Evolution of DNA replication origin specification and gene silencing mechanisms.

DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove. Using a massively parallel origin selection assay coupled with a custom mutual-information-based modeling approach, and a separate analysis of whole-genome replication profiling, here we show that the Orc4 α-helix contributes to the DNA sequence-specificity of origins in S. cerevisiae and Orc4 α-helix mutations change genome-wide origin firing patterns. The DNA sequence specificity of replication origins, mediated by the Orc4 α-helix, has co-evolved with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.

Mechanism of origin unwinding: sequential binding of DnaA to double- and single-stranded DNA.

The initiator protein DnaA of Escherichia coli binds to a 9mer consensus sequence, the DnaA box (5'-TT(A/T)TNCACA). If complexed with ATP it adopts a new binding specificity for a 6mer consensus sequence, the ATP-DnaA box (5'-AGatct). Using DNase footprinting and surface plasmon resonance we show that binding to ATP-DnaA boxes in the AT-rich region of oriC of E.coli requires binding to the 9mer DnaA box R1. Cooperative binding of ATP-DnaA to the AT-rich region results in its unwinding. ATP-DnaA subsequently binds to the single-stranded region, thereby stabilizing it. This demonstrates an additional binding specificity of DnaA protein to single-stranded ATP-DnaA boxes. Binding affinities, as judged by the DnaA concentrations required for site protection in footprinting, were approximately 1 nM for DnaA box R1, 400 nM for double-stranded ATP-DnaA boxes and 40 nM for single-stranded ATP-DnaA boxes, respectively. We propose that sequential recognition of high- and low-affinity sites, and binding to single-stranded origin DNA may be general properties of initiator proteins in initiation complexes.

Bacterial replication initiator DnaA. Rules for DnaA binding and roles of DnaA in origin unwinding and helicase loading.

We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.

Comparison of assays to detect cytomegalovirus shedding in the semen of HIV-infected men.

We sought to determine the optimal assays for cytomegalovirus (CMV) shedding in semen. Over a 2-month period, 149 HIV-1-infected men who have sex with men each provided up to three semen specimens. Specimens were tested for CMV by culture, rapid assay (shell vial) and polymerase chain reaction (PCR). By culture, 30% of seminal plasma and 28% of seminal cell specimens grew CMV. By rapid assay, results were 38 and 33%, respectively. By PCR, 56% of seminal cell specimens demonstrated CMV: 20% in a single semen specimen; 33% in two specimens; and 34% in all three specimens. Overall, 69% of men had CMV detected by PCR in at least one seminal cell specimen. By quantitative PCR, 14% had ten, 14% had 100, 16% had 1000, and 12% had 10000 copies in 6.25 microl of semen analyzed. Adjusting for initial CD4+ cell count, men with CMV shedding demonstrated by PCR at the first visit were approximately four times as likely to shed CMV at a subsequent visit (RR 4.28, CI: 2.30-7.95). CMV shedding was associated with decreased CD4+ cell counts in peripheral blood (P=0.05). It is concluded that the PCR assay provided the greatest sensitivity among the three detection methods.

The Escherichia coli SeqA protein binds specifically and co-operatively to two sites in hemimethylated and fully methylated oriC.

The Escherichia coli SeqA protein has been found to affect initiation of replication negatively, both in vivo and in vitro. The mechanism of inhibition is, however, not known. SeqA has been suggested to affect the formation and activity of the initiation complex at oriC, either by binding to DNA or by interacting with the DnaA protein. We have investigated the binding of SeqA to oriC by electron microscopy and found that SeqA binds specifically to two sites in oriC, one on each side of the DnaA binding site R1. Specific binding was found for fully and hemimethylated but not unmethylated oriC in good agreement with earlier mobility shift studies. The affinity of SeqA for hemi-methylated oriC was higher than for fully methylated oriC. The binding was in both cases strongly cooperative. We suggest that SeqA binds to two nucleation sites in oriC, and by the aid of protein-protein interaction spreads to adjacent regions in the same oriC as well as recruiting additional oriC molecules and/or complexes into larger aggregates.

Identification of the chromosomal replication origin from Thermus thermophilus and its interaction with the replication initiator DnaA.

The chromosomal replication origin oriC and the gene encoding the replication initiator protein DnaA from Thermus thermophilus have been identified and cloned into an Escherichia coli vector system. The replication origin is composed of 13 characteristically arranged DnaA boxes, binding sites for the DnaA protein, and an AT-rich stretch, followed by the dnaN gene. The dnaA gene is located upstream of the origin and expresses a typical DnaA protein that follows the division into four domains, as with other members of the DnaA protein family. Here, we report the purification of Thermus-DnaA (Tth-DnaA) and characterize the interaction of the purified protein with the replication origin, with regard to the binding kinetics and stoichiometry of this interaction. Using gel retardation assays, surface plasmon resonance (SPR) and electron microscopy, we show that, unlike the E. coli DnaA, Tth-DnaA does not recognize a single DnaA box, instead a cluster of three tandemly repeated DnaA boxes is the minimal requirement for specific binding. The highest binding affinities are observed with full-length oriC or six clustered, tandemly repeated DnaA boxes. Furthermore, high-affinity DNA-binding of Tth-DnaA is dependent on the presence of ATP. The Thermus DnaA/oriC interaction will be compared with oriC complex formation generated by other DnaA proteins.

ATP- and ADP-dnaA protein, a molecular switch in gene regulation.

DnaA protein functions by binding to asymmetric 9mer DNA sites, the DnaA boxes. ATP-DnaA and ADP-DnaA bind to 9mer DnaA boxes with equal affinity, but only ATP-DnaA protein binds in addition to an as yet unknown 6mer site, the ATP-DnaA box AGATCT, or a close match to it. ATP-DnaA protein binding to ATP-DnaA boxes is restricted to sites located in close proximity to DnaA boxes, suggesting that protein-protein interaction is required for its stabilization. We show that ATP-DnaA represses dnaA transcription much more efficiently than ADP-DnaA. DnaA is thus a regulatory molecule that, depending on the adenosine nucleotide bound, can bind to different sequences and thereby fulfill distinct functions.

Functional domains of DnaA proteins.

Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.

Evidence for distinct substrate specificities of importin alpha family members in nuclear protein import.

Importin alpha plays a pivotal role in the classical nuclear protein import pathway. Importin alpha shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin beta-dependent import pathway. In contrast to what is found for importin beta, several isoforms of importin alpha, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin alpha7. To analyze specific functions of the distinct importin alpha proteins, we recombinantly expressed and purified five human importin alpha's along with importin alpha from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin alpha proteins from humans or Xenopus bind their import receptor (importin beta) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin alpha proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin alpha3, whereas most of the other substrates were imported by all importin alpha proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin alpha proteins were revealed when two substrates were offered simultaneously.

Risk factors for HIV-1 shedding in semen.

Semen is the body fluid most commonly associated with sexual transmission of human immunodeficiency virus type-1 (HIV-1). Because the male genitourinary tract is distinct immunologically from blood, compartment-dependent factors may determine HIV-1 shedding in semen. To identify these factors, the authors obtained 411 semen and blood specimens from 149 men seen up to three times. Seminal plasma was assayed for HIV-1 RNA and semen was cocultured for HIV-1 and cytomegalovirus (CMV), which may up-regulate HIV-1 replication. The best multivariate model for predicting a positive semen HIV-1 coculture included two local urogenital factors, increased seminal polymorphonuclear cell count (odds ratio (OR) = 12.6 for each log10 increase/mL, 95% confidence interval (CI) 12.2, 134.5) and a positive CMV coculture (OR = 3.0, 95% CI 1.2, 7.7). The best multivariate model for predicting semen HIV-1 RNA included two systemic host factors, CD4+ cell counts <200/microliter (OR = 3.0, 95 percent CI 1.3, 6.9) and nucleoside antiretroviral therapy (monotherapy: OR = 0.5, 95% CI 0.3, 1.0; combination therapy: OR = 0.4, 95% CI 0.2, 0.9), and a positive CMV coculture (OR = 1.7, 95% CI 1.0, 3.0). Thus, both systemic and local genitourinary tract factors influence the risk of semen HIV-1 shedding. These findings suggest that measures of systemic virus burden alone may not predict semen infectivity reliably.

Interaction of the Escherichia coli DnaA protein with bacteriophage lambda DNA.

Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage lambda DNA demonstrated differential binding of DnaA along the whole lambda DNA. Interaction of DnaA with the lambda replication region (from the promoter pR to the origin of replication, orilambda) demonstrated a strong binding of DnaA to the region around the p(o) promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the lambda DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.

Modification of the clinical course of intestinal microsporidiosis in acquired immunodeficiency syndrome patients by immune status and anti-human immunodeficiency virus therapy.

The clinical course of 37 Enterocytozoon bieneusi-infected acquired immunodeficiency syndrome patients with diarrhea was studied. Parasite clearance was seen in 15 patients (40.5%). Clearance of E. bieneusi resulted in a 25-100% reduction in episodes of diarrhea, suggesting that microsporidia are true pathogens. Univariate and multivariate proportional hazards analyses revealed that peripheral blood CD4 cell counts > or = 100/mm3, the use of two or more antiretroviral medications, and use of a protease inhibitor were statistically associated with decreased time to clearance of E. bieneusi. Specific anti-microsporidial therapy (albendazole) was not associated with parasite eradication. Factors related to immunocompetence and human immunodeficiency virus suppression appeared to be important in the clearance of E. bieneusi.

Examination of the prevalence and seasonal variation of intestinal microsporidiosis in the stools of persons with chronic diarrhea and human immunodeficiency virus infection.

The epidemiology of human microsporidiosis is poorly understood and environmental factors affecting transmission of the organism have not been fully elucidated. Temporal variation in the prevalence of microsporidia in the stool of patients with human immunodeficiency virus (HIV) infection and diarrhea was studied to evaluate the role of water-borne transmission. From January 1993 to December 1996, 8,439 stools from HIV-infected individuals were examined for microsporidia spores in southern California. Yearly positivity rates were 8.8% in 1993, 9.7% in 1994, 6.6% in 1995, and 2.9% in 1996. An analysis for linear trend showed a statistically significant decrease in stool positivity rates over time (chi2 = 81.9, P = 0.001). No significant seasonal variation in the prevalence of microsporidiosis was seen over that time period. These results suggest the constant presence of microsporidia in the environment, rather than a seasonal association with recreational water use or seasonal contamination of the water supply, and a real decrease in yearly prevalence of microsporidia related diarrhea. Factors related to a progressive decrease in prevalence are subjects of future investigation.

Association between culturable human immunodeficiency virus type 1 (HIV-1) in semen and HIV-1 RNA levels in semen and blood: evidence for compartmentalization of HIV-1 between semen and blood.

Both qualitative and quantitative virologic measurements were compared between blood and genital compartments for 128 men infected with human immunodeficiency virus type 1 (HIV-1) to address several controversial issues concerning HIV-1 shedding in semen and to obtain further information about the distribution of virus between these two compartments. Evidence for viral compartmentalization was suggested by earlier studies that noted the poor correlation between blood and seminal virus load, phenotype, and genotype. Further support for this viral compartmentalization was based on the following observations between semen and blood: lack of association between culturability of virus in semen and viral RNA level in blood, discordant distribution of viral phenotypes, discordant viral RNA levels, a weak correlation between viral RNA level in semen and CD4 cell count in blood, differences in the biologic variability of viral RNA levels, and differences in the virus load response to antiretroviral therapy.

[Evaluation of the introductory course for nursing licensure. Faculty of Medical Sciences, February-June 1996]. / Valoración del curso introductorio de la licenciatura en enfermería. Facultad de ciencias médicas. Febrero-junio, 1996.

A cross sectional study of 20 nursing baccalaureates, 14 heads of department, 4 methodologists and 2 professors from the faculties of medical sciences of the country was conducted with the objective of assessing the efficiency of the introductory course for future nursing baccalaureates, and of suggesting new topics for its development. A formulary was applied by using the survey method. Primary data were taken and processed by a traditional statistical method. It was proved that 95% of the sample considered this course as important, whereas 85% expressed that students were little motivated, and 35% recommended to make it langer.

Absence of detectable human herpesvirus 8 in the semen of human immunodeficiency virus-infected men without Kaposi's sarcoma.

The prevalence of human herpesvirus 8 (HHV-8)/Kaposi's sarcoma (KS)-associated herpesvirus was investigated in the semen of 99 human immunodeficiency virus (HIV)-infected men (median CD4 cell count, 357/mm3) by use of a polymerase chain reaction (PCR) assay capable of detecting <10 copies of HHV-8 DNA. Of the subjects, 95 (96%) self-identified as men who have sex with men (MSM), and 3 had a history of clinical KS. Seminal cell specimens were negative for HHV-8 in 98 subjects. None of the 26 without KS (27.1% of 96 tested) who were seropositive for HHV-8 by IFA for latency-associated nuclear antigens had HHV-8 detected in their semen. The only subject with any evidence for seminal HHV-8 DNA was seropositive for HHV-8 and had active KS. HHV-8 was detected in 10 (10.4%) of 96 peripheral blood mononuclear cell specimens. The prevalence of HHV-8 DNA by PCR in semen of HIV-infected MSM without KS is low.

From footprint to toeprint: a close-up of the DnaA box, the binding site for the bacterial initiator protein DnaA.

The Escherichia coli DnaA protein binds as a monomer to the DnaA box, a 9 bp consensus sequence: 5'-TTA/TTNCACA. To assess the contribution of individual bases to protein binding we probed the DnaA-DnaA box complex with the uracil-DNA glycosylase (UDG) footprinting technique. (i) dU at the positions of T2, T4, T7' or T9' completely inhibits DnaA binding to the DnaA box. At these positions the methyl groups of the thymine residues are essential for successful DnaA binding, indicating protein contact with the major groove. Additionally they are positioned exactly on one side of the helix. (ii) dU at the position of T1 or at three T residues adjacent to the 9 bp core sequence of the DnaA box allows DnaA binding. These positions are protected from UDG digestion as revealed by the footprint assay. (iii) dU at the position of T3' on the complementary strand of teh box 5'-TTATCCACA was not protected from UDG digestion in DNA-DnaA complexes. Therefore, DnaA cannot contact the major groove at this position. In addition, a slight bend of the DnaA box towards UDG would help the enzyme to access this site.

History of depression as a risk factor for Alzheimer's disease.

Research regarding the possible association between Alzheimer's disease and a history of depression has been inconclusive. Using a case-control design, we assessed the strength of the association between reported history of depression and onset of Alzheimer's disease. We enrolled probable Alzheimer's disease cases (N = 294), who were ascertained and diagnosed by our Alzheimer's Disease Patient Registry, and randomly selected nondemented controls (N = 300) of similar age and gender from the same base population. The mean age (for cases) was 78.5 years. Informants provided data regarding history of depression. "Treated depression" was defined as depression for which a physician/psychologist consultation, medication, or hospitalization had occurred. Restricting treated depression to exclude primary loss or grief reactions, we found a modest association with Alzheimer's disease [odds ratio (OR) = 1.8; 95% confidence interval (CI) = 0.9-3.5] after adjusting for gender, age, education, and type of informant. When these data were stratified by depression onset year, we observed an odds ratio of 2.0 (95% CI = 0.9-4.6) for depression occurring more than 10 years before the onset of dementia symptoms, and an OR of 0.9 (95% CI = 0.2-3.0) for depression onset within 10 years of the onset of dementia symptoms. Thus, depressive episodes occurring well before dementia symptom onset appear to increase the risk of Alzheimer's disease.

Seminal shedding of human immunodeficiency virus type 1 and human cytomegalovirus: evidence for different immunologic controls.

Contact with semen of seropositive men is important for sexual transmission both human immunodeficiency virus (HIV) type 1 and human cytomegalovirus (CMV), but the factors that determine shedding of either virus in semen are poorly understood. HIV was cultured from 36 (17%) of 215 semen specimens from 56 seropositive men, and CMV was cultured from 42 (30%) of 139 specimens. In logistic regression models, the CD8+ cell count in peripheral blood was the best predictor of HIV shedding in semen. Shedding of HIV was more closely associated with concomitant shedding of CMV than with CD4+ cell count, and antiretroviral therapy had minimal influence on shedding of HIV. In contrast, CD4+ cell count was the best predictor of CMV shedding in semen. Factors that determine shedding of viruses in semen may differ substantially from those that influence virus levels in the systemic immune compartment. Likewise, immunologic factors that determine shedding of HIV appear to differ from those that control shedding of CMV in semen.