Recombinant plasmids capable of integration into the chromosome of the cyanobacterium Anacystis nidulans R2

Recombinant plasmids, series pIAB and pIAH, have been constructed by insertion of BamHI or HindIII chromosomal fragments from Anacystis nidulans R2 into the tet gene of plasmid pACYC184. Plasmids pIAB and pIAH are stably maintained in Escherichia coli cells and transfer the CmR marker in transformation of Anacystis nidulans. Blot hybridization technique has shown the formation of CmR clones in transformation to result from integration of plasmid pACYC184 with the chromosome of cyanobacterium.     

Cloning the alpha-amylase gene of Bacillus amyloliquefaciens in cyanobacteria cells

The recombinant plasmids of pIAH4amy series were constructed containing the alpha-amylase gene of Bacillus amyloliquefaciens A50 with its own promoter and leading sequence within an integrative vector plasmid pIAH4 (CmR) for cyanobacterium Anacystis nidulans R2. At Anacystis nidulans transformation the hybrid plasmids integrate into cyanobacterium chromosome with high efficiency and all CmR transformants produce alpha-amylase. Expression of bacillar alpha-amylase gene in cyanobacterium cells is independent of the cloned gene orientation in the vector plasmid. Secretion of alpha-amylase into the cyanobacterial periplasm has been demonstrated.     

Transgenic mice carrying the human poliovirus receptor: new animal models for study of poliovirus neurovirulence.

Recombinant viruses between the virulent Mahoney and attenuated Sabin 1 strains of poliovirus type 1 were subjected to neurovirulence tests using a transgenic (Tg) mouse line, ICR-PVRTg1, that carried the human poliovirus receptor gene. The Tg mice were inoculated intracerebrally with these recombinant viruses and observed for clinical signs, histopathological lesions, and viral antigens as parameters of neurovirulence of the viruses. These parameters observed in the Tg mice were different for different inoculated viruses. Dose-dependent incidences of paralysis and of death were observed in the Tg mice inoculated with any viruses used. This indicates that values of 50% lethal dose are useful to score a wide range of neurovirulence of poliovirus. The neurovirulence of individual viruses estimated by the Tg mouse model had a strong correlation with those estimated by monkey model. Consequently, the mouse tests identified the neurovirulence determinants on the genome of poliovirus that had been identified by monkey tests. In addition, the mouse tests revealed new neurovirulence determinants, that is, different nucleotides between the two strains at positions 189 and 21 and/or 935 in the 5'-proximal 1,122 nucleotides. The Tg mice used in this study may be suitable for replacing monkeys for investigating poliovirus neurovirulence.     

Construction of a mobilizable cloning vector for site-directed mutagenesis of gram-negative bacteria: application to Rhizobium leguminosarum

A mobilizable cloning vector was constructed from defined fragments to serve as a suicide plasmid for site-directed mutagenesis. The new vector, pKOK4, closely resembles plasmid pBR325. However, the inverted duplication existing in the latter was not introduced. The useful cloning sites of pBR325 (EcoRI, HindIII, EcoRV, BamHI, SalI, PstI and PvuI) were retained and are located in one of the three resistance markers, ApR, CmR or TcR, respectively. Also, in pKOK4 the CmR gene retains its own promoter. The mob site of plasmid RP4 was introduced as a 760-bp fragment at a defined location. The mobilization frequency of pKOK4 within Escherichia coli strains is approx. 4 x 10(-2) per recipient cell. The size of pKOK4, deduced from the construction, is 6368 bp. We used pKOK4 for site-directed mutagenesis of hup-specific DNA from Rhizobium leguminosarum B10. Integration of the vector could be distinguished reliably from marker exchange by screening for the antibiotic resistance(s) of the plasmid. This reduced the number of clones to be retested by colony and Southern hybridization to approx. 1% of the original number. Of these, almost 70% contained the desired marker exchange.     

An efficient method of constructing homologous recom binant baculovirus with PCR-amplified fragments

Baculovirus is a rod-shaped virus containing a large circular dsDNA genome with the size of 80—180 kb[1]. Baculoviruses have been used as insecticides for biological control of forest and agricultural pests[2]. In addition, baculovirus is of great interest as it can be used as efficient eukaryotic expression vector[3], surface display vector[4], and gene therapy vector[5]. Till April 2002, the complete genome sequences of 13 baculoviruses have been reported. The functional genomics has now…     

Identification of a thyroiditogenic sequence within the thyroglobulin molecule.

Thyroglobulin (Tg)-specific T cells are important in the induction of experimental autoimmune thyroiditis (EAT), but the nature and the number of the Tg T cell epitopes involved in the disease process are unknown. Through the use of computerized algorithms that search for putative T cell epitopes, a 17-mer peptide (TgP1) was identified within the known portion of the rat Tg sequence (corresponding to amino acids 2495 to 2511 of the human Tg sequence) that induced strong mononuclear cell infiltration of the thyroid in classic EAT-susceptible murine strains such as SJL, C3H, and B10.BR and low or undetectable infiltration in EAT-resistant strains such as BALB/c and B10. TgP1 appears to be phylogenetically conserved since it is completely homologous to its bovine counterpart and differs at a single amino acid position from its human analogue. After priming with TgP1 in vivo, significant proliferative T cell responses to TgP1 in vitro were observed only with lymphocytes from susceptible (high responder) strains, thus correlating proliferative capacity with EAT induction. TgP1-primed T cells did not respond to intact mouse Tg (MTg) or rat Tg in vitro and, conversely, T cells primed in vivo with MTg or rat Tg did not respond to TgP1 in culture, suggesting that TgP1 is comprised of non-immunodominant T cell determinants. TgP1 was defined as a serologically nonimmunodominant epitope as well, since in vivo priming of all strains with MTg led to strong MTg-specific IgG responses but no TgP1-specific responses in ELISA assays. This was not due to lack of immunogenic B cell determinants on TgP1, however, because peptide challenge of EAT-susceptible strains elicited TgP1-specific IgG that also cross-reacted with MTg and rat, human, bovine, and porcine Tg. The data demonstrate that TgP1 delineates nonimmunodominant but highly immunogenic determinants at both the T and B cell level, which may play an important role in the development of autoimmune thyroiditis.     

Use of the thermoinducible temperate phage Phi 105cts139 for cloning in Bacillus subtilis

The gene for alpha-amylase from Bacillus amyloliquefaciens having a foreign promoter providing gene expression in logarithmic growth phase and the cat gene of plasmid pC194 (AC fragment) were inserted into thermoinducible prophage phi 105 cts139. Possibility of amylolytic activity enhancement was studied after thermoinduction. When AC fragment and random PstI restricts of phage DNA were ligated and used to transform Bacillus subtilis 1A289 (phi 105 cts139) the Amy+ CmR transformants were obtained having the different levels of increased amylolytic activity (maximum--26 fold). Numerous phages without insert found in induced lysates suggest that insertions were unstable and (or) the clones were double lysogens for hybrid and original type phages. Stable insertion of AC fragment replacing the PstI-H-fragment of phage DNA revealed that all Amy+ CmR transformants were double lysogens. Inducibility depended on the insertion orientation.     

Evaluation of the human transmission risk of an atypical bovine spongiform encephalopathy prion strain

Bovine spongiform encephalopathy (BSE), the prion disease in cattle, was widely believed to be caused by only one strain, BSE-C. BSE-C causes the fatal prion disease named new variant Creutzfeldt-Jacob disease in humans. Two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H, have been discovered in several countries since 2004; their transmissibility and phenotypes in humans are unknown. We investigated the infectivity and human phenotype of BASE strains by inoculating transgenic (Tg) mice expressing the human prion protein with brain homogenates from two BASE strain-infected cattle. Sixty percent of the inoculated Tg mice became infected after 20 to 22 months of incubation, a transmission rate higher than those reported for BSE-C. A quarter of BASE strain-infected Tg mice, but none of the Tg mice infected with prions causing a sporadic human prion disease, showed the presence of pathogenic prion protein isoforms in the spleen, indicating that the BASE prion is intrinsically lymphotropic. The pathological prion protein isoforms in BASE strain-infected humanized Tg mouse brains are different from those from the original cattle BASE or sporadic human prion disease. Minimal brain spongiosis and long incubation times are observed for the BASE strain-infected Tg mice. These results suggest that in humans, the BASE strain is a more virulent BSE strain and likely lymphotropic.     

Detected microsatellite polymorphisms in genetically altered inbred mouse strains

Microsatellites are 50–200 repetitive DNA sequences composed of 1- to 6-base-pair-long reiterative motifs within the genome. They are vulnerable to DNA modifications, such as recombination and/or integration, and are recognized as “sentinel” DNA. Our previous report indicated that the genotypes of the microsatellite loci could change from mono- to poly-morphisms (CMP) in gene knockout (KO) mice, implying that genetic modification induces microsatellite mutation. However, it is still unclear whether the random insertion of DNA fragments into mice genomes produced via transgene (Tg) or N-ethyl-N-nitrosourea (ENU) would also result in microsatellite mutations or microsatellite loci genotypes changes. This study was designed to find possible clues to answer this question. In brief, 198 microsatellite loci that were distributed among almost all of the chromosomes (except for the Y) were examined through polymerase chain reaction to screen possible CMPs in six Tg strains. First, for each strain, the microsatellite sequences of all loci were compared between Tg and the corresponding background strain to exclude genetic interference. Simultaneously, to exclude spontaneous mutation-related CMPs that might exist in the examined six strains, mice from five spontaneously mutated inbred strains were used as the negative controls. Additionally, the sequences of all loci in these spontaneous mutated mice were compared to corresponding genetic background controls. The results showed that 40 of the 198 (20.2 %) loci were identified as having CMPs in the examined Tg mice strains. The CMP genotypes were either homozygous or heterozygous compared to the background controls. Next, we applied the 40 CMP positive loci in ENU-mutated mice and their corresponding background controls. After that, a general comparison of CMPs that exist among Tg, ENU-treated and KO mouse strains was performed. The results indicated that four (D11mit258, D13mit3, D14mit102 and DXmit172) of the 40 (10 %) CMP loci were shared by Tg and KO mice, two (D15mit5 and D14mit102) (5 %) by Tg and ENU-treated mice, and one (D14mit102) (2.5 %) by all three genetic modifications. Collectively, our study implies that genetic modifications by KO, Tg or chemical mutant can trigger microsatellite CMPs in inbred mouse strains. These shared microsatellite loci could be regarded as “hot spots” of microsatellite mutation for genetic monitoring in genetic modified mice.     

A chloramphenicol-streptomycin-resistance plasmid from a clinical strain of Staphylococcus sciuri and its structural relationships to other staphylococcal resistance plasmids

A 5.1-kb plasmid, designated pSCS12, isolated from a naturally occurring Staphylococcus sciuri conferred resistance to chloramphenicol (CmR) and streptomycin (SmR). Restriction endonuclease analyses of pSCS12 revealed partial structural homologies to the CmR-plasmids pC221 from S. aureus and pSCS1 from S. intermedius, to the SmR-plasmids pSAI-1 from S. hyicus and pS194 from S. aureus, as well as to the CmR/SmR plasmid pSK68 from S. aureus. Southern-blot hybridization with specific CmR- and SmR-gene probes confirmed these similarities and allowed the mapping of the CmR- and SmR-determinants in the S. sciuri plasmid pSCS12. These observations lead to the suggestion that CmR/SmR-plasmids, such as pSCS12, may have evolved from CmR- and SmR-plasmids by interplasmidic recombination.     

Transmission barriers for bovine, ovine, and human prions in transgenic mice

Transgenic (Tg) mice expressing full-length bovine prion protein (BoPrP) serially propagate bovine spongiform encephalopathy (BSE) prions without posing a transmission barrier. These mice also posed no transmission barrier for Suffolk sheep scrapie prions, suggesting that cattle may be highly susceptible to some sheep scrapie strains. Tg(BoPrP) mice were also found to be susceptible to prions from humans with variant Creutzfeldt-Jakob disease (CJD); on second passage in Tg(BoPrP) mice, the incubation times shortened by 30 to 40 days. In contrast, Tg(BoPrP) mice were not susceptible to sporadic, familial, or iatrogenic CJD prions. While the conformational stabilities of bovine-derived and Tg(BoPrP)-passaged BSE prions were similar, the stability of sheep scrapie prions was higher than that found for the BSE prions but lower if the scrapie prions were passaged in Tg(BoPrP) mice. Our findings suggest that BSE prions did not arise from a sheep scrapie strain like the one described here; rather, BSE prions may have arisen spontaneously in a cow or by passage of a scrapie strain that maintains its stability upon passage in cattle. It may be possible to distinguish BSE prions from scrapie strains in sheep by combining conformational stability studies with studies using novel Tg mice expressing a chimeric mouse-BoPrP gene. Single-amino-acid substitutions in chimeric PrP transgenes produced profound changes in incubation times that allowed us to distinguish prions causing BSE from those causing scrapie.     

Cloning and sequence analysis of a plasmid-encoded chloramphenicol acetyltransferase gene from Staphylococcus intermedius.

The chloramphenicol acetyltransferase gene (cat) of a 3.9 kb chloramphenicol resistance (CmR) plasmid from Staphylococcus intermedius, designated pSCS1, was cloned into an Escherichia coli plasmid vector. Sequence analysis revealed a high degree of base similarity with the cat gene of the S. aureus CmR plasmid pC221 but there were several differences in the regulatory region. A lesser degree of similarity was observed between the cat gene of the S. intermedius plasmid and the cat gene of the S. aureus plasmid pC194.     

Construction of a novel gene bank of Bacillus subtilis using a low copy number vector in Escherichia coli

Low copy number vector plasmid pCT571 was constructed to clone Bacillus subtilis genomic fragments in Escherichia coli. pCT571 confers KmR, TcR and CmR in E. coli and CmR in B. subtilis. It has unique restriction sites within the KmR and TcR markers to allow screening for recombinant plasmids by insertional inactivation of these genes. It contains the pSC101 replicon and replicates normally at six to eight copies per chromosome equivalent in E. coli. It also contains oriVRK2, which when supplied with the product of the trfA gene of RK2 in trans, allows pCT571 to replicate at 35-40 copies per chromosome equivalent. A B. subtilis gene bank was created by cloning partially Sau3A-digested and size-fractionated fragments of B. subtilis chromosomal DNA into the BamHI site of pCT571. DNA from 1097 KmR TcS transformants was extracted and analysed electrophoretically as supercoiled DNA and after digesting with EcoRI or EcoRI and SalI. Approximately 1000 hybrid plasmids were found with reasonably sized B. subtilis fragments. The mean size of the inserts in pCT571 is 8 kb, ranging from 4 to 20 kb in different plasmids. The gene bank covers most of the B. subtilis chromosome, as demonstrated by the results of screening the gene bank for selectable nutritional markers in E. coli and B. subtilis. Hybrid plasmids which complement E. coli mutants for arg, his, lys, met, pdx, pyr and thr markers were identified from the gene bank. In B. subtilis the presence of argC, cysA, dal, hisA, ilvA, leuA, lys, metB, metC, phe, purA, purB, thr and trpC was established by transformation experiments. The effects of copy number on cloning and long-term maintenance in the bacterial strains were also investigated. At high copy number some hybrid plasmids cannot be maintained at all, while others show an increased rate of structural deletions and rearrangements.     

Scrapie strain transmission studies in ovine PrP transgenic mice reveal dissimilar susceptibility

The Tg(OvPrP4) mouse line, expressing the sheep prion protein, is a sensitive model crucial for the identification of the bovine spongiform encephalopathy agent possibly present in natural sheep spongiform encephalopathies. It was also previously demonstrated as susceptible to infection with natural scrapie isolates from sheep harbouring various genotypes. The performance of this new transgenic mouse line in scrapie strain characterization was further assessed by intracranial inoculation of five groups of Tg(OvPrP4) mice with brain homogenate of the wild type mouse-adapted scrapie strains, C506M3, 22A, 79A, 87V, or Chandler. The Tg(OvPrP4) mice were susceptible to the scrapie agent transmitted using mouse-adapted scrapie strains but not equivalently. Strains 87V and Chandler were most readily transmissible followed by 79A and C506M3. Strain 22A was the least transmissible. Clinical signs, survival data, spongiosis, and PrP^sc distribution were also reported. These various data demonstrate the possibility of distinguishing between scrapie strains. Our findings are discussed with regard to agent strain and host factors and already demonstrate the dissimilar susceptibilities of Tg(OvPrP4) mice to the different murine strains studied, thus, reinforcing their potential use in strain typing studies.     

High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection

Three types of alpha-complementation plasmid vectors were constructed which contain a chloramphenicol- or kanamycin-resistance (CmR or KmR) gene and polylinker cloning sites within the coding region of lacZ'. These vectors are essentially based on high- or low-copy-number replicons. The low-copy-number vectors, 3.61 kb in size, confer CmR and contain the pSC101 replicon and pUC8-/pUC9-type polylinker. On the other hand, the high-copy-number vectors, 2.21 to 2.68 kb in size, confer either CmR or KmR, and contain the pBR322 replicon and pUC18-/pUC19-type or other modified polylinkers. All cloning sites except HindIII and SmaI sites in the KmR vectors are unique in each plasmid. Since almost all frequently used plasmid vectors confer ampicillin resistance, these vectors may be useful to simplify the subcloning/DNA joining experiments due to unnecessity of radioisotope labelling, size fractionation and purification of foreign DNA segments.     

Failure of rearranged TCR transgenes to prevent age-associated thymic involution.

After puberty, the thymus undergoes a dramatic loss in volume, in weight and in the number of thymocytes, a phenomenon termed age-associated thymic involution. Recently, it was reported that age-associated thymic involution did not occur in mice expressing a rearranged transgenic (Tg) TCRalphabeta receptor. This finding implied that an age-associated defect in TCR rearrangement was the major, if not the only, cause for thymic involution. Here, we examined thymic involution in three other widely used MHC class I-restricted TCRalphabeta Tg mouse strains and compared it with that in non-Tg mice. In all three TCRalphabeta Tg strains, as in control mice, thymocyte numbers were reduced by approximately 90% between 2 and 24 mo of age. The presence or absence of the selecting MHC molecules did not alter this age-associated cell loss. Our results indicate that the expression of a rearranged TCR alone cannot, by itself, prevent thymic involution. Consequently, other presently unknown factors must also contribute to this phenomenon.     

Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli.

The chimeric plasmid pBN183 was first constructed in Escherichia coli by ligating the BamHI-digested E. coli plasmid pBR322 and a Bg/II-linearized streptococcal plasmid, pNZ18. The pBN183 transformed E. coli to ApR at a frequency of (8.2 +/- 1.2) x 10(5) colony forming units (CFU)/microgram DNA. Electrotransformation of Streptococcus thermophilus with pBN183 yielded CmR, ApS clones at a frequency of (2.6 +/- 0.3) x 10(1) CFU/microgram DNA. Plasmid screening with pBN183-transformed S. thermophilus clones revealed that ca. 70% of these transformants contained deleted plasmids. Plasmid pBN183A, a pBN183 deletion mutant lacking one copy of a tandemly arranged, highly homologous DNA sequence, was isolated for further study. It transformed E. coli to ApR and S. thermophilus to CmR with frequencies of (4.8 +/- 0.1) x 10(5) and (8.1 +/- 0.2) x 10(2) CFU/microgram DNA, respectively. Screening of S. thermophilus transformants did not show the presence of deleted plasmids. Based on the structure of pBN183A, a new shuttle plasmid, pDBN183, was constructed from pBN183 by removal of the small (1.2 kb) Sa/I fragment. Transformation frequencies of pDBN183 were (5.0 +/- 1.3) x 10(5) and (4.6 +/- 0.2) x 10(2) CFU/microgram DNA with E. coli and S. thermophilus, respectively. In contrast to the parent pBN183, only 17% of the pDBN183-transformed S. thermophilus contained deleted plasmids. Plasmid copy numbers of the three vectors in E. coli were estimated at 17-18 per chromosome. The three plasmids conferred ApR and CmR to E. coli, but only CmR to S. thermophilus. The insertion of a Streptomyces cholesterol oxidase gene (choA) into pDBN183 did not affect the plasmid's stability in Lactobacillus casei, but resulted in deletion of the recombinant DNA in S. thermophilus.