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E K Jagusztyn-Krynicka

Publications and source records attributed to E K Jagusztyn-Krynicka.

14 recordsLinked to original sources
PubMed2000

Genetic diversity of the Campylobacter genes coding immunodominant proteins.

Three Campylobacter jejuni 72Dz/92 genes (cjaA (ompH1), cjaC (hisJ) and cjaD (omp18)) encoding immunodominant proteins are considered to be potential chicken vaccine candidates. The presence and conservation of cjaA, cjaC and cjaD genes among different Campylobacter clinical isolates were determined. The genes were detected in thirty Campylobacter strains using hybridization as well as Western blot analysis. However, PCR products of the predicted size were amplified only from ten out of thirty examined strains regardless of the employed primer pair. The nucleotide sequence of the C. jejuni 72Dz/92 genes was compared with the nucleotide sequences of their homologs cloned from other Campylobacter strains as well as with the whole genome sequence of C. jejuni NCTC 11168. The examined sequences revealed 0 to 16% divergence. Strain-dependent levels of divergence were observed. The polymorphism detected in cjaC was mainly within the 5' region of the gene, while the nucleotide substitutions in cjaA and cjaD are distributed uniformly along the whole genes. Most of the observed nucleotide substitutions occurred at the third base of the codons. This observation is consistent with the results of Western blot experiments.

ATP-Binding Cassette Transporters↗
PubMed1998

Campylobacter jejuni 72Dz/92 cjaC gene coding 28 kDa immunopositive protein, a homologue of the solute-binding components of the ABC transport system.

Screening of the Campylobacter jejuni 72Dz/92 cosmid gene bank enabled identification of the cjaC (Camp. jejuni antigen C) gene encoding a highly immunogenic protein of apparent molecular mass 28 kDa. Gene bank searches indicated significant overall homology of the cjaC gene product, mainly to the Gram-negative periplasmic solute-binding proteins of the ABC transport system which recognize polar amino acids and opines. CjaC protein contains the motif LVAC at the end of the putative 19 amino acid signal sequence, which suggests that it might be a lipoprotein. In the 5' flanking region of the cjaC gene, two potential promoter regions were observed. The cjaC gene is conserved among some isolates of three serotypes commonly isolated from humans (HL serotypes 1,4,71).

ATP-Binding Cassette Transporters↗
PubMed1997

Cloning and characterization of a Campylobacter jejuni 72Dz/92 gene encoding a 30 kDa immunopositive protein, component of the ABC transport system; expression of the gene in avirulent Salmonella typhimurium.

Three gene libraries of Campylobacter jejuni 72Dz/92 DNA were prepared using lambda gt11, pSupercos and pWSK129 cloning vectors. Screening of the libraries with Escherichia coli absorbed antiserum generated against whole C. jejuni revealed several immunoreactive clones of apparent molecular masses 19, 28, 30 and 50 kDa. The most commonly isolated clones expressed 30 kDa protein. The nucleotide sequence of the 1768 bp C. jejuni DNA yielded one complete (ORF2) and two partial open reading frames (ORF1 and ORF3). ORF2 encoded CjaA protein exhibits relevant overall homology to several prokaryotic solute binding proteins (family 3), components of the ABC transport system, while the product of the truncated ORF3 (CjaB protein) shows extensive homology to Gram-negative bacterial proteins, members of the sugar transporter family. The genetic organization of the putative cjaAB operon was studied. The cjaA gene fragment (616 bp) was amplified from three C. jejuni strains isolated from patients with acute bloody diarrhea, whereas it was not amplified from strains which caused acute diarrhea with no blood in the stools. The gene was introduced into avirulent Salmonella typhimurium vaccine strain where it is expressed at a reasonably high level.

ATP-Binding Cassette Transporters↗
PubMed1995

Characterization of Campylobacter jejuni asd gene cloned in Escherichia coli.

asd gene of the human enteric pathogen, Campylobacter jejuni Dz72/92, has been isolated from a genomic library constructed in the expression plasmid vector pUC8-2 in Escherichia coli. The gene has been fished out by complementation of the asd gene deletion present in the genome of E. coli chi 6097. The smallest recombinant plasmid (pUWM3) able to confer Asd+ phenotype contains a 1.8 kb insert cloned into HindIII site located within the multi-cloning site of the pUC8-2 vector. The origin of the insert has been confirmed by hybridization. Several pieces of evidence indicate that the expression of the cloned house-keeping gene is driven from its own promoter, which can be recognised by E. coli RNA polymerase. The asd gene promoter has been located on 300 kb HindIII-DraI fragment of pUWM3. Recombinant plasmid pUWM3 specifies a new 38 kDa protein which we believe is the asd gene product. Overproduction of the 38 kDa protein due to the transcription originating from the vector lacZ gene promoter is toxic for the cells.

Aspartate-Semialdehyde Dehydrogenase↗
PubMed1993

Escherichia coli heat-labile toxin subunit B fusions with Streptococcus sobrinus antigens expressed by Salmonella typhimurium oral vaccine strains: importance of the linker for antigenicity and biological activities of the hybrid proteins.

A set of vectors possessing the genes for aspartate semialdehyde dehydrogenase (asd) and the B subunit of the heat-labile enterotoxin of Escherichia coli (LT-B) has been developed. These vectors allow operon or gene fusions of foreign gene epitopes at the C-terminal end of LT-B. Two groups of vectors have been constructed with and without leader sequences to facilitate placing of the foreign antigen in different cell compartments. Two Streptococcus sobrinus genes coding for principal colonization factors, surface protein antigen A (SpaA), and dextranase (Dex), have been fused into the 3' end of the LT-B gene. Resulting protein fusions of approximately 120 to 130 kDa are extremely well recognized by antibodies directed against both SpaA and Dex as well as against LT-B domains and retain the enzymatic activity of dextranase and the biological activity of LT-B in that they bind to GM1 gangliosides. Maximum antigenicity was obtained with the vector possessing an intervening linker of at least six amino acids with two proline residues. Some of the fusion proteins also exhibited another property of LT-B in that they were exported into the periplasm where they oligomerized. LT-B-SpaA and LT-B-Dex hybrid proteins are expressed stably and at a high level in avirulent Salmonella typhimurium vaccine strains which are being used to investigate their immunogenicity and types of induced immune responses. The fusion vectors will also be useful for production and purification of LT-B fusion antigens to be used and evaluated in other vaccine compositions.

Amino Acid Sequence↗
PubMed1993

Immune responses to Streptococcus sobrinus surface protein antigen A expressed by recombinant Salmonella typhimurium.

In this study, we used a vaccine strain of Salmonella typhimurium to express antigenic determinants of the SpaA antigen of Streptococcus sobrinus, which is involved in the caries-forming process. We cloned either a single repeat (pYA2901) or three tandem repeats (pYA2905) of the 0.48-kb fragment of the spaA gene, which codes for an important component of the SpaA protein, plus a 1.2-kb minor antigenic determinant and measured the resulting immune responses to SpaA in orally immunized BALB/c mice. The single or triple repeat of the spaA gene fragment was inserted into the Asd+ vector pYA292 and was transformed into the S. typhimurium delta cya delta crp vaccine strain chi 4072 containing delta asd in the chromosome. Female BALB/c mice were then orally immunized with two doses of the S. typhimurium containing either of the two SpaA constructs, and the immune responses to the expressed SpaA protein were assessed. Significant serum immunoglobulin G (IgG) anti-SpaA titers were detected in mice immunized with chi 4072(pYA2905) but not chi 4072(pYA2901). Salivary anti-SpaA IgA titers were minimal and were only detected in mice immunized with S. typhimurium expressing the SpaA encoded by pYA2905. Intestinal anti-SpaA IgA titers, however, were detected in both groups of mice, particularly in mice immunized with chi 4072(pYA2905). An oral booster 26 weeks after the initial series of immunizations resulted in increased serum IgG titers in both chi 4072(pYA2901)- and chi 4072(pYA2905)-immunized animals, particularly in the chi 4072(pYA2905)-immunized animals. No anamnestic IgA response was detected in the saliva following the booster immunization.

Animals↗
PubMed1992

Streptococcus mutans serotype c tagatose 6-phosphate pathway gene cluster.

DNA cloned into Escherichia coli K-12 from a serotype c strain of Streptococcus mutans encodes three enzyme activities for galactose utilization via the tagatose 6-phosphate pathway: galactose 6-phosphate isomerase, tagatose 6-phosphate kinase, and tagatose-1,6-bisphosphate aldolase. The genes coding for the tagatose 6-phosphate pathway were located on a 3.28-kb HindIII DNA fragment. Analysis of the tagatose proteins expressed by recombinant plasmids in minicells was used to determine the sizes of the various gene products. Mutagenesis of these plasmids with transposon Tn5 was used to determine the order of the tagatose genes. Tagatose 6-phosphate isomerase appears to be composed of 14- and 19-kDa subunits. The sizes of the kinase and aldolase were found to be 34 and 36 kDa, respectively. These values correspond to those reported previously for the tagatose pathway enzymes in Staphylococcus aureus and Lactococcus lactis.

Aldehyde-Lyases↗
PubMed1990

Restriction map of Thiobacillus versutus plasmid pTAV1.

A restriction map of Thiobacillus versutus plasmid pTAV1 was constructed using EcoRI, BamHI and SalI restriction enzymes. Knowledge of the restriction map is an obligatory starting point for genetic and molecular studies of this, so far cryptic, plasmid.

Deoxyribonuclease BamHI↗
PubMed1990

Cloning and expression of Thiobacillus versutus cryptic plasmid pTAV-1 DNA in Escherichia coli.

pTAV-1 is an approximately 100 kb Thiobacillus versutus cryptic plasmid. pTAV-1 DNA was cloned in Escherichia coli. Nine recombinant plasmids containing pTAV-1 DNA inserted into the EcoRI restriction site of pACYC184 were constructed. The origin of DNA inserts was confirmed by Southern blot hybridization. The expression of mixotrophic T. versutus plasmid genes was demonstrated in E. coli.

Bacterial Proteins↗
PubMed1989

Cloning and expression of Thiobacillus versutus aspartate-semialdehyde dehydrogenase gene in Escherichia coli.

The Thiobacillus versutus asd gene coding for aspartate-semialdehyde dehydrogenase was cloned in Escherichia coli cells using pBR322 as a vector. The gene was expressed independently of its orientation, suggesting that E. coli RNA polymerase recognized T. versutus promoter sequence. The T. versutus DNA coded protein, of the molecular weight 44,000, was identified by the analysis of the proteins produced by minicells.

Aspartate-Semialdehyde Dehydrogenase↗
PubMed1982

Expression of Streptococcus mutans aspartate-semialdehyde dehydrogenase gene cloned into plasmid pBR322.

Streptococcus mutans chromosomal DNA cloned into the vector plasmid pBR322 in Escherichia coli is able to complement the metabolic defect of an aspartate-semialdehyde dehydrogenase (EC 1.2.1.11) gene (asd) deletion in the host strain. We constructed two Asd+ recombinant plasmids, pYA570 and pYA571, containing 4.7 and 4.5 kilobases, respectively, of S. mutans chromosomal DNA inserted into the HindIII restriction endonuclease site of pBR322 in the same orientation. The S. mutans UAB62 Asd+ DNA did not hybridize with E. coli DNA which contained an intact asd gene, but did not hybridize with S. mutans UAB62 chromosomal DNA. Derivative Asd+ plasmids were then constructed from pYA570. One, pYA574, had a 4.5 kilobase S. mutans insert DNA in the opposite direction from pYA570. In another pYA575, the S. mutans insert DNA was reduced in size to 1.3 kilobases. It was seen that the orientation of the S. mutans DNA fragment inserted into the promotor region of the pBR322 tetracycline resistance (Tcr) gene affected expression of Tcr. Orientation of the S. mutans insert also affected the stability of the plasmid in certain E. coli strains. Restriction maps for pYA570, pYA571, pYA574 and pYA575 using the endonucleases EcoRI, BamHI, HindIII, PstI and SalI were determined, Asd+ plasmid-directed protein synthesis was studied in E. coli minicells. The plasmids pYA570, pYA574 and pYA575 each produced large amounts of a protein with a monomeric molecular weight of about 45000, that was distinct from both pBR322 and E.coli specified proteins: this protein is the S. mutans asd gene product. Smaller derivatives of recombinant plasmid pYA575 that were Asd- allowed the location of the S. mutans asd gene promotor and the direction of transcription to be determined.

Aspartate-Semialdehyde Dehydrogenase↗
PubMed1982

Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12.

Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.

Cloning, Molecular↗