogs. Age at radiographic examination was significantly associated with osteoarthritis in all three breeds. The heritability estimates in Retrievers were high enough to warrant including FCP findings in the breeding policy, but until the biomechanical and genetic background of elbow dysplasia are better understood, correct phenotyping with a sensitive technique is essential.

Expression and mutation analysis of Tpit in the canine pituitary gland and corticotroph adenomas.

Pituitary-dependent hyperadrenocorticism (PDH) in dogs is caused by a pituitary corticotroph adenoma. Although PDH is a common disorder in dogs, little is known about the underlying pathogenesis. In the pituitary glands of humans and mice, the pro-opiomelanocortin (POMC)-expressing cell lineages, the corticotrophs and melanotrophs, have a specific marker in common, the T-box transcription factor Tpit (Tbx19), which is obligate for POMC expression. Tpit also regulates the late differentiation of the corticotrophs and melanotrophs, and therefore may contribute to the pathogenesis of the corticotroph adenomas. The aim of this study was to perform an expression and mutation analysis of Tpit in the normal canine pituitary and in corticotroph adenomas. The distribution of the Tpit protein in the pituitary gland was studied with immunohistochemistry and the expression of the gene with RT-PCR. The coding region of Tpit cDNA from 14 dogs with PDH was screened for mutations. Tpit was expressed in corticotroph and melanotroph cells of the normal and adenomatous canine pituitary, and remained present in non-adenomatous corticotrophs of pituitaries from PDH dogs. No tumor-specific mutation in the Tpit cDNA from the corticotroph adenomas was found. However, a missense polymorphism in the highly conserved DNA-binding domain, the T-box, was discovered in one dog. It is concluded that Tpit can be used as a reliable marker for the corticotroph and melanotroph cells in the canine pituitary tissue and that mutations in the Tpit gene are unlikely to play a major role in the pathogenesis of canine corticotroph adenomas.

A novel susceptibility locus for Hirschsprung's disease maps to 4q31.3-q32.3.

We report on a multigenerational family with isolated Hirschsprung's disease (HSCR). Five patients were affected by either short segment or long segment HSCR. The family consists of two main branches: one with four patients (three siblings and one maternal uncle) and one with one patient. Analysis of the RET gene, the major gene involved in HSCR susceptibility, revealed neither linkage nor mutations. A genome wide linkage analysis was performed, revealing suggestive linkage to a region on 4q31-q32 with a maximum parametric multipoint LOD score of 2.7. Furthermore, non-parametric linkage (NPL) analysis of the genome wide scan data revealed a NPL score of 2.54 (p = 0.003) for the same region on chromosome 4q (D4S413-D4S3351). The minimum linkage interval spans a region of 11.7 cM (12.2 Mb). No genes within this chromosomal interval have previously been implicated in HSCR. Considering the low penetrance of disease in this family, the 4q locus may be necessary but not sufficient to cause HSCR in the absence of modifying loci elsewhere in the genome. Our results suggest the existence of a new susceptibility locus for HSCR at 4q31.3-q32.3.

The leukemia inhibitory factor receptor gene is not involved in the etiology of pituitary dwarfism in German shepherd dogs.

Pituitary dwarfism in German shepherd dogs is characterized by combined pituitary hormone deficiency (CPHD) and intrapituitary cyst formation. Activation of the leukemia inhibitory factor (LIF)-LIF receptor (LIFR) signal transduction pathway results in a similar phenotype in (transgenic) mice. We therefore assessed the role of the LIFR in the etiology of pituitary dwarfism in German shepherd dogs. A polymorphic microsatellite marker (UULIFR) was used to analyze the segregation of the LIFR gene in 22 German shepherd dogs from 4 pedigrees, each including one dwarf. There was no allelic association between UULIFR and the dwarfism phenotype. Based on our findings LIFR was excluded as a candidate gene for CPHD.

Arg113His mutation in eIF2Bepsilon as cause of leukoencephalopathy in adults.

Vanishing white matter is a leukoencephalopathy that usually affects young children. Five genes were found recently for this disease, allowing a DNA-based diagnosis. The authors describe six patients homozygous for the Arg113His mutation in eIF2Bepsilon. Only one had a childhood onset; four had a later onset and a protracted disease course; one adult still has no symptoms. Our data suggest that the Arg113His mutation is particularly mild and should be considered in the differential diagnosis of adult diffuse leukoencephalopathies, independent of whether there are associated clinical signs, an episodic course, or MRI shows white matter rarefaction/cystic degeneration.

Clinical and genetic heterogeneity in megalencephalic leukoencephalopathy with subcortical cysts (MLC).

BACKGROUND: Megalencephalic leukoencephalopathy with subcortical cysts (MLC) was recently localized on chromosome 22q (tel) and 26 different mutations of the gene MLC1 have been found. We report three siblings of non-consanguineous parents who presented with characteristic features of MLC, but did not have MLC1 mutations. MEYHODS: Clinical, laboratory and neuro-imaging findings of the siblings are described and similar patients with MLC are reviewed. RESULTS: All three siblings suffered from ataxia, progressive severe tetraparesis, dysarthria, dysphagia and epilepsy. Generalized dystonia occurred in one patient. Mental deterioration progressed more slowly than motor deterioration. The youngest male was the most severely affected and died at the age of 23 years. The two older females are now 34 and 35 years old. Our patients are among the oldest described with this clinical entity. No mutation of the MLC1 gene was found in our siblings and linkage with the MLC1 locus was excluded. CONCLUSIONS: The genetic findings in our patients suggest at least a second gene locus for MLC.

Identification of novel mutations in MLC1 responsible for megalencephalic leukoencephalopathy with subcortical cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an inherited neurologic disorder with macrocephaly before the age of one and slowly progressive deterioration of motor functions. Magnetic resonance imaging shows diffusely abnormal and swollen white matter of the cerebral hemispheres and the presence of subcortical cysts in the anterior-temporal region and often also in the frontoparietal region. Mutations in the MLC1 gene, encoding a putative membrane protein, have been recently identified as a cause for MLC. Here, we describe 14 new mutations in 18 patients. Two identified polymorphisms lead to alterations of amino acid residues. The role, suggested by others, of a mutation in the MLC1gene in catatonic schizophrenia and the possible function of the MLC1 protein as a cation channel are discussed.

Mutations of MLC1 (KIAA0027), encoding a putative membrane protein, cause megalencephalic leukoencephalopathy with subcortical cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive disorder characterized by macrocephaly, deterioration of motor functions with ataxia, and spasticity, eventuating in mental decline. The brain appears swollen on magnetic resonance imaging, with diffuse white-matter abnormalities and the invariable presence of subcortical cysts. MLC was recently localized on chromosome 22q(tel). We have narrowed down the critical region by linkage analysis of 11 informative families with MLC to a region of approximately 250 kb, containing four known genes. One family with two patients who were siblings did not display linkage between the MLC phenotype and any of the analyzed microsatellite markers on chromosome 22q(tel), suggesting genetic heterogeneity and the existence of at least a second MLC locus. The maximum two-point LOD score for the 11 families was 6.6 at recombination fraction .02. Twelve different mutations in seven informative and six uninformative families were found in one of the candidate genes, KIAA0027, which we renamed "MLC1." The gene encodes a putative membrane protein with eight predicted transmembrane domains. The patients of one family were compound heterozygotes for mutations that both introduced stop codons. The mutations further included frameshifts, splice-acceptor mutations, a putative splice-donor mutation, and amino acid substitutions of residues in predicted transmembrane domains. These data provide strong evidence that mutations of MLC1 cause the disease.

The Fanconi anemia group E gene, FANCE, maps to chromosome 6p.

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive disease with bone marrow failure and predisposition to cancer as major features, often accompanied by developmental anomalies. The cells of patients with FA are hypersensitive to DNA cross-linking agents in terms of cell survival and chromosomal breakage. Of the eight complementation groups (FA-A to FA-H) distinguished thus far by cell fusion studies, the genes for three-FANCA, FANCC, and FANCG-have been identified, and the FANCD gene has been localized to chromosome 3p22-26. We report here the use of homozygosity mapping and genetic linkage analysis to map a fifth distinct genetic locus for FA. DNA from three families was assigned to group FA-E by cell fusion and complementation analysis and was then used to localize the FANCE gene to chromosome 6p21-22 in an 18.2-cM region flanked by markers D6S422 and D6S1610. This study shows that data from even a small number of families can be successfully used to map a gene for a genetically heterogeneous disorder.

Analysis of DNA methylation of the 5' region of the deoxycytidine kinase gene in CCRF-CEM-sensitive and cladribine (CdA)- and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA)-resistant cells.

DNA methylation of the CpG-rich 5' region of the deoxycytidine kinase (dCK) gene is potentially involved in the suppression of the gene and the resistance of tumour cells to arabinosylcytosine (ara-C). 2-Chlorodeoxyadenosine (cladribine, CdA) and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA) are purine nucleoside analogues which are also phosphorylated by dCK. We observed a reduction in dCK activity in a number of CCRF-CEM-derived cell lines that are resistant to these drugs and hypothesized that this reduction is due to DNA methylation of the 5' region of the dCK gene. The DNA methylation state was analyzed at the DNA sequence level after bisulfite modification of genomic DNA. The investigated region included 0.3 kb of DNA upstream to the start site of transcription, exon 1 and part of intron 1. Sensitive cells (CCRF-CEM/0) and three resistant cell lines (CCRF-CEM/CdA4000, CCRF-CEM/CAFdA100 and CCRF-CEM/CAFdA4000) were investigated. The region that was analyzed contained no methylated cytosine residues in the parental cell line CCRF-CEM/0 or in the resistant cell lines. Therefore, it is highly unlikely that DNA methylation plays a role in the suppression of dCK gene expression in these cell lines.

Inhibition of DNA methylation in malignant MOLT F4 lymphoblasts by 6-mercaptopurine.

Treatment of MOLT F4 lymphoblasts with 6-mercaptopurine (6-MP) resulted in a decrease of ATP and a depletion of S-adenosylmethionine (AdoMet). To investigate whether this might affect the methylation of DNA, we treated MOLT F4 lymphoblasts with increasing concentrations of 6-MP, followed by labeling with [methyl-14C]methionine and [methyl-3H]thymidine. After DNA isolation, we measured the incorporated radioactivity and determined the 14C/3H ratio as a measure for the methylation of newly formed DNA. The 14C/3H ratio was decreased by 17% with 1 mumol/L 6-MP; treatment with increasing concentrations of 6-MP up to 10 mumol/L showed a further decrease to 70%, in comparison with untreated cells. To demonstrate that the methylation of deoxycytidine residues in DNA was reduced, we quantified hydrolyzed DNA by HPLC. The 14C/3H ratio showed a decrease with increasing 6-MP concentrations, indicating that treatment with 6-MP resulted in hypomethylation of DNA.

DNA methylation patterns in the calcitonin gene region at first diagnosis and at relapse of acute lymphoblastic leukemia (ALL).

Aberrant DNA methylation can occur early in neoplastic transformation and may lead to the development of cancer. We describe the alterations of methylation patterns at the DNA sequence level which occurred in the 5' region of the calcitonin gene in lymphoblasts from 14 pediatric patients with acute lymphoblastic leukemia (ALL). The DNA methylation status of 25 CpG sites was determined by sequence analysis after bisulfite treatment of the DNA. This method showed that 13 out of 14 patients had increased numbers of methylated CpG sites in the calcitonin gene region at initial diagnosis when compared to control DNA from healthy individuals. The 5' region of the calcitonin gene appears to be methylated to a significantly higher degree in T lineage ALL compared to B lineage ALL (P < 0.01). Each of six ALL patients who were investigated at initial diagnosis and at relapse showed alterations in DNA methylation between the two stages. These six cases were also investigated by Southern blot analysis with methylcytosine-sensitive restriction enzymes and this method showed an increase in DNA methylation in only four of the six cases. The DNA sequencing method thus appears to be better suited to assess alterations of DNA methylation than Southern blot analysis. There are marked regional differences in the frequency of methylation of individual CpG sites and in the frequency of alterations between the two stages. Our results show that alterations in DNA methylation continue to occur from the initial stage to the relapse stage of ALL, suggesting that aberrant DNA methylation may play a role in tumor progression.

Thiopurine methyltransferase: a review and a clinical pilot study.

Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of 6-mercaptopurine (6MP), which is used in the treatment of acute lymphoblastic leukemia (ALL). TPMT catalyzes the formation of methylthioinosine monophosphate (MetIMP), which is cytotoxic for cultured cell lines, and it plays a role in detoxification of 6MP. Population studies show a genetic polymorphism for TPMT with both high and low activity alleles. About 1 of 300 subjects is homozygous for the low activity. The function TPMT plays in detoxification or therapeutic efficacy of 6MP in vivo is not clear. In this article the genetic polymorphism of TPMT is reviewed and the contribution of TPMT to the cytotoxic action, or detoxification, of 6MP in children with ALL is discussed. Induction of TPMT activity has been described during the treatment for ALL. We performed a pilot study on the influence of high-dose 6MP infusions (1300 mg/m2 in 24 h) on TPMT activity of peripheral blood mononuclear cells (pMNC) of eleven patients with ALL. The TPMT activities were in, or, above the normal range. There was no statistically significant difference between the TPMT activities before and after the 6MP infusions. MetIMP levels in pMNC increased during successive courses. This might be explained by TPMT induction, but other explanations are plausible as well. Twenty five percent of the TPMT assays failed, because less than the necessary 5.10(6) pMNC could be isolated from the blood of leukopenic patients. Red blood cells can not be used for TPMT measurements, since transfusions are frequently required during the treatment with 6MP infusions. Therefore, the influence of high-dose 6MP infusions on TPMT activity can only be investigated further when a TPMT assay which requires less pMNC has been developed.

Adenovirus DNA replication in vitro: duplication of single-stranded DNA containing a panhandle structure.

Adenovirus DNA replicates by displacement of one of the parental strands followed by duplication of the displaced parental single strand (complementary strand synthesis). Displacement synthesis has been performed in a reconstituted system composed of viral and cellular proteins, employing either the viral DNA-terminal protein complex as template or linearized plasmids containing the origin. Previously, evidence was obtained that in vivo complementary strand synthesis requires formation of a panhandle structure originating from hybridization of the inverted terminal repeats. To study the conditions for complementary strand synthesis in vitro, we have constructed an artificial panhandle molecule that contains a double-stranded inverted terminal repetition (ITR) region and a single-stranded loop derived from the left and right terminal XmaI fragments of Ad2. Such a molecule appeared to be an efficient template and could initiate by the same protein-priming mechanism as double-stranded DNA, employing the precursor terminal protein. The efficiency of both types of template was comparable. Like for replication of the duplex molecule initiation of panhandle replication was stimulated by nuclear factors I and III, proteins that bind to specific double-stranded regions of the ITR. The Ad DNA-binding protein is essential and the 39 kDa C-terminal domain of this protein that harbors the DNA-binding properties is sufficient for its function. These results support the hypothesis that panhandle formation is required for duplication of the displaced strand.