The Fontan patient: inconsistencies in medication therapy across seven pediatric heart network centers.

Patients who have undergone the Fontan procedure are at risk for thrombosis, ventricular dysfunction, and valve regurgitation, but data to guide the medical treatment and prevention of these adverse outcomes in this population are lacking. This analysis examined medication usage among Fontan patients by putative indication and by study center. The medical history and current medications of 546 Fontan subjects, ages 6-18 years, were assessed in a Pediatric Heart Network multicenter cross-sectional study. Cardiac imaging was performed within 3 months of enrollment. The majority of the subjects (64%) were taking two or more medications. Antithrombotics were taken by 86% of those with a history of stroke, thrombosis, or both and 67% of those without such a history (P = 0.01). Conversely, 14% of those with a history of stroke, thrombosis, or both were taking no antithrombotic. Angiotensin-converting enzyme inhibitor (ACEi) therapy was independently associated with moderate or severe atrioventricular valve regurgitation (P = 0.004), right ventricular morphology (P < 0.001), and shorter time since Fontan (P = 0.004) but not with ventricular systolic dysfunction. Glycoside therapy and diuretic therapy each was associated with older age at Fontan (P = 0.001 and P = 0.023, respectively) and a history of post-Fontan arrhythmia (P < 0.001 and P = 0.003, respectively) but not with ventricular systolic dysfunction. Medication use rates varied widely among the centers, even with controls for center differences in patient characteristics. Prospective therapeutic trials are needed to guide the medical treatment of Fontan patients.

The cohesion protein MEI-S332 localizes to condensed meiotic and mitotic centromeres until sister chromatids separate.

The Drosophila MEI-S332 protein has been shown to be required for the maintenance of sister-chromatid cohesion in male and female meiosis. The protein localizes to the centromeres during male meiosis when the sister chromatids are attached, and it is no longer detectable after they separate. Drosophila melanogaster male meiosis is atypical in several respects, making it important to define MEI-S332 behavior during female meiosis, which better typifies meiosis in eukaryotes. We find that MEI-S332 localizes to the centromeres of prometaphase I chromosomes in oocytes, remaining there until it is delocalized at anaphase II. By using oocytes we were able to obtain sufficient material to investigate the fate of MEI-S332 after the metaphase II-anaphase II transition. The levels of MEI-S332 protein are unchanged after the completion of meiosis, even when translation is blocked, suggesting that the protein dissociates from the centromeres but is not degraded at the onset of anaphase II. Unexpectedly, MEI-S332 is present during embryogenesis, localizes onto the centromeres of mitotic chromosomes, and is delocalized from anaphase chromosomes. Thus, MEI-S332 associates with the centromeres of both meiotic and mitotic chromosomes and dissociates from them at anaphase.

Stopping and starting the meiotic cell cycle.

The meiotic cell cycle arrests in response to both perturbations and developmental signals. Recent research suggests that meiosis has checkpoints to monitor the completion of meiotic recombination and the attachment of chromosomes to the spindle. New insights have been gained into how meiosis resumes after normal developmental arrests, and new genes have been identified that are required for proper meiotic progression.

Drosophila PLUTONIUM protein is a specialized cell cycle regulator required at the onset of embryogenesis.

Unfertilized eggs and fertilized embryos from Drosophila mothers mutant for the plutonium (plu) gene contain giant polyploid nuclei resulting from unregulated S-phase. The PLU protein, a 19-kDa ankyrin repeat protein, is present in oocytes and early embryos but is not detectable after the completion of the initial rapid S-M cycles of the embryo. The persistence of the protein during the early embryonic divisions is consistent with a direct role in linking S-phase and M-phase. When ectopically expressed in the eye disc, PLU did not perturb the cell cycle, suggesting that PLU regulates S-phase only in early embryonic development. The pan gu (png) and giant nuclei (gnu) genes also affect the S-phase in the unfertilized egg and early embryo. We show that functional png is needed for the presence of PLU protein. By analyzing png mutations of differing severity, we find that the extent of the png mutant phenotype inversely reflects the level of PLU protein. Our data suggest that PLU protein is required at the time of egg activation and the completion of meiosis.

Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase.

Mammalian DNA cytosine-5-methyltransferase (MTase, EC 2.1.1.37) is an essential component for establishing and maintaining cell-type specific methylation patterns in the genome. The cDNA for the murine enzyme was previously cloned in segments. We have reconstructed the entire gene, encoding a protein of 1517 amino acids, from a set of overlapping cDNA clones. We report the assembly of two expression constructs in bacterial/mammalian shuttle vectors. Transcription in the first construct (pEMT) is driven by the cytomegalovirus enhancer/promoter and encodes a fusion protein with 15 additional aa at the N terminus, while the second construct (pJMT) is driven by the simian virus 40 early promoter/enhancer upstream from the natural ATG codon. Immunofluorescence microscopy and immunoblot analysis have shown that both constructs direct the synthesis of MTase in COS-1 cells. Enzyme activity in whole-cell lysates of transfected COS-1 cells transfected with pEMT and pJMT are on average tenfold and fivefold higher than in controls, respectively. The specific activities of the recombinant and endogenous mouse-cell enzyme are similar. These expression constructs will be of use in studies of DNA methylation in mammals.

Battered wife syndrome: overview of dynamics and treatment.

The authors review some pertinent features of the battered wife syndrome, including definitions, incidence, characteristics of wives, injury profiles, setting of the violence, psychodynamics, role of alcohol, family backgrounds, characteristics of husbands, coping responses of wives, seeking help, social support systems, recognition of the battering syndrome, prevention and treatment, and group therapy. Although force has long been used as a resource in family politics, we have just started to understand some of its precipitants, social and clinical consequences, and treatment options. The battering syndrome is not a disease per se because it cuts across socioeconomic and diagnostic categories. Controlled studies and national attention are required in order to understand more about this costly problem, which poses a treatment dilemma to clinical psychiatry.

Pharmacokinetics and safety of TP10, soluble complement receptor 1, in infants undergoing cardiopulmonary bypass.

BACKGROUND: Increase in vascular permeability and multiorgan dysfunction after cardiopulmonary bypass (CPB) are barriers to successful cardiac surgery in infants. Complement inhibition with TP10, a C3/C5 convertase inhibitor (AVANT Immunotherapeutics, Needham, Mass), blunts post-CPB organ dysfunction in the neonatal pig. Methods and results The pharmacokinetics and safety of TP10 in infants (age <1 year, n = 15) undergoing CPB were examined in a phase I/II open-label prospective trial. TP10 (10 mg/kg) was given intravenously before CPB and also added (10 mg/100 mL prime volume) to the CPB circuit. TP10 plasma levels correlated with C3a levels and measures of clinical course. All infants survived. No adverse events were attributed to TP10. TP10 plasma concentration fell to < or =60 microg/mL 12 hours after CPB. A 2-compartment model was fit to the TP10 blood levels as a function of time. Based on this model, an initial dose of 10 mg/kg over 0.5 hours followed by 10 mg/kg over 23.5 hours is the most appropriate for maintaining TP10 concentration between 100 microg/mL and 160 microg/mL for 24 hours after CPB. C3a was lower 12 hours after CPB than before CPB and still lower 24 hours after CPB. TP10 concentration was inversely correlated with the 12-hour post-CPB to pre-CPB ratio of C3a (Spearman rho -0.76, P = -.016), and with total (rho -0.56, P =.047) and net (rho -0.85, P =.0016) fluid and blood product administration/kg >24 hours after CPB. CONCLUSIONS: TP10 administration to infants appears safe. Pharmacokinetic analysis generated an optimal dosing strategy to achieve effective TP10 levels for 24 hours after CPB. In the infant, TP10 appears to decrease CPB-induced complement activation and protect vascular function. These results support a phase III trial of TP10 in infants requiring CPB.

Cardiac troponin T forms a tetramer in vitro.

Cardiac troponin T (cTnT) is a myofibrillar protein essential for calcium-dependent contraction. This property has led to functional studies of developmentally expressed cTnT isoforms and mutants identified in patients with hypertrophic cardiomyopathy. The release of cTnT into the serum following myocardial infarction has led to the development of antibody-based assays for measuring cTnT serum concentration. We examined the behavior of cTnT in solution. Recombinant human cTnT3, the dominant isoform in the adult human heart, was used. The protein was pure and functional, as demonstrated by SDS-PAGE and surface plasmon resonance. cTnT3 was found to bind specifically and in a concentration-dependent manner to cTnC. Routine size exclusion chromatography suggested a higher-than-expected MW for cTnT. Using analytical ultracentrifugation, we found cTnT3 in solution to be mainly in the form of a tightly bound tetramer at concentrations as low as 4 micromol/L. Our sedimentation velocity and transmission electron microscopy results indicate that the tetramer's shape is elongated rather than globular. CTnT's self-association in solution is an important consideration in the design and interpretation of experiments with the aim of understanding the biochemical and biophysical properties of cTnT, its isoforms, and its mutants.

Contemporary outcomes after the Fontan procedure: a Pediatric Heart Network multicenter study.

OBJECTIVES: We characterized a large cohort of children who had a Fontan procedure, with measures of functional health status, ventricular size and function, exercise capacity, heart rhythm, and brain natriuretic peptide (BNP). BACKGROUND: The characteristics of contemporary Fontan survivors are not well described. METHODS: We enrolled 546 children (age 6 to 18 years, mean 11.9 years) and compared them within pre-specified anatomic and procedure subgroups. History and outcome measures were obtained within a 3-month period. RESULTS: Predominant ventricular morphology was 49% left ventricular (LV), 34% right ventricular (RV), and 19% mixed. Ejection fraction (EF) was normal for 73% of subjects; diastolic function grade was normal for 28%. Child Health Questionnaire mean summary scores were lower than for control subjects; however, over 80% of subjects were in the normal range. Brain natriuretic peptide concentration ranged from <4 to 652 pg/ml (median 13 pg/ml). Mean percent predicted peak O2 consumption was 65% and decreased with age. Ejection fraction and EF Z score were lowest, and semilunar and atrioventricular (AV) valve regurgitation were more prevalent in the RV subgroup. Older age at Fontan was associated with more severe AV valve regurgitation. Most outcomes were not associated with a superior cavopulmonary connection before Fontan. CONCLUSIONS: Measures of ventricular systolic function and functional health status, although lower on average in the cohort compared with control subjects, were in the majority of subjects within 2 standard deviations of the mean for control subjects. Right ventricular morphology was associated with poorer ventricular and valvular function. Effective strategies to preserve ventricular and valvular function, particularly for patients with RV morphology, are needed.

Calcium signals induce liver stem cells to acquire a cardiac phenotype.

Heart failure is a major cause of premature death and disability in the United States. Stem cell therapy has attracted great interest for the treatment of myocardial infarction and heart failure. Some tissue-specific adult-derived stem cells demonstrate plasticity in that they are multipotent, react to inductive signals provided by a new micro-environment, and acquire the phenotype of cells endogenous to the new micro-environment. The mechanism through which this phenotype is acquired is unknown. We have demonstrated that a liver-derived clonal stem cell line, WB F344, differentiate into cardiomyocytes in vivo and in vitro. Using a coculture model of neonatal heart cells and WB F344 cells, we have found that cytosolic communication between the two cell types results in calcium-induced transcription of cardiac transcription factors and appears to usher in the cardiac phenotype. Functional gap junctions and IP3 receptors appear to be required for this process. We propose that the observed low frequency of stem cell differentiation into cardiomyocytes when transplanted into the injured heart is due, in part, to their inability to establish functioning intercellular communications with healthy cardiomyocytes and receive instructive signals needed to activate a cardiac gene program.

Complement factor 1 inhibitor improves cardiopulmonary function in neonatal cardiopulmonary bypass.

BACKGROUND: The inflammatory insult associated with cardiopulmonary bypass (CPB) continues to result in morbidity for neonates undergoing complex repair of congenital cardiac defects. Complement and contact activation are important mediating processes involved in this injury. Complement factor 1 esterase inhibitor (C1-inh), a natural inhibitor of complement, kallikrein, and coagulation pathways, may be decreased in children undergoing cardiac operations requiring CPB. We tested the hypothesis that C1-inh supplementation will ameliorate the cardiac and pulmonary dysfunction in a model of neonatal CPB. METHODS: Fifty-two neonatal pigs were randomly assigned to receive 0 IU (n = 22), 500 IU (n = 15), 1,000 IU (n = 8), or 1,500 IU (n = 7) of C1-inh. Doses were delivered 5 minutes before starting 90 minutes of normothermic CPB. Pulmonary and cardiovascular measures were taken before and 5, 30, and 60 minutes after CPB. RESULTS: Five animals did not survive CPB. The C1-inh concentration post-CPB increased monotonically with increasing dose (p < 0.001). Weight gain was significantly less in the 1,500 IU group (0.24 +/- 0.10 kg versus 0.38 +/- 0.09 kg, p = 0.001). Dynamic compliance increased with C1-inh dose from 0 to 500 IU by 23% +/- 4% (p < 0.001), but the increase leveled off at the higher doses. Alveolar-arterial O2 gradient decreased with C1-inh dose (p = 0.009). Time derivative of left ventricular pressure (dP/dt(max)) increased significantly with increasing dose (p = 0.016). At the highest dose of C1-inh, the time constant of isovolumic relaxation was increased (p = 0.018). CONCLUSIONS: The C1-inh supplementation results in improved pulmonary and systolic cardiac function in a model of neonatal CPB. The negative effect on diastolic function requires further investigation.

Mechanisms controlling the acquisition of a cardiac phenotype by liver stem cells.

The mechanisms underlying stem cell acquisition of a cardiac phenotype are unresolved. We studied early events during the acquisition of a cardiac phenotype by a cloned adult liver stem cell line (WB F344) in a cardiac microenvironment. WB F344 cells express a priori the transcription factors GATA4 and SRF, connexin 43 in the cell membrane, and myoinositol 1,4,5-triphosphate receptor in the perinuclear region. Functional cell-cell communication developed between WB F344 cells and adjacent cocultured cardiomyocytes in 24 h. De novo cytoplasmic [Ca(2+)](c) and nuclear [Ca(2+)](nu) oscillations appeared in WB F344 cells, synchronous with [Ca(2+)](i) transients in adjacent cardiomyocytes. The [Ca(2+)] oscillations in the WB F344 cells, but not those in the cardiomyocytes, were eliminated by a gap junction uncoupler and reappeared with its removal. By 24 h, WB F344 cells began expressing the cardiac transcription factors Nkx2.5, Tbx5, and cofactor myocardin; cardiac proteins 24 h later; and a sarcomeric pattern 4-6 days later. Myoinositol 1,4,5-triphosphate receptor inhibition suppressed WB F344 cell [Ca(2+)](nu) oscillations but not [Ca(2+)](c) oscillations, and L-type calcium channel inhibition eliminated [Ca(2+)] oscillations in cardiomyocytes and WB F344 cells. The use of these inhibitors was associated with a decrease in Nkx2.5, Tbx5, and myocardin expression in the WB F344 cells. Our findings suggest that signals from cardiomyocytes diffuse through shared channels, inducing [Ca(2+)] oscillations in the WB F344 cells. We hypothesize that the WB F344 cell [Ca(2+)](nu) oscillations activate the expression of a cardiac specifying gene program, ushering in a cardiac phenotype.

The use of TP10, soluble complement receptor 1, in cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) for cardiac surgery or lung transplantation initiates a systemic inflammatory response characterized by increased vascular permeability, generalized edema, abnormal lung function and oxygenation and impaired ventricular function. This post-CPB syndrome significantly contributes to postoperative morbidity and mortality. Activation of complement during CPB is a key component that initiates and augments this process. TP10, soluble complement receptor 1, is a novel complement inhibitor that is a potent inhibitor of C3 and C5 convertases, blocking activation of the complement cascade at the nexus of all three complement pathways. Recent controlled trials in humans have demonstrated that TP10 effectively inhibits complement activation during CPB. In high-risk adult patients, TP10 decreases the incidence of mortality and myocardial infarction in males but not in females following cardiac surgery. TP10 is also well tolerated and protects vascular function in infants undergoing CPB. In addition, TP10 leads to early extubation in adult lung transplant recipients. TP10 is currently positioned for clinical development in a male-only indication of cardiac surgery on CPB.

Acquired cell-to-cell coupling and "cardiac-like" calcium oscillations in adult stem cells in a cardiomyocyte microenvironment.

Adult-derived stem cells have recently been found to respond in vivo to inductive signals from the microenvironment and to differentiate into a phenotype that is characteristic of cells in that microenvironment. We examined the differentiation potential of an adult liver stem cell line (WBF344) in a cardiac microenvironment in vitro. WBF344 cells were established from a single cloned non-parenchymal epithelial cell isolated from a normal male adult rat liver. Genetically modified, WBF344 cells that express beta-galactosidase, green fluorescent protein (GFP) or mitochondrial red fluorescent protein (DsRed) were co-cultured with rat neonatal cardiac cells. After 4-14 days, we identified WBF344-derived cardiomyocytes that were elongated, binucleated and expressed the cardiac specific proteins cardiac troponin T, cardiac troponin I and N cadherin. These WBF344-derived cardiomyocytes also exhibited myofibrils, sarcomeres, and a nascent sarcoplasmic reticulum. Furthermore, rhythmically beating WBF344-derived cardiomyocytes displayed "cardiac-like" calcium transients similar to the surrounding neonatal cardiomyocytes. Fluorescent recovery after photobleaching demonstrated that WBF344-derived cardiomyocytes were electrically coupled with adjacent neonatal cardiomyocytes through gap junctions (GJs). Collectively, these results support the conclusion that these adult-derived liver stem cells respond to signals generated in a cardiac microenvironment in vitro acquiring a cardiomyocyte phenotype and function. The identification of micro-environmental signals that appear to cross germ layer and species specificities should prove valuable in understanding the regulation of normal development and stem cell differentiation in vivo.

Development and cardiac contractility: cardiac troponin T isoforms and cytosolic calcium in rabbit.

Cardiac contractility depends on calcium sensitivity of the myofilaments and cytosolic free calcium concentration ([Ca(2+)](i)) during activation. During development, the cardiac troponin T isoform cTnT(1) is replaced by shorter cTnT isoforms, including cTnT(4), and changes occur in other myofibrillar proteins and in calcium regulation. We expressed rabbit recombinant (r)cTnT(1) and rcTnT(4) in Spodoptera frugiperda cells and determined their effect on calcium binding to TnC in solution and on the calcium sensitivity of myofilaments in skinned rabbit ventricular fibers in vitro. We measured [Ca(2+)](i) and L-type calcium current (I(Ca)) in ventricular myocytes from 3-wk-old and adult rabbits. The dissociation constant (K(d)) of Ca-Tn(cTnT1) in solution was smaller than that of Ca-Tn(cTnT4) (mean +/- SE: 0.52 +/- 0.08 mumol/L versus 0.83 +/- 0.09 mumol/L). The Ca(2+) sensitivity of force development was greater in fibers reconstituted with rcTnT(1) (pCa(50) 6.07 +/- 0.04) than those reconstituted with rcTnT(4) (pCa(50) 5.75 +/- 0.07). Systolic [Ca](i) was lower in 3-wk-old than adult cells (443 +/- 35 nmol/L versus 882 +/- 88 nmol/L) as was I(Ca) (5.8 +/- 0.9 pA/pF versus 14.2 +/- 1.6 pA/pF). The higher calcium sensitivity of Tn-Ca binding and of force development conferred by rcTnT(1) suggest that higher neonatal cTnT(1) expression may partially compensate for the lower systolic [Ca(2+)](i).

cTnT1, a cardiac troponin T isoform, decreases myofilament tension and affects the left ventricular pressure waveform.

Four isoforms of cardiac troponin T (cTnT), a protein essential for calcium-dependent myocardial force development, are expressed in the human; they differ in charge and length. Their expression is regulated developmentally and is affected by disease states. Human cTnT (hcTnT) isoform effects have been examined in reconstituted myofilaments. In this study, we evaluated the modulatory effects of overexpressing one cTnT isoform on in vitro and in vivo myocardial function. A hcTnT isoform, hcTnT(1), expressed during development and in heart disease but not in the normal adult heart, was expressed in transgenic (TG) mice (1-30% of total cTnT). Maximal active tension measured in skinned myocardium decreased as a function of relative hcTnT(1) expression. The pCa at half-maximal force development, Hill coefficient, and rate of redevelopment of force did not change significantly with hcTnT(1) expression. In vivo maximum rates of rise and fall of left ventricular pressure decreased, and the half-time of isovolumic relaxation increased, with hcTnT(1) expression. Substituting total cTnT charge for hcTnT(1) expression resulted in similar conclusions. Morphometric analysis and electron microscopy revealed no differences between wild-type (non-TG) and TG myocardium. No differences in isoform expression of tropomyosin, myosin heavy chain, essential and regulatory myosin light chains (MLC), TnI, or in posttranslational modifications of mouse cTnT, cTnI, or regulatory MLC were observed. These results support the hypothesis that cTnT isoform amino-terminal differences affect myofilament function and suggest that hcTnT(1) expression levels present during human development and in human heart disease can affect in vivo ventricular function.

Activation of the meiotic divisions in Drosophila oocytes.

Key meiotic events in many organisms are controlled at the translational level. In this study, we examine the role of translational regulation in the meiotic cell cycle of Drosophila. In order to address this question, we developed a system for activating Drosophila oocytes in vitro. With this method, hundreds of mature oocytes can be activated to resume and complete meiosis. The stages of meiosis are normal by cytological criteria, and the timing of the meiotic divisions is similar to that of eggs activated in vivo. We use this system to examine the role of protein synthesis in regulating the progression of meiosis and the maintenance of the metaphase I arrest. We find that synthesis of new proteins after metaphase I is not required for anaphase I, meiosis II, or the decondensation of the meiotic products. Also, continued protein synthesis is not required to maintain the metaphase I arrest. New protein synthesis is required, however, for proper chromatin recondensation after meiosis.

Properties and localization of DNA methyltransferase in preimplantation mouse embryos: implications for genomic imprinting.

Preimplantation mouse embryos contain very high levels of DNA methyltransferase activity. We show here that the form of DNA methyltransferase (DNA MTase) in early embryos differs from the form found in other cells and tissues by a slightly higher mobility on gel electrophoresis. Levels of DNA MTase were found to be very high throughout preimplantation development even though levels of 5-methylcytosine (m5C) in nuclear DNA are known to undergo a substantial decline in the same period. Confocal laser scanning microscopy of mouse embryos stained with DNA MTase-specific antibodies showed striking developmentally regulated changes in the distribution of DNA MTase. From the oocyte stage to the four-cell-stage, most DNA MTase was concentrated in peripheral cytoplasm, and nuclei did not contain detectable DNA MTase. In four- and eight-cell embryos, DNA MTase was seen in cytoplasmic granules; and in eight-cell embryos, DNA MTase was also present in large amounts in nuclei. Nuclei of blastocysts stained only faintly, whereas the cytoplasmic granules remained prominent. Paradoxically, DNA MTase was found to be at its highest levels in nuclei at a developmental stage where levels of m5C in DNA are decreasing most rapidly. Changes in methylation patterns in preimplantation embryos are therefore proposed to be under the control of unidentified regulatory factors rather than DNA MTase itself; these regulatory factors could be members of the group that contains the products of the Ssm-1 and Imp-1 genes, which are involved in the regulation of genomic imprinting.

The Drosophila genes grauzone and cortex are necessary for proper female meiosis.

In Drosophila, normal female meiosis arrests at metaphase I. After meiotic arrest is released by egg activation, the two meiotic divisions are rapidly completed, even in unfertilized eggs. Since little is known about the regulation of the meiotic cell cycle after the meiotic arrest, we screened for mutants that arrest in meiosis. Here we describe the phenotype of eggs laid by sterile mothers mutant for either grauzone or cortex. These eggs arrest in metaphase of meiosis II, and although they can enter into an aberrant anaphase II, they never exit meiosis. Prolonged sister-chromatid cohesion is not the cause of this arrest, since a premature release of sister cohesion does not rescue the meiotic arrest of cortex eggs. Aberrant chromosome segregation at meiosis I was the earliest observable defect, suggesting that grauzone and cortex are first required immediately after egg activation. The cortical microtubules are also defective, remaining in a pre-activated state in activated mutant eggs. The mutations had no observable effect on either male meiosis or mitosis. We believe these genes will provide insight into the developmental regulation of meiosis in a genetically tractable organism.

A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei.

Tissue-specific patterns of methylated deoxycytidine residues in the mammalian genome are preserved by postreplicative methylation of newly synthesized DNA. DNA methyltransferase (MTase) is here shown to associate with replication foci during S phase but to display a diffuse nucleoplasmic distribution in non-S phase cells. Analysis of DNA MTase-beta-galactosidase fusion proteins has shown that association with replication foci is mediated by a novel targeting sequence located near the N-terminus of DNA MTase. This sequence has the properties expected of a targeting sequence in that it is not required for enzymatic activity, prevents proper targeting when deleted, and, when fused to beta-galactosidase, causes the fusion protein to associate with replication foci in a cell cycle-dependent manner.