Homology Polymerase Chain Reaction Research Articles

Quorum sensing LuxS/autoinducer-2 inhibits Enterococcus faecalis biofilm formation ability.

Objective:To investigate the relation between biofilm formation ability and quorum sensing gene LuxS/AI-2.Materials and Methods: Enterococcus faecalis (E. faecalis) standard strain ATCC 29212 was used in the study. Long flanking homology polymerase chain reaction method was used to build the LuxS gene knockout strain. Sequential culture turbidity measurement and CFU counting were used to assess the proliferation ability of E. faecalis after the depletion of LuxS. 96-well plate assay was used to quantify the biofilm formation ability; CLSM was used to observe the attached bacteria areas, while scanning electron microscopy (SEM) was performed to observe biofilm microstructure conditions.Results:LuxS gene knockout strains were successfully constructed and identified. The results showed that proliferation ability of E. faecalis was not affected by the depletion of the luxS gene, and the biofilm formation ability of ΔLuxS 29212 significantly decreased (P<0.05).Conclusions:Collectively, our studies provide the LuxS gene's key role in controlling biofilm formation of E. faecalis, which presented a negative regulation, and furthermore, providing us a possible way to conquer the persistent apical periodontitis.

Substitution of Nitrite Reductase of Thermosynechococcus elongatus BP-1 by the Homologous Gene of Phormidium laminosum

Even though the nitrate assimilation operon has been extensively studied in Phormidium laminosum, some aspects still remain unclear. The genetic manipulation of this cyanobacterium is problematic that hinders the elucidation of further aspects of nitrogen metabolism. To circumvent this, Thermosynechococcus elongatus BP-1 was selected as a surrogate host and its nirA gene was substituted by the homologous gene of P. laminosum. This process, based on Long Flanking Homology Polymerase Chain Reaction and the natural competence of T. elongatus BP-1, required an intermediate T. elongatus BP-1 ΔnirA::kat mutant, which carries a gene encoding a thermostable kanamycin nucleotidyl transferase in place of nirA_Te. In the presence of nirA_Pl, nirA defective mutants of T. elongatus BP-1 recovered the ability to grow with nitrate as the sole nitrogen source, and showed a phenotype similar to that observed in wild-type cells. The procedure could be useful to substitute other genes from T. elongatus BP-1 with the homologues from P. laminosum in order to study this particular operon. Furthermore, it may be used as a general tool to explore phenotypic changes due to the exchange of a single gene between cyanobacteria.

Construction of ply gene-deletion mutant of Streptococcus pneumoniae and research of its virulence change

Objective To lay the foundation for further exploration on parasitifer's defence reaction to pneumolysin through constructing ply gene-deletion strain of Streptococcus pneumoniae and researching on its virulence change. Methods A linker fragment with erm gene in middle and homologous upstream and downstream fragment of ply gene at both sides was prepared by long flanking homology-polymerase chain reaction(LFH-PCR). The linker fragment was transformed into Streptococcus pneumoniae. ply-deficient strain was then screened out from blood plate which contains erythromycin and identified by PCR. ply-deficient strain growth in vitro was observed and virulence change was observed through infecting mouse model. Results PCR results showed that ply gene was replaced completely by erm gene. The ply deficient strain was successfully constructed. The growth of single strain culture medium showed that ply genetic defect made no influence on bacterial's external growth. While in the mice nasal cavity infecting experiment, deficient strain enter into blood after 6 h from infecting which obviously slower than that did wild-type(2 h). And the number of bacteria at each point was much smaller than that of wild-type(P ＜0. 01 ). The mice peritonaeum infecting experiment showed that median lethal time of wild-type was 3 d, while that of deficient strain was 18 d(P＜0. 01). Conclusion It is a good way to completely substitute ply gene using LFH-PCR. ply deletion made no influence on baterial's growth in vitro, but it resulting in reduction of bacterial virulence in vivo. Key words: Streptococcus pneumoniae; Pneumolysin; LFH-PCR; Virulence

CHARACTERIZATION OF SUBTILASE PROTEASE IN CRYPTOSPORIDIUM PARVUM AND C. HOMINIS

Cryptosporidium spp., enteropathogens of humans and other animals, are members of the Apicomplexa. In parasites belonging to this phylum, proteases have been shown to play a key role in the invasion of host cells, organelle biogenesis, and intracellular survival. The subtilases constitute a family of serine proteases present in prokaryotes, eukaryotes, and viruses. The C. parvum subtilase gene, CpSUB1, encodes a transcript of 3972 base pairs (bp) and 1324 amino acids. Using homologous polymerase chain reaction primers, a similar gene, ChSUB1, which has 98% (4007 bp/4050 bp) identity to CpSUB1, was found in C. hominis. The alignment of the CpSUB1 and ChSUB1 nucleotide sequences identified primarily silent substitutions, consistent with the absence of diversifying selection. The catalytic domain of CpSUB1 is very similar to that of other Apicomplexa (> 38% amino acid identity and >57% similarity) and to the bacterial subtilisin BPN from B. subtilis (36 and 47%). Transcriptional upregulation during merozoite development was observed in cell culture, and a predicted 76-bp intron located near the 3' end of the open reading frame was confirmed experimentally. Cryptosporidium parvum infection in cell culture was significantly inhibited by subtilisin inhibitor III and other serine protease inhibitors, emphasizing the importance of the parasite's subtilase for intracellular development and the enzyme's potential as a drug target.

Multiplex Polymerase Chain Reaction Method for Detection of Bovine Materials in Foodstuffs

Rapid identification of bovine materials in animal foodstuffs is essential for effective control of a potential source of bovine spongiform encephalophathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a bovine-specific genomic DNA sequence in foodstuffs. Simultaneously the assay assessed the DNA quality of the experiment system by amplification of a highly conserved eucaryotic DNA region of the 18-S ribosomal gene, helping to check the reliability of the test result. The amplified bovine-specific PCR product was a genomic DNA fragment of lactoferrin, a low copy gene that was different from a commonly used bovine-specific mitochondria sequence for identification of bovine materials. The specificity of this method was confirmed by the absence of detectable homologous PCR product using reference foodstuff samples that lacked bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method could detect the presence of bovine material in foodstuffs when the samples contained > 0.02% bovine-derived meat and bone meal. Furthermore, it was not affected by prolonged heat treatment. The specificity, convenience, and sensitivity of this method suggest that it can be used for the routine detection of bovine-derived materials.

FAT is a component of glomerular slit diaphragms

Slit diaphragms are intercellular junctions of podocytes of the renal glomerulus. The molecular composition of slit diaphragms is still elusive. Slit diaphragms are characterized by the presence of a wide intercellular space. The morphological feature is shared by desmosomes and adherens junctions, which contain members of the cadherin superfamily. Thus, we have hypothesized that some components of slit diaphragms belong to the cadherin superfamily. Consequently, we have isolated cDNA encoding FAT from reverse-transcribed (RT) glomerular cDNA by homology polymerase chain reaction (PCR) using primers based on conserved sequences in cadherin molecules. FAT is a novel member of the cadherin superfamily with 34 tandem cadherin-like extracellular repeats, and it closely resembles the Drosophila tumor suppressor fat. Expression of FAT was examined in glomeruli of the adult rat kidney by the ribonuclease protection assay and in situ hybridization. To localize the FAT protein in podocytes minutely, we prepared affinity-purified antibody against FAT by immunizing rabbits against an oligopeptide corresponding to the C-terminal 20 amino acids. Expression of FAT mRNA was detected in total RNA from glomeruli. In situ hybridization revealed significant signals in podocytes. Western blot analysis using solubilized glomeruli showed a single band, in which the molecular weight was more than 500 kD. Immunostaining of cultured epithelial cells from rat kidney (NRK52E) revealed FAT accumulation in cell-cell contact sites. In the glomerulus, FAT staining was observed distinctly along glomerular capillary walls. Double-label immunostaining using monoclonal antibody against slit diaphragms (mAb 5-1-6) showed identical localization of anti-FAT antibody and mAb 5-1-6. Furthermore, the double-label immunogold technique with ultrathin cryosections demonstrated that gold particles for FAT cytoplasmic domain were located at the base of slit diaphragms labeled by mAb 5-1-6 and that the cytoplasmic domain of FAT colocalized with ZO-1, a cytoplasmic component associated with slit diaphragms. The molecular structure of FAT and its colocalization with 5-1-6 antigen and ZO-1 indicate that FAT is a component of slit diaphragms.

The αBβC Integrin Is Expressed on the Surface of the Sea Urchin Egg and Removed at Fertilization

Integrins are expressed on the surface of some vertebrate eggs where they are thought to have a role in fertilization. The objective of this study is to determine if integrins are expressed on sea urchin eggs. The αB and βC subunits were cloned using the homology polymerase chain reaction. Monoclonal and polyclonal antibodies were developed against bacterially expressed fragments of the extracellular domains of the βC subunit and the αB subunit. As well, a monoclonal antibody was developed against a synthesized peptide corresponding to part of the cytoplasmic domain of βC. Analysis of biotinylated egg cortex extracts immunoprecipitated with either anti-βC or anti-αB yields bands of 130 and 225 kDa. Immunoblots confirm that βC is part of the complex immunoprecipitated with anti-αB. Confocal immunofluorescence and immunogold electron microscopy show that βC is present on the surface of the unfertilized egg at the tips of microvilli and in cortical granules. During the cortical reaction, immunoreactivity with antibodies to the extracellular domains of βC and αB disappears from the egg surface, and microvillar casts on the fertilization envelope become immunoreactive. With antibodies to the cytoplasmic domain of βC, immunoreactivity is lost from the surface of the egg, but the fertilization envelope does not immediately become immunoreactive. In immunoblots of egg cortex there are immunoreactive bands of the predicted sizes for αB and βC. However, in fertilization envelopes, a second band that is slightly lower in molecular weight is also present. Eggs fertilized in the presence of soybean trypsin inhibitor have elongated microvilli that remain bound to the elevating fertilization envelope and immunoreactive to anti-βC antibodies. Eggs fertilized in the presence of an ovoperoxidase inhibitor, 3-amino-1,2,4-triazole, have a patchy distribution of βC immunoreactivity in fertilization envelopes. Together, these data suggest that αBβC integrins are expressed on the surface of unfertilized eggs and, during the cortical reaction, the extracellular domains are cleaved by proteases and cross-linked into the fertilization envelope by ovoperoxidase. The αBβC integrin receptors may have several potential functions prior to their removal at fertilization, including attachment of the vitelline envelope to the egg surface and anchoring the cortical cytoskeleton.

Detection of Bovine Mitochondrial DNA in Ruminant Feeds: A Molecular Approach to Test for the Presence of Bovine-Derived Materials

A ban on ruminant-derived proteins in ruminant feeds has been introduced as a preventive measure to avoid the spread of bovine spongiform encephalopathy (BSE), as well as to minimize any potential risk of BSE transmission from bovines to humans. In the absence of commercially available efficient methods for identification of bovine-derived proteins in animal feeds, we developed a rapid and sensitive polymerase chain reaction (PCR)-based assay which allows detection and identification of a bovine-specific mitochondrial DNA sequence from feedstuffs. The amplified product encodes for the whole ATPase subunit 8 and the amino-terminal portion of the ATPase subunit 6 proteins, which are known to exhibit a relatively low degree of conservation among vertebrates. The specific amplification of such a bovine mitochondrial sequence from reference feedstuff samples was demonstrated by means of both direct sequencing and single-strand conformational analysis of the PCR product. Specificity was also confirmed by the absence of detectable homologous PCR product when using reference feedstuff samples lacking bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method allows detection of the presence of bovine mitochondrial DNA in feedstuffs containing less than 0.125% of bovine-derived meat and bonemeals. Furthermore, it does not appear to be considerably affected by prolonged heat treatment. DpnII and SspI restriction endonuclease digestions of the unpurified PCR product may be used routinely to confirm the bovine origin of the amplified sequence. Since this method is specific, rapid, and sensitive, it could be successfully utilized as a routine control assay to evaluate the presence of bovine-derived meat and bonemeals in ruminant feeds.

Identification of pathogenic yeasts of the imperfect genus Candida by polymerase chain reaction fingerprinting.

With the increase in the number of immunocompromised hosts, the number of fungal pathogens has increased markedly. Identification and classification, especially of yeast species and strains, is often difficult when based solely on phenotypic characteristics. Since it became clear that different fungal pathogens require specific treatment strategies, there is a need for simple, rapid and reliable methods to identify fungal isolates. Polymerase chain reaction (PCR) fingerprinting was successfully applied here to identify yeast isolates. Microsatellite [(GTG)5; (GACA)4] and minisatellite [(5'GAGGGTGGCGGTTCT 3'), derived from the core-sequence of the phage M13] specific primers were used as single primers in the PCR to amplify hypervariable interrepeat DNA sequences from over 200 European, American and Australian clinical isolates within the genus Candida. Each species, represented by its type strain, could be identified by a specific multilocus pattern, allowing for the assignment of all the isolates to the appropriate species. Intra-species variation in the multilocus profiles was about 20% compared to inter-species variation, which was up to 80%. Anamorph-teleomorph pairs could be identified by highly homologous PCR fingerprint patterns. PCR fingerprinting was more discriminatory when compared with routinely used biochemical tests (Vitek YBC and API ID 32C). PCR fingerprinting has proven to be a powerful tool for the identification of medically important yeasts. It is rapid, sensitive, reliable, highly reproducible, stable in vitro and in vivo, and applicable to large scale experiments. Potential applications include: yeast taxonomy, epidemiology, environmental surveys, and improvement of the diagnosis of mycotic diseases.

Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis

Liver fibrosis results from a relative imbalance between synthesis and degradation of matrix proteins. We have previously described release of the protein collagenase inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), by culture-activated human hepatic stellate cells (HSCs). In this study, we have investigated the relative expression of TIMP-1 and interstitial collagenase in culture-activated rat HSCs and rat models of liver injury and fibrosis. The complementary DNA (cDNA) for rat TIMP-1 was obtained by homology polymerase chain reaction (PCR) and sequenced. By Northern analysis using this probe, TIMP-1 messenger RNA (mRNA) expression was up-regulated with HSC activation by culture on plastic as defined by cellular expression of procollagen-1. Interstitial collagenase mRNA was expressed in early 1. Interstitial collagenase mRNA was expressed in early culture (<4 days) but became undetectable in more activated cells (7-21 days). By activity assay of serum-free cell-conditioned media, TIMP-1 was found to be released in increasingly concentrations with duration of culture on plastic. Expression of TIMP-1 interstitial collagenase, and procollagen-1 mRNAs were studied in rat models of biliary and parenchymal injury (bile duct ligation and CC14 administration) by ribonuclease protein assay. TIMP-1 mRNA expression was increased at 6, 24 hours, and 3 days after bile duct ligation and was also shown to rise in acute CC14 liver injury and remain elevated as the liver became fibrotic. TIMP-1 expression preceded procollagen-1 expression in both models. In contrasts, interstitial collagenase mRNA levels remained similar to control values throughout both models of liver injury. Total cellular RNA from hepatocytes, HSCs, and kupffer cells freshly isolated from livers after acute CC14 injury was subjected to Northern analysis. TIMP-1 transcripts were observed in nonparenchymal cells only. We suggest that increased expression of TIMP-1 relative to interstitial collagenase by HSCs may promote progression of liver fibrosis in these rat models by preventing degradation of secreted collagens. (Hepatology 1996 Jul;24(1):176-84)

Novel Integrin α and β Subunit cDNAs Identified in Airway Epithelial Cells and Lung Leukocytes Using the Polymerase Chain Reaction

The integrins are a large group of cell surface glycoproteins that mediate cell-matrix and cell-cell adhesive interactions. Integrins play a role in normal lung development, in host defense against pulmonary infection, and in the pathogenesis of the adult respiratory distress syndrome. Integrins are heterodimers consisting of one alpha subunit and one beta subunit. We identified consensus sequences within integrin subunits and used oligonucleotide primers based on these sequences to amplify cDNA by the polymerase chain reaction (PCR). We previously reported the use of this homology PCR technique for the identification of one novel integrin beta subunit, beta 6, from guinea pig airway epithelial cells. Here we demonstrate that primers based on alpha subunit consensus sequences can also be used for homology PCR. We have used the alpha and beta subunit primers to amplify and clone a large variety of integrin partial cDNAs from several cell types and species. Comparison of the deduced amino acid sequences reveals a high degree of cross-species conservation (86 to 98% identity). One alpha subunit (identified in guinea pig airway epithelial cells) and one beta subunit (identified in rabbit leukocytes obtained by bronchoalveolar lavage and in human and mouse leukocyte cell lines) have novel sequences that are related to but clearly distinct from all previously reported integrin sequences (24 to 61% identity). These novel cDNAs are very likely to encode previously unsequenced integrin subunit proteins that are expressed in the lung. Homology PCR is a powerful technique for the identification of known and novel integrin alpha and beta subunit cDNAs in cells from the lung and other organs.