Developing neocortical progenitors express transcription factors in gradients that induce programs of region-specific gene expression. Our previous work identified anteriorly upregulated expression gradients of a number of corticofugal neuron-associated gene probe sets along the anterior–posterior axis of the human neocortex (8-12 postconceptional weeks [PCW]). Here, we demonstrate by real-time polymerase chain reaction, in situ hybridization and immunohistochemistry that 3 such genes, ROBO1, SRGAP1, and CTIP2 are highly expressed anteriorly between 8-12 PCW, in comparison with other genes (FEZF2, SOX5) expressed by Layer V, VI, and subplate neurons. All 3 were prominently expressed by early postmitotic neurons in the subventricular zone, intermediate zone, and cortical plate (CP) from 8 to 10 PCW. Between 12 and 15 PCW expression patterns for ER81 and SATB2 (Layer V), TBR1 (Layer V/VI) and NURR1 (Layer VI) revealed Layer V forming. By 15 PCW, ROBO1 and SRGAP1 expression was confined to Layer V, whereas CTIP2 was expressed throughout the CP anteriorly. We observed ROBO1 and SRGAP1 immunoreactivity in medullary corticospinal axons from 11 PCW onward. Thus, we propose that the coexpression of these 3 markers in the anterior neocortex may mark the early location of the human motor cortex, including its corticospinal projection neurons, allowing further study of their early differentiation.
cerebral cortex; corticospinal tract; regionalization
We have employed immunohistochemistry for multiple markers to investigate the structure and possible function of the different compartments of human cerebral wall from the formation of cortical plate at 8 postconceptional weeks (PCW) to the arrival of thalamocortical afferents at 17 PCW. New observations include the subplate emerging as a discrete differentiated layer by 10 PCW, characterized by synaptophysin and vesicular gamma-aminobutyric acid transporter expression also seen in the marginal zone, suggesting that these compartments may maintain a spontaneously active synaptic network even before the arrival of thalamocortical afferents. The subplate expanded from 13 to 17 PCW, becoming the largest compartment and differentiated further, with NPY neurons located in the outer subplate and KCC2 neurons in the inner subplate. Glutamate decarboxylase and calretinin-positive inhibitory neurons migrated tangentially and radially from 11.5 PCW, appearing in larger numbers toward the rostral pole. The proliferative zones, marked by Ki67 expression, developed a complicated structure by 12.5 PCW reflected in transcription factor expression patterns, including TBR2 confined to the inner subventricular and outer ventricular zones and TBR1 weakly expressed in the subventricular zone (SVZ). PAX6 was extensively expressed in the proliferative zones such that the human outer SVZ contained a large reservoir of PAX6-positive potential progenitor cells.
cell migration; cortical development; immunohistochemistry; synaptogenesis
In a previous study, Staphylococcus aureus purified cell walls (PCW), consisting of peptidoglycan (PG) plus covalently linked teichoic acid (TA), were found to be more active in complement consumption than isolated PG. Isolated TA has now been shown to be capable of activating complement. Mild sonication markedly increased the ability of PG to activate complement but had essentially no effect on the activities of PCW and TA. Optimal sonication of PG did not yield activities equal to those of PCW in dose-response and kinetic studies, which may imply that TA plays some role in complement consumption. Sonication did not lead to solubilization of PCW or PG but may have enhanced the activity of PG in complement consumption by better dispersing PG particles, thereby exposing more surface area. Lysostaphin solubilization of PCW and PG markedly decreased their activities in complement consumption. The PCW of an S. aureus TA-deficient mutant, which were mostly PG, caused similar amounts of complement consumption as the parent strain PCW. Of the treatments of PCW commonly used to isolate PG, formamide and periodate extractions in particular led to PG preparations with lower activities in complement consumption than the PCW from which they were prepared, although these activities were stimulated by sonication. When whole organisms were studied by using a TA-deficient mutant, a mutant with an additional cell surface polymer, and the TA-containing parent strains and complement consumption by these strains was compared, no difference was found in either the rate or the degree of complement activation. This led to experiments demonstrating that both material released extracellularly from staphylococci and the cytoplasmic fraction of S. aureus were active in complement consumption. The results of these experiments indicate that both physical and chemical factors must be considered in studies of complement activation by isolated bacterial cell wall components. Under certain conditions, staphylococcal TA may enhance complement activation, but studies with whole organisms clearly show that this cell wall constituent does not play an essential role in this process. In addition, studies of complement consumption with intact organisms have demonstrated that there may be contributions both from cell surface components and from material released by the cells.
The conjugative tetracycline resistance plasmid pCW3 is the paradigm conjugative plasmid in the anaerobic gram-positive pathogen Clostridium perfringens. Two closely related FtsK/SpoIIIE homologs, TcpA and TcpB, are encoded on pCW3, which is significant since FtsK domains are found in coupling proteins of gram-negative conjugation systems. To develop an understanding of the mechanism of conjugative transfer in C. perfringens, we determined the role of these proteins in the conjugation process. Mutation and complementation analysis was used to show that the tcpA gene was essential for the conjugative transfer of pCW3 and that the tcpB gene was not required for transfer. Furthermore, complementation of a pCW3ΔtcpA mutant with divergent tcpA homologs provided experimental evidence that all of the known conjugative plasmids from C. perfringens use a similar transfer mechanism. Functional genetic analysis of the TcpA protein established the essential role in conjugative transfer of its Walker A and Walker B ATP-binding motifs and its FtsK-like RAAG motif. It is postulated that TcpA is the essential DNA translocase or coupling protein encoded by pCW3 and as such represents a key component of the unique conjugation process in C. perfringens.
The abilities of intact Staphylococcus aureus H, crude cell walls (CCW), purified cell walls (PCW, peptidoglycan [PG] and covalently linked teichoic acid), peptidoglycan, and cell membranes (CM) to activate the complement system in normal human serum, C2-deficient serum, and immunoglobulin-deficient serum were compared. On a weight basis, PCW was the most active fraction; intact organisms and CCW were about equally effective; and PG was least active in causing complement consumption in normal serum. CM also activated complement but did not give a clear dose-response relationship in the concentrations used. Kinetic studies revealed that C3-C9 consumption occurred at a significantly slower rate in C2-deficient serum, indicating that intact organisms, PCW, and PG may activate the complement system via the classical and alternative pathways in normal serum. C3-C9 consumption was also slower in immunoglobulin-deficient serum than in normal serum, implying that immunoglobulins play a role in attaining maximum rates of complement activation. In all sera studied, PG was less active in complement activation than PCW. These results indicate that a number of cell surface components of S. aureus can play a role in complement activation by this organism and that the presence of teichoic acid has a significant enhancing effect in this regard.
Mutations in PLA2G6, which encodes calcium-independent phospholipase A2 group VIA (iPLA2-VIA), underlie the autosomal recessive disorder infantile neuroaxonal dystrophy (INAD). INAD typically presents in the first year of life, and leads to optic atrophy and psychomotor regression. We have examined PLA2G6 expression in early human embryonic development by in situ hybridization. At Carnegie Stage (CS) 19 (approximately 7 post conception weeks [PCW]), strong expression is evident in the ventricular zone (VZ) of midbrain and forebrain suggestive of expression in neural stem and progenitor cells. At CS23 (8 PCW) expression is also detectable in the VZ of the hindbrain and the subventricular zone (SVZ) of the developing neocortex, ganglionic eminences and diencephalon. By 9 PCW strong expression in the post-mitotic cells of the cortical plate can be seen in the developing neocortex. In the eye, expression is seen in the lens and retina at all stages examined. PLA2G6 expression is also evident in the alar plate of the spinal cord, dorsal root ganglia, the retina and lens in the eye and and several non-neuronal tissues, including developing bones, lung, kidney and gut. These findings suggest a role for PLA2G6 in neuronal proliferation throughout the developing brain and in maturing neurons in the cortical plate and hindbrain. Although widespread PLA2G6 expression is detected in neuronal tissues, the pattern shows dynamic changes with time and indicates that INAD pathogenesis may begin prior to birth.
PLA2G6; INAD; neurodegeneration; development; in-situ hybridization
Growth of methicillin-resistant Staphylococcus aureus DU4916 in the presence of methicillin yielded crude cell walls that showed an increased rate of autolysis and purified cell walls (PCW) and peptidoglycan (PG) that had increased susceptibilities to autolysin extracted with LiCl and to lysozyme. The PG of cells grown in the presence of methicillin had markedly decreased cross-linking and O acetylation. Growth of the methicillin-susceptible strain H in the presence of subinhibitory concentrations of cefoxitin, a specific inhibitor of penicillin-binding protein (PBP) 4, caused a substantial decrease in PG cross-linking and O acetylation and increased susceptibilities of PCW and PG to LiCl-extracted autolysin and to lysozyme. Strain DU4916 cells grown in the presence of methicillin did not show an increased rate of autolysis or an increased susceptibility to vancomycin- or D-cycloserine-induced lysis, even though their PG was hypo-cross-linked. This implies that the potential for increased autolysis is controlled in intact cells and that this regulation may be involved in the methicillin resistance phenomenon. Growth of the methicillin-susceptible strain DU4916S in the presence of methicillin yielded PCW and PG that showed small increases in susceptibilities to LiCl-extracted autolysin and to lysozyme and a small decrease in PG cross-linking. Comparison of the PBPs of a penicillinase-nonproducing derivative of strain DU4916 (DU4916-K7) with those of strain DU4916S in intact cells and isolated membranes revealed that PBPs 1 to 4 had similar high beta-lactam antibiotic affinities in both strains and identified an additional PBP, PBP2(1), with low beta-lactam affinity in the methicillin-resistant strain DU4916-K7. The low degree of cross-linking of PG in strain DU4916 cells grown with methicillin was probably due mainly to inhibition of the secondary cross-linking function of PBP 4.
The inflammatory response in bacterial meningitis is mediated by cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1), which are produced in the subarachnoid space by different cells, e.g., leukocytes, astrocytes, and microglia. The recruitment of leukocytes into the cerebrospinal fluid (CSF) has been shown to contribute to the neurological damage in this disease, a process which could be enhanced by treatment with antibiotics. In this study, we have used a rabbit meningitis model for two sets of experiments with intracisternal (i.c.) injections of Streptococcus pneumoniae. First, pneumococcal cell wall (PCW) components were injected i.c., inducing an inflammatory response with pleocytosis and increased levels of CSF TNF-α) and IL-1 at 6 and 12 h after PCW injection. Treatment with fucoidin, known to inhibit leukocyte rolling, abolished pleocytosis and inhibited the release of TNF-α and IL-1. In the second experiment, live pneumococcal bacteria were injected i.c. and treatment with one dose of ampicillin (40 mg/kg of body weight intravenously) was given 16 h after induction of meningitis, causing a sevenfold increase in CSF leukocytes over a 4-h period. CSF IL-1 levels at 16 h were high but did not increase further at 20 h. Also, CSF TNF-α levels were high at 16 h and tended to increase at 20 h. Fucoidin treatment prevented the antibiotic-induced increase of CSF leukocytes but had no effect on the TNF-α and IL-1 levels. Taken together, fucoidin reduced CSF TNF-α and IL-1 levels in acute bacterial meningitis induced by PCW fragments but had no effect later in the course of the disease, when live bacteria were used and an inflammatory increase was caused by a dose of antibiotics.
Woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) are closely similar with respect to genomic organization, host antiviral responses, and pathobiology of the infection. T-cell immunity against viral nucleocapsid (HBcAg or WHcAg) has been shown to play a critical role in viral clearance and protection against infection. Here we show that vaccination of healthy woodchucks by gene gun bombardment with a plasmid coding for WHcAg (pCw) stimulates proliferation of WHcAg-specific T cells but that these cells do not produce significant levels of gamma interferon (IFN-γ) upon antigen stimulation. In addition, animals vaccinated with pCw alone were not protected against WHV inoculation. In order to induce a Th1 cytokine response, another group of woodchucks was immunized with pCw together with another plasmid coding for woodchuck interleukin-12 (IL-12). These animals exhibited WHcAg-specific T-cell proliferation with high IFN-γ production and were protected against challenge with WHV, showing no viremia or low-level transient viremia after WHV inoculation. In conclusion, gene gun immunization with WHV core generates a non-Th1 type of response which does not protect against experimental infection. However, steering the immune response to a Th1 cytokine profile by IL-12 coadministration achieves protective immunity. These data demonstrate a crucial role of Th1 responses in the control of hepadnavirus replication and suggest new approaches to inducing protection against HBV infection.
The transcription factors Emx2 and Pax6 are expressed in the proliferating zones of the developing rodent neocortex, and gradients of expression interact in specifying caudal and rostral identities. Pax6 is also involved in corticoneurogenesis, being expressed by radial glial progenitors that give rise to cells that also sequentially express Tbr2, NeuroD and Tbr1, genes temporally downstream of Pax6. In this study, using in situ hybridization, we analysed the expression of EMX2, PAX6, TBR2, NEUROD and TBR1 mRNA in the developing human cortex between 8 and 12 postconceptional weeks (PCW). EMX2 mRNA was expressed in the ventricular (VZ) and subventricular zones (SVZ), but also in the cortical plate, unlike in the rodent. However, gradients of expression were similar to that of the rodent at all ages studied. PAX6 mRNA expression was limited to the VZ and SVZ. At 8 PCW, PAX6 was highly expressed rostrally but less so caudally, as has been seen in the rodent, however this gradient disappeared early in corticogenesis, by 9 PCW. There was less restricted compartment-specific expression of TBR2, NEUROD and TBR1 mRNA than in the rodent, where the gradients of expression were similar to that of PAX6 prior to 9 PCW. The gradient disappeared for TBR2 by 10 PCW, and for NEUROD and TBR1 by 12 PCW. These data support recent reports that EMX2 but not PAX6 is more directly involved in arealization, highlighting that analysis of human development allows better spatio-temporal resolution than studies in rodents.
arealization; development; neurogenesis; subventricular zone
Microarray technology makes it possible to identify changes in gene expression of an organism, under various conditions. Data mining is thus essential for deducing significant biological information such as the identification of new biological mechanisms or putative drug targets. While many algorithms and software have been developed for analysing gene expression, the extraction of relevant information from experimental data is still a substantial challenge, requiring significant time and skill.
MADIBA (MicroArray Data Interface for Biological Annotation) facilitates the assignment of biological meaning to gene expression clusters by automating the post-processing stage. A relational database has been designed to store the data from gene to pathway for Plasmodium, rice and Arabidopsis. Tools within the web interface allow rapid analyses for the identification of the Gene Ontology terms relevant to each cluster; visualising the metabolic pathways where the genes are implicated, their genomic localisations, putative common transcriptional regulatory elements in the upstream sequences, and an analysis specific to the organism being studied.
MADIBA is an integrated, online tool that will assist researchers in interpreting their results and understand the meaning of the co-expression of a cluster of genes. Functionality of MADIBA was validated by analysing a number of gene clusters from several published experiments – expression profiling of the Plasmodium life cycle, and salt stress treatments of Arabidopsis and rice. In most of the cases, the same conclusions found by the authors were quickly and easily obtained after analysing the gene clusters with MADIBA.
Although excystation is crucial to the initiation of infection by Giardia lamblia, little is known about the regulation of this important process. We have been able to reliably induce excystation in vitro by mimicking cyst passage through the stomach and upper small intestine by the exposure of in vitro-derived cysts to an acidic, reducing environment (stage I) followed by protease treatment at a slightly alkaline pH (stage II). Preexposure of cysts to polyclonal rabbit antiserum against purified cyst walls (PCWs) or to wheat germ agglutinin (WGA) inhibited excystation by > 90%. Adsorption of either ligand with PCWs eliminated inhibition, demonstrating specificity for cyst wall epitopes. Inhibition by WGA was reversed by either chitotriose or sialic acid, while inhibition by polyclonal antibodies against PCWs (anti-PCW) was reversed only by sialic acid, which also inhibited binding of both ligands to intact cysts and to cyst wall antigens in immunoblots. Binding of anti-PCW did not affect acidification of cyst cytoplasm during stage I. Exposure of cysts to anti-PCW and WGA prior to, but not after, stage II was sufficient to inhibit excystation, and inhibition could be partially reversed by increasing the protease concentration during stage II. A 7- to 10-fold higher proportion of WGA- and anti-PCW-treated cysts than control cysts remained intact after stage II. Our results suggest that these ligands, which bind cyst wall epitopes, inhibit excystation, most likely by interfering with proteolysis of cyst wall glycoproteins during stage II.
The aim of the study is to describe the work pattern of personal care workers (PCWs) in nursing homes. This knowledge is important for staff performance appraisal, task allocation and scheduling. It will also support funding allocation based on activities.
A time-motion study was conducted in 2010 at two Australian nursing homes. The observation at Site 1 was between the hours of 7:00 and 14:00 or 15:00 for 14 days. One PCW was observed on each day. The observation at Site 2 was from 10:00 to 17:00 for 16 days. One PCW working on a morning shift and another one working on an afternoon shift were observed on each day. Fifty-eight work activities done by PCWs were grouped into eight categories. Activity time, frequency, duration and the switch between two consecutive activities were used as measurements to describe the work pattern.
Personal care workers spent about 70.0% of their time on four types of activities consistently at both sites: direct care (30.7%), indirect care (17.6%), infection control (6.4%) and staff break (15.2%). Oral communication was the most frequently observed activity. It could occur independently or concurrently with other activities. At Site 2, PCWs spent significantly more time than their counterparts at Site 1 on oral communication (Site 1: 47.3% vs. Site 2: 63.5%, P = 0.003), transit (Site 1: 3.4% vs. Site 2: 5.5%, P < 0.001) and others (Site 1: 0.5% vs. Site 2: 1.8%, P < 0.001). They spent less time on documentation (Site 1: 4.1% vs. Site 2: 2.3%, P < 0.001). More than two-thirds of the observed activities had a very short duration (1 minute or less). Personal care workers frequently switched within or between oral communication, direct and indirect care activities.
At both nursing homes, direct care, indirect care, infection control and staff break occupied the major part of a PCW’s work, however oral communication was the most time consuming activity. Personal care workers frequently switched between activities, suggesting that looking after the elderly in nursing homes is a busy and demanding job.
Four chloramphenicol resistance (Cm) and four tetracycline resistance (Tc) plasmids from Staphylococcus aureus were characterized by restriction endonuclease mapping. All four Tc plasmids had molecular masses of 2.9 megadaltons (Mdaltons) and indistinguishable responses to seven different restriction endonucleases. The four Cm plasmids (pCW6, pCW7, pCW8, and pC221) had molecular masses of 2.6, 2.8, 1.9, and 2.9 Mdaltons, respectively. The four Cm plasmids also differed both in the level of resistance to Cm and in susceptibility to retriction endonucleases. Single restriction endonuclease sites contained within each plasmid included the following: in pCW6 for HindIII, XbaI, HpaII, and BstEII; in pCW7 for HindIII, BstEII, BglII, HaeIII, and HpaII; in pCW8 for HindIII, HaeIII, and HpaII; in pC221 for HindIII, BstEII, and EcoRI. The molecular cloning capabilities of pCW8 and pC221 were determined. Cm and erythromycin resistance (Em) recombinant plasmids pCW12, PCW13, and pCW14 were constructed and used to transform S. aureus 8325-4. A 2.8-Mdalton HindIII fragment from plasmid pI258 was found to encode Em resistance and contain single sites for the retriction endonucleases BglII, PstI, HaeIII, and HpaII. The largest EcoRI fragment (8 Mdaltons) from pI258 contained the HindIII fragment encoding Em resistance intact. Cloning of DNA into the BglII site of pCW14 did not alter Em resistance. Cloning of DNA into the HindIII site of pCW8 and the HindIII and EcoRI sites of pC221 did not disrupt either plasmid replication of Cm resistance.
Canal wall down and canal wall up mastoidectomy represent two surgical approaches to middle ear cleft pathology. Very few studies have examined the effects of these procedures both on the patients' well being and on the resources needed to maintain that state. In this study the authors report the outpatient attendance pattern of canal wall down mastoidectomy patients
This is a retrospective case-note review of 101 patients who underwent a CWD mastoidectomy at Derriford Hospital, Plymouth, UK. All surgery was performed by the senior author (PCW-T) between 1985 and 1997. The main outcome measures were the frequency of outpatients' visits, clinical problems at visit and the percentage of discharged patients.
The studied patients made a total of 1341 outpatient visits between November 1985 and December 1998 with an average of 13.3 visits per patient (median of 11 visits). Almost two thirds of the group still attend for regular follow up. The greatest number of visits occurred in the first 24 months after surgery. The commonest reasons for outpatient visits were the removal of the clinical features of chronic cavity inflammation. Residual/recurrent cholesteatoma, residual perforations and structural cavity problems were infrequent.
CWD mastoidectomy carries an intrinsic morbidity resulting in a long term attendance in the outpatients.
The diversity of gene cassette promoters in class 1 integrons was investigated in 47 strains isolated from wastewaters. The weak PcW and PcH1 variants predominated, suggesting that, similar to clinical environments, high rates of gene cassette recombination, rather than high expression of gene cassettes, have been preferentially selected in wastewaters.
Macrophages and granulocytes seem to play a key role in the pathogenesis of bacterial meningitis. Transforming growth factor beta (TGF-beta) leads to macrophage deactivation, as well as to inhibition of cytokine production and of endothelial granulocyte adhesion. We have investigated the influence of TGF-beta on regional cerebral blood flow (rCBF), intracranial pressure (ICP), and brain edema formation during the early phase of experimental meningitis. Rats which were inoculated intracisternally with live pneumococci or with pneumococcal cell wall hydrolyzed by the M1 muramidase (PCW-M) developed an increase of rCBF and ICP within 4 h postintracisternal challenge. A single intraperitoneal injection of TGF-beta 2 but not of TGF-beta 2 vehicle- control prevented the changes of rCBF. Furthermore, TGF-beta 2 significantly reduced the increase of ICP in rats inoculated with PCW- M. Likewise, the elevation of brain water content after intracisternal injection of pneumococci or PCW-M was blocked by pretreatment of rats with TGF-beta 2. TGF-beta 1 exhibited similar inhibitory effects in PCW- M-injected rats. The beneficial effects of TGF-beta 2 on the initial phase after pneumococcal inoculation seem to be tumor necrosis factor alpha- (TNF-alpha) independent since (a) intracisternal or intraperitoneal injection of neutralizing anti-TNF-alpha antibodies did not significantly influence rCBF, ICP, and brain water content in PCW-M- induced meningitis; and (b) TNF-alpha was only occasionally detected at low levels in cerebrospinal fluid at 4 h after PCW-M application.
Analyses of gene expression data from microarray experiments has become a central tool for identifying co-regulated, functional gene modules. A crucial aspect of such analysis is the integration of data from different experiments and different laboratories. How to weigh the contribution of different experiments is an important point influencing the final outcomes. We have developed a novel method for this integration, and applied it to genome-wide data from multiple Arabidopsis microarray experiments performed under a variety of experimental conditions. The goal of this study is to identify functional globally co-regulated gene modules in the Arabidopsis genome.
Following the analysis of 21,000 Arabidopsis genes in 43 datasets and about 2 × 108 gene pairs, we identified a globally co-expressed gene network. We found clusters of globally co-expressed Arabidopsis genes that are enriched for known Gene Ontology annotations. Two types of modules were identified in the regulatory network that differed in their sensitivity to the node-scoring parameter; we further showed these two pertain to general and specialized modules. Some of these modules were further investigated using the Genevestigator compendium of microarray experiments. Analyses of smaller subsets of data lead to the identification of condition-specific modules.
Our method for identification of gene clusters allows the integration of diverse microarray experiments from many sources. The analysis reveals that part of the Arabidopsis transcriptome is globally co-expressed, and can be further divided into known as well as novel functional gene modules. Our methodology is general enough to apply to any set of microarray experiments, using any scoring function.
The Tet P determinant from the conjugative Clostridium perfringens R plasmid pCW3 two functional overlapping tetracycline resistance genes, tetA(P) and tetB(P). The tetA(P) gene encodes a putative 46-kDa transmembrane protein which mediates active efflux of tetracycline from the cell, while tetB(P) encodes a putative 72.6-kDa protein which has significant similarity to Tet M-like tetracycline resistance proteins (J. Sloan, L.M. McMurry, D. Lyras, S. B. Levy, and J. I. Rood, Mol. Microbiol. 11:403-415, 1994). In the present study, hybridization and PCR analysis of 81 tetracycline-resistant isolates of C. perfringens showed that they all carried the tetA(P) gene. Most of these isolates (93%) carried a second tetracycline resistance gene, with 53% carrying tetB(P) and 40% carrying a tet(M)-like gene. Despite the wide distribution of the tetB(P) and tet(M) genes, no isolate which carried both of these determinants was detected. In isolates that carried both tetA(P) and tetB(P) these genes overlapped, as in pCW3. Isolates carrying this combination of genes originated from diverse geographical locations and environmental sources. The single Clostridium paraputrificum isolate examined carried tetA(P), indicating that this gene is not confined to C.perfringens. However, neither tetA(P) nor tetB(P) was detected in the nine Clostridium difficile isolates tested. Nucleotide sequence analysis of isolates lacking tetB(P) revealed that they contained the tetA408(P) gene, which lacked the codons for the 12 carboxy-terminal amino acids of the TetA(P) protein.
The nephropathogenic Escherichia coli strain P673 was shown to harbor two plasmids with molecular sizes of 70 and 41 megadaltons, respectively. The 70-megadalton plasmid, pCW1, coded for tetracycline resistance, whereas hemolysin production was coded by the 41-megadalton plasmid, pCW2. Plasmid pCW1 proved to be self-transmissible, in contrast to pCW2. Transfer of the hemolysin character was associated with the appearance of a 110-megadalton plasmid, pCW3. The incompatibility of pCW3 with both native plasmids and restriction enzyme analysis led to the conclusion that pCW3 is a cointegrate of pCW1 and pCW2, pCW2, carrying the hemolytic determinant, is involved in the nephropathogenic character of strain P673, because (i) elimination of pCW2 from P673 was associated with a loss of virulence and (ii) the nephropathogenicity of the avirulent mutant could be restored by reintroduction of pCW2 DNA as part of a cointegrate structure.
Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes’ genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.
Nitrogen (N) is an essential macronutrient for plant growth and development. Plants adapt to changes in N availability partly by changes in global gene expression. We integrated publicly available root microarray data under contrasting nitrate conditions to identify new genes and functions important for adaptive nitrate responses in Arabidopsis thaliana roots. Overall, more than 2000 genes exhibited changes in expression in response to nitrate treatments in Arabidopsis thaliana root organs. Global regulation of gene expression by nitrate depends largely on the experimental context. However, despite significant differences from experiment to experiment in the identity of regulated genes, there is a robust nitrate response of specific biological functions. Integrative gene network analysis uncovered relationships between nitrate-responsive genes and 11 highly co-expressed gene clusters (modules). Four of these gene network modules have robust nitrate responsive functions such as transport, signaling, and metabolism. Network analysis hypothesized G2-like transcription factors are key regulatory factors controlling transport and signaling functions. Our meta-analysis highlights the role of biological processes not studied before in the context of the nitrate response such as root hair development and provides testable hypothesis to advance our understanding of nitrate responses in plants.
meta-analysis; root hairs; nitrate; systems biology; gene co-expression analysis; transcription factors; Gene Ontology (GO)
Current experimental evidence indicates that functionally related genes show coordinated expression in order to perform their cellular functions. In this way, the cell transcriptional machinery can respond optimally to internal or external stimuli. This provides a research opportunity to identify and study co-expressed gene modules whose transcription is controlled by shared gene regulatory networks.
We developed and integrated a set of computational methods of differential gene expression analysis, gene clustering, gene network inference, gene function prediction, and DNA motif identification to automatically identify differentially co-expressed gene modules, reconstruct their regulatory networks, and validate their correctness. We tested the methods using microarray data derived from soybean cells grown under various stress conditions. Our methods were able to identify 42 coherent gene modules within which average gene expression correlation coefficients are greater than 0.8 and reconstruct their putative regulatory networks. A total of 32 modules and their regulatory networks were further validated by the coherence of predicted gene functions and the consistency of putative transcription factor binding motifs. Approximately half of the 32 modules were partially supported by the literature, which demonstrates that the bioinformatic methods used can help elucidate the molecular responses of soybean cells upon various environmental stresses.
The bioinformatics methods and genome-wide data sources for gene expression, clustering, regulation, and function analysis were integrated seamlessly into one modular protocol to systematically analyze and infer modules and networks from only differential expression genes in soybean cells grown under stress conditions. Our approach appears to effectively reduce the complexity of the problem, and is sufficiently robust and accurate to generate a rather complete and detailed view of putative soybean gene transcription logic potentially underlying the responses to the various environmental challenges. The same automated method can also be applied to reconstruct differentially co-expressed gene modules and their regulatory networks from gene expression data of any other transcriptome.
Gene co-expression module; Gene regulatory network; Transcription factor; Microarray; Soybean
Conjugative plasmids encode antibiotic resistance determinants or toxin genes in the anaerobic pathogen Clostridium perfringens. The paradigm conjugative plasmid in this bacterium is pCW3, a 47-kb tetracycline resistance plasmid that encodes the unique tcp transfer locus. The tcp locus consists of 11 genes, intP and tcpA-tcpJ, at least three of which, tcpA, tcpF, and tcpH, are essential for the conjugative transfer of pCW3. In this study we examined protein-protein interactions involving TcpA, the putative coupling protein. Use of a bacterial two-hybrid system identified interactions between TcpA and TcpC, TcpG, and TcpH. This analysis also demonstrated TcpA, TcpC, and TcpG self-interactions, which were confirmed by chemical cross-linking studies. Examination of a series of deletion and site-directed derivatives of TcpA identified the domains and motifs required for these interactions. Based on these results, we have constructed a model for this unique conjugative transfer apparatus.
Plant growth promotion by rhizobacteria is a known phenomenon but the underlying mechanisms are poorly understood. We searched for plant growth-promoting rhizobacteria that are naturally associated with Arabidopsis thaliana to investigate the molecular mechanisms that are involved in plant growth-promotion. We isolated a Pseudomonas bacterium (Pseudomonas sp. G62) from roots of field-grown Arabidopsis plants that has not been described previously and analyzed its effect on plant growth, gene expression and the level of sugars and amino acids in the host plant. Inoculation with Pseudomonas sp. G62 promoted plant growth under various growth conditions. Microarray analysis revealed rapid changes in transcript levels of genes annotated to energy-, sugar- and cell wall metabolism in plants 6 h after root inoculation with P. sp. G62. The expression of several of these genes remained stable over weeks, but appeared differentially regulated in roots and shoots. The global gene expression profile observed after inoculation with P. sp. G62 showed a striking resemblance with previously described carbohydrate starvation experiments, although plants were not depleted from soluble sugars, and even showed a slight increase of the sucrose level in roots 5 weeks after inoculation. We suggest that the starvation-like transcriptional phenotype - while steady state sucrose levels are not reduced - is induced by a yet unknown signal from the bacterium that simulates sugar starvation. We discuss the potential effects of the sugar starvation signal on plant growth promotion.