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Biomedical subjects

W J Meijer

Publications and source records attributed to W J Meijer.

At least 19 recordsLinked to original sources
PubMed2005

Judging relative positions across saccades.

When components of a shape are presented asynchronously during smooth pursuit, the retinal image determines the perceived shape, as if the parts belong to the moving object that the eyes are pursuing. Saccades normally shift our gaze between structures of interest, so there is no reason to expect anything to have moved with the eyes. We therefore decided to examine how people judge the separation between a target flashed before and another flashed after a saccade. Subjects tracked a jumping dot with their eyes. Targets were flashed at predetermined retinal positions, with a 67-242 ms interval between the flashes. After each trial subjects indicated where they had seen the targets. We selected the trials on which subjects made a complete saccade between the presentations of the two targets. For short inter-target intervals, subjects' judgements depended almost exclusively on the retinal separation, even when there were conspicuous visual references nearby. Even for the longest intervals, only part of the change in eye orientation was taken into consideration. These findings cannot simply be accounted for on the basis of the mislocalisation of individual targets or a compression of space near saccades. We conclude that the retinal separation determines the perceived separation between targets presented with a short interval between them, irrespective of any intervening eye movements.

Discrimination, Psychological↗
PubMed2001

Structural and functional analysis of a bipolar replication terminus. Implications for the origin of polarity of fork arrest.

We have delineated the amino acid to nucleotide contacts made by two interacting dimers of the replication terminator protein (RTP) of Bacillus subtilis with a novel naturally occurring bipolar replication terminus by converting RTP to a site-directed chemical nuclease and mapping its cleavage sites on the terminus. The data show a relatively symmetrical arrangement of the amino acid to base contacts, and a comparison of the bipolar contacts with that of a normal unipolar terminus suggests that the DNA-protein contacts play an important determinative role in generating polarity from structurally symmetrical RTP dimers. The amino acid to nucleotide contacts provided distance constraints that enabled us to build a three-dimensional model of the protein-DNA complex. The model is consistent with features of the bipolar Ter.RTP complex derived from mutational and cross-linking data. The bipolar terminus arrested Escherichia coli DNA replication and DnaB helicase and T7 RNA polymerase in vitro in both orientations. RTP arrested the unwinding of duplex DNA on the bipolar Ter DNA substrate regardless of the length of the duplex DNA. The latter result suggested further that the terminus arrested authentic DNA unwinding by the helicase rather than just translocation of helicase on DNA.

Bacillus subtilis↗
PubMed2001

Characterization of the bacteriophage phi29-encoded protein p16.7: a membrane protein involved in phage DNA replication.

An early expressed operon, located at the right end of the linear bacteriophage phi29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in phi29-related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N-terminally located transmembrane-spanning domain is required for its membrane localization. The variant p16.7A, in which the N-terminal membrane anchor was replaced by a histidine-tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a phi29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivo phi29 DNA replication is discussed.

Amino Acid Sequence↗
PubMed2001

Analysis of early promoters of the Bacillus bacteriophage GA-1.

Bacteriophage GA-1, which infects Bacillus sp. strain G1R, is evolutionarily related to phage phi29, which infects Bacillus subtilis. We report the characterization of several GA-1 promoters located at either end of its linear genome. Some of them are unique for GA-1 and drive the expression of open reading frames that have no counterparts in the genome of phi29 or related phages. These unique promoters are active at early infection times and are repressed at late times. In vitro transcription reactions revealed that the purified GA-1-encoded protein p6 represses the activity of these promoters, although the amount of p6 required to repress transcription was different for each promoter. The level of protein p6 produced in vivo increases rapidly during the first stage of the infection cycle. The protein p6 concentration may serve to modulate the expression of these early promoters as infection proceeds.

Bacillus Phages↗
PubMed2001

Phi29 family of phages.

Continuous research spanning more than three decades has made the Bacillus bacteriophage phi29 a paradigm for several molecular mechanisms of general biological processes, such as DNA replication, regulation of transcription, phage morphogenesis, and phage DNA packaging. The genome of bacteriophage phi29 consists of a linear double-stranded DNA (dsDNA), which has a terminal protein (TP) covalently linked to its 5' ends. Initiation of DNA replication, carried out by a protein-primed mechanism, has been studied in detail and is considered to be a model system for the protein-primed DNA replication that is also used by most other linear genomes with a TP linked to their DNA ends, such as other phages, linear plasmids, and adenoviruses. In addition to a continuing progress in unraveling the initiation of DNA replication mechanism and the role of various proteins involved in this process, major advances have been made during the last few years, especially in our understanding of transcription regulation, the head-tail connector protein, and DNA packaging. Recent progress in all these topics is reviewed. In addition to phi29, the genomes of several other Bacillus phages consist of a linear dsDNA with a TP molecule attached to their 5' ends. These phi29-like phages can be divided into three groups. The first group includes, in addition to phi29, phages PZA, phi15, and BS32. The second group comprises B103, Nf, and M2Y, and the third group contains GA-1 as its sole member. Whereas the DNA sequences of the complete genomes of phi29 (group I) and B103 (group II) are known, only parts of the genome of GA-1 (group III) were sequenced. We have determined the complete DNA sequence of the GA-1 genome, which allowed analysis of differences and homologies between the three groups of phi29-like phages, which is included in this review.

Amino Acid Sequence↗
PubMed2000

The putative coiled coil domain of the phi 29 terminal protein is a major determinant involved in recognition of the origin of replication.

The linear double-stranded genome of phage phi29 contains a terminal protein (TP) covalently linked at each 5' DNA end, called parental TP. Initiation of phi29 DNA replication starts with the recognition of the origins of replication, constituted by the parental TP-containing DNA ends, by a heterodimer containing phi29 DNA polymerase and primer TP. It has been argued that origin recognition involves protein-protein interactions between parental and primer TP. Analysis of the TP sequence revealed that the region between amino acids 84 and 118 has a high probability to form an amphipatic alpha-helix that could be involved in the interaction between parental and primer TP. Therefore, this TP region may be important for origin recognition. To test this hypothesis we introduced various mutations in the predicted amphipatic alpha-helix and analyzed the functionality of the corresponding purified TP mutants. The results obtained show that the identified putative amphipatic alpha-helix of TP is an important determinant involved in origin recognition.

Amino Acid Sequence↗
PubMed2000

Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7.

We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.

Bacillus Phages↗
PubMed1999

Phage phi29 terminal protein residues Asn80 and Tyr82 are recognition elements of the replication origins.

Initiation of phage phi29 DNA replication starts with the recognition of the origin of replication, located at both ends of the linear DNA, by a heterodimer formed by the phi29 terminal protein (TP) and the phi29 DNA polymerase. The parental TP, covalently linked to the DNA ends, is one of the main components of the replication origin. Here we provide evidence that recognition of the origin is mediated through interactions between the TP of the TP/DNA polymerase heterodimer, called primer TP, and the parental TP. Based on amino acid sequence comparisons, various phi29 TP mutants were generated at conserved amino acid residues from positions 61 to 87. In vitro phi29 DNA amplification analysis revealed that residues Asn80 and Tyr82 are essential for functional interaction between primer and parental TP required for recognition of the origin of replication. Although these mutant TPs can form functional heterodimers with phi29 DNA polymerase that are able to recognize the origin of replication, these heterodimers are not able to recognize an origin containing a mutant TP.

Amino Acid Sequence↗
PubMed1999

The plasmid-encoded signal peptidase SipP can functionally replace the major signal peptidases SipS and SipT of Bacillus subtilis.

The gram-positive eubacterium Bacillus subtilis is the organism with the largest number of paralogous type I signal peptidases (SPases) known. These are specified both by chromosomal and plasmid-borne genes. The chromosomally encoded SPases SipS and SipT have a major function in precursor processing, and cells depleted of SipS and SipT stop growing and die. In this study, we show that the SPase SipP, specified by the B. subtilis plasmid pTA1015, can functionally replace SipS and SipT, unlike the three chromosomally encoded SPases with a minor function in protein secretion (i.e., SipU, SipV, and SipW). Unexpectedly, SipP is not specifically required for the processing and secretion of Orf1p, which is specified by a gene that is cotranscribed with sipP. These two genes form a conserved structural module of rolling-circle plasmids from B. subtilis. As previously shown for the chromosomal sipS and sipT genes, the transcription of plasmid-borne copies of sipP is temporally controlled, reaching maximal levels during the post-exponential growth phase when the cells secrete proteins at high levels. However, increased transcription of sipP starts at the end of exponential growth, about 2 h earlier than that of sipS and sipT. These data suggest that SipP fulfills a general role in the secretory precursor processing of pTA1015-containing cells.

Bacillus subtilis↗
PubMed1998

DNA polymerase template switching at specific sites on the phi29 genome causes the in vivo accumulation of subgenomic phi29 DNA molecules.

The accumulation of subgenomic phage phi29 DNA molecules with specific sizes was observed after prolonged infection times with delayed lysis phage mutants. Whereas the majority of the molecules had a size of 4 kb, additional DNA species were observed with sizes of 8.2, 6.5, 2.3, 2 and 1 kb. Most of the molecules were shown to originate from the right end of the linear Bacillus subtilis phage phi29 genome. The nature of the 4, 2.3, 2 and 1 kb molecules was studied. The 2 kb molecules were shown to be single-stranded self-complementary strands forming hairpin structures. The other molecules consisted of palindromic linear double-stranded DNA molecules. Most probably, the subgenomic DNA molecules were formed when the moving phage replication fork from the right origin encountered a block that induces the DNA polymerase to switch template. Once formed, the subgenomic molecules are then amplified in vivo. Determination of the centres of symmetry of the 4 and 1 kb molecules revealed that both contained the almost 16 bp perfect dyad symmetry element (DSE): 5'-TGTTtCAC-GTGg-AACA-3' being a likely candidate for a protein binding site. Database analysis showed that this sequence occurs four times in the phi29 genome. In addition, the almost identical sequence 5'-TgGTTTCAC-GTGGAAtCA-3' was found once. These five DSEs are all located in the right half of the phi29 genome, and the same sequences are also present in the linear DNA of related B. subtilis phages. Most interestingly, this sequence is also found in the spoOJ gene of the B. subtilis chromosome. Recently, it has been shown that the SpoOJ protein is associated in vivo with the same DSE. As the same subgenomic phi29 DNA molecules accumulate after infection of B. subtilis spoOJ deletion strains, it is likely that, in addition to and/or independently of SpoOJ, other protein(s) bind to DSE.

Bacillus Phages↗
PubMed1998

Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from gram-positive bacteria.

Most small plasmids of Gram-positive bacteria use the rolling-circle mechanism of replication and several of these have been studied in considerable detail at the DNA level and for the function of their genes. Although most of the common laboratory Bacillus subtilis 168 strains do not contain plasmids, several industrial strains and natural soil isolates do contain rolling-circle replicating (RCR) plasmids. So far, knowledge about these plasmids was mainly limited to: (i) a classification into seven groups, based on size and restriction patterns; and (ii) DNA sequences of the replication region of a limited number of them. To increase the knowledge, also with respect to other functions specified by these plasmids, we have determined the complete DNA sequence of four plasmids, representing different groups, and performed computer-assisted and experimental analyses on the possible function of their genes. The plasmids analyzed are pTA1015 (5.8 kbp), pTA1040 (7.8 kbp), pTA1050 (8.4 kbp), and pTA1060 (8.7 kbp). These plasmids have a structural organization similar to most other known RCR plasmids. They contain highly related replication functions, both for leading and lagging strand synthesis. pTA1015 and pTA1060 contain a mobilization gene enabling their conjugative transfer. Strikingly, in addition to the conserved replication modules, these plasmids contain unique module(s) with genes which are not present on known RCR plasmids of other Gram-positive bacteria. Examples are genes encoding a type I signal peptidase and genes encoding proteins belonging to the family of response regulator aspartate phosphatases. The latter are likely to be involved in the regulation of post-exponential phase processes. The presence of these modules on plasmids may reflect an adaptation to the special conditions to which the host cells were exposed.

Bacillus subtilis↗
PubMed1997

A comprehensive model of cooperation between caregivers related to quality of care.

BACKGROUND: The system and delivery of health care tend to suffer from fragmentation, resulting in discontinuous and costly care. Local cooperation between caregivers is essential to achieve appropriate, timely, continuous and efficient care. The article develops a general comprehensive patient-centered model of quality of care related to local cooperation between caregivers. The model can be used in quality improvement and research. THE PROPOSED MODEL: Within the framework of Donabedian's triad (structure-->process-->outcome), cooperation between caregivers is divided into two parts. Firstly, client related cooperation (multiple caregivers to one patient) is a part of the process of health care and relevant for important quality aspects: appropriateness, timeliness, continuity of care, effectiveness and efficiency. Secondly, local cooperation between caregivers, which is not restricted to one patient, is called non client related cooperation. Such non client related cooperation creates conditions for optimal patient care. Non client related cooperation has different degrees, dependent on the existence of agreements between the caregivers (such as protocols) and evaluation of these agreements. The highest degree of non client related cooperation is continuous quality improvement. To achieve a certain degree of non client related cooperation, conditions must be fulfilled in several fields (organization, information, financing and participants). APPLICATION OF THE MODEL: A "how to use the model in eight steps" is given and possible operationalizations of key concepts in the model are outlined. Finally, the use of the model is illustrated by two examples.

Caregivers↗
PubMed1996

Obstetric care: competition or co-operation.

OBJECTIVE: The aim of this study was to determine the feasibility of co-operation within maternity and obstetric care between midwives, general practitioners (GPs) and obstetricians. DESIGN: descriptive correlational study. SETTING: The Netherlands. Policy is towards more co-operation between primary and secondary health care. However, in Dutch health care midwives, GPs and obstetricians may also have conflicts of interests. PARTICIPANTS: members of obstetric co-operation groups (91 midwives, 53 GPs, 31 obstetricians) completed a questionnaire. MEASUREMENTS AND FINDINGS: in the questionnaire information was collected on what members of obstetric co-operative groups expect from co-operation. Findings indicated that consensus existed about experienced advantages and disadvantages of co-operation, tasks that should be achieved, and how obstetric co-operative groups could be set up. Nevertheless, there was evidence of competition and there were also conflicting ideas about co-operation. KEY CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: it is concluded that the findings have implications for the organisation of an obstetric co-operative group. If professionals want to start such a group it is preferable to start with topics that benefit all participants (win-win situation) and motivate them to participate actively. A second step may be an attempt to reach agreement about how to communicate with each other in the case of referrals and consultations. During this phase mutual trust and respect may grow, so that finally more difficult problems can be discussed (mixed-motives situation).

Attitude of Health Personnel↗
PubMed1996

Identification and characterization of a novel type of replication terminator with bidirectional activity on the Bacillus subtilis theta plasmid pLS20.

We have sequenced and analysed a 3.1 kb fragment of the 55 kb endogenous Bacillus subtilis plasmid pLS20 containing its replication functions. Just outside the region required for autonomous replication, a segment of 18 bp was identified as being almost identical to part of the major B. subtilis chromosomal replication terminator. Here, we demonstrate that this segment is part of a functional replication terminator. This newly identified element, designated TerLS20, is the first replication terminator identified on a theta plasmid from a Gram-positive bacterium. TerLS20 is distinct from other known replication terminators in the sense that it is functional in both orientations. The region required for bipolar functionality of TerLS20 was delineated to a sequence of 29 bp, which is characterized by an imperfect dyad symmetry.

Bacillus subtilis↗
PubMed1995

Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01.

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 bp region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR I) showed some homology with other previously described SSOs of the SSOA type, as well as with the conversion signal of the Escherichia coli phage phi X174. This IR was shown to have a partial, RNA polymerase-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerase-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).

Bacteriophage M13↗
PubMed1995

Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a novel type of replicon.

A 3.1 kb fragment of the large (approximately 55 kb) Bacillus subtilis plasmid pLS20 containing all the information for autonomous replication was cloned and sequenced. In contrast to the parental plasmid, derived minireplicons were unstably maintained. Using deletion analysis the fragment essential and sufficient for replication was delineated to 1.1 kb. This 1.1 kb fragment is located between two divergently transcribed genes, denoted orfA and orfB, neither of which is required for replication. orfA shows homology to the B.subtilis chromosomal genes rapA (spoOL, gsiA) and rapB (spoOP). The 1.1 kb fragment, which is characterized by the presence of several regions of dyad symmetry, contains no open reading frames of more than 85 codons and shows no similarity with other known plasmid replicons. The structural organization of the pLS20 minimal replicon is entirely different from that of typical rolling circle plasmids from Gram-positive bacteria. The pLS20 minireplicons replicate in polA5 and recA4 B.subtilis strains. Taken together, these results strongly suggest that pLS20 belongs to a new class of theta replicons.

Amino Acid Sequence↗
PubMed1995

Characterization of single strand origins of cryptic rolling-circle plasmids from Bacillus subtilis.

In this paper we describe the isolation and characterization of single strand origins (SSOs) of several cryptic Bacillus subtilis plasmids which use the rolling-circle mechanism of replication. The plasmids used in this study involved pTA1015, pTA1020, pTA1030, pTA1040, pTA1050 and pTA1060. The SSO of pTA1015 was isolated by shotgun cloning in a specially designed vector, pWM100, which has no SSO of its own. Sequence analysis revealed that the SSO of pTA1015 is almost identical to formerly described palT type SSOs. Also pTA1020 and pTA1060 were shown to contain SSOs highly homologous to palT. Using Southern hybridization with the palT of pTA1015 as a probe, the SSO of pTA1040 was cloned. Sequence analysis revealed a region of 200 bp which is 77% identical to the palT of pTA1015. The plasmids pTA1030 and pTA1050 contain an SSO which is highly homologous to the SSO of pTA1040. The majority of the SSOs of rolling-circle plasmids from B.subtilis seem to belong to two related families which we denote as palT1 (present on pTA1015, pTA1020 and pTA1060) and palT2 (present on pTA1030, pTA1040 and pTA1050). Both families of SSOs are highly efficient single-strand-conversion signals in B.subtilis.

Bacillus subtilis↗