Temporal order of gene replication in Chinese hamster ovary cells.

To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-K1 cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTPP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [alpha-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific genes revealed a number of exceptions. Approximately 25% of total poly(A) RNA was transcribed from the late-replicating DNA.

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.

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.

[The incidence of heat tolerant Campylobacter in rivers and lakes of the Warsaw region]. / Wystepowanie termotolerancyjnych bakterii rodzaju Campylobacter w rzekach i jeziorach okolic Warszawy.

The presence of thermotolerant Campylobacter in rivers and lakes of Warsaw region was examined with the detectability of 1 c.f.u./ml. Samples were taken from depth of water and from the surface of different objects deposited on the bottom. The results indicate that about 70% of water samples are contaminated with Campylobacter, whereas the contamination of the underwater objects is less prevalent. The species distribution was as follows: C. jejuni-65%, C. coli-22%, C. lari-13%. In vitro experiment was also performed to test the ability of Campylobacter to create biofilms on the surface of wood, metal and plastic, however no such property was revealed. From the analysis of presented results it was established that localization of the highest contamination is connected mainly with presence of municipal sewage and in less extent with the presence of the droppings of wild animals. The samples of water give the better reflection of the examined reservoir contamination than solid samples.

Mn2+ dependent transformation of Bacillus subtilis.

The present study shows that transformation of Bacillus subtilis can proceed in the presence of Mn2+ instead of Mg2+ with equal efficiency. The crucial condition seems to be the Mn2+ concentration which should not exceed 0.025 mM. Binding, uptake and breakdown of donor DNA as well as its physicochemical fate in Mn2+ dependent transformation was investigated. No changes as compared to the standard procedure with Mg2+ were noticed. The possible source of errors in this kind of experiments is discussed.

Detection of the coccoid form of Campylobacter jejuni with the use of polymerase chain reaction.

The incubation of Campylobacter jejuni in aerobic conditions results in transformation of spiral cells into coccoid form. It is shown in presented paper that the assay of coccoid of C. jejuni with PCR is limited. Its detectability decreases with the increase of time and temperature of cell storage.

An unstable donor-recipient DNA complex in transformation of Bacillus subtilis.

In re-extracted DNA obtained shortly after uptake of transforming DNA by Bacillus subtilis, increased amounts of donor DNA radioactivity banding at the position of donor-recipient DNA complex (DRC) are observed in CsCl gradients, if the cells are irradiated with high doses of UV prior to reextraction of the DNA. Qualitatively, the same phenomenon is observed if lysates of transforming cells are irradiated. UV-irradiation of lysates of competent cells to which single-stranded DNA is added after lysis, does not result in linkage of this DNA to the chromosomal DNA. Two observations argue in favour of the formation of a specific labile complex between donor and resident DNA during transformation. Firstly, heterologous donor DNA from Escherichia coli, although being processed to single-stranded DNA in competent B. subtilis, does not seem to be linked to the recipient chromosome upon UV-irradiation, and secondly, the labile complex of donor and recipient DNA can be stabilized by means of treatment of the lysates of transforming cells with 4, 5(1), 8-trimethylpsoralen in conjuction with long-wave ultra violet light irradiation. This indicates that basepairing is involved in the formation of the complex. On the basis of these results we assume that the unstable complex of donor and recipient DNA is an early intermediate in genetic recombination during transformation.

Campylobacter jejuni in coccoid form does not reverse into spiral form in chicken guts.

The incubation of Campylobacter jejuni suspension in aerobic conditions results in the conversion of spiral cells into coccoid form. The present paper shows that such forms introduced into the alimentary tract of two-day-old chicks are not able to convert back into cultivable, infectious spiral forms, as has been suggested by some authors. Technical problems connected with this type of experiment are also discussed.

Antibiotic resistance of Campylobacter jejuni with reference to plasmid profiles of clinical and chicken isolates.

A total of 47 clinical isolates and 52 poultry isolates of Campylobacter jejuni were characterized by their resistance to 16 antimicrobial agents and by plasmid profiles on agarose gel electrophoresis. Almost all isolates were susceptible to erythromycin, chloramphenicol, gentamycin and nitrofuratoin. Plasmids were detected in 19% of C. jejuni strains isolated from feces of children patients and in 36% of strains isolated from chicken. The presence of plasmid DNA was not found to be correlated with any definite resistance, despite of the number of resistant strains was also higher among poultry isolates. Plasmids, as well, does not seem to be essential for colonization of alimentary tract of pathogenic activity.

Bacteriophage T7 mRNA is polyadenylated.

To determine whether the RNA of bacterial viruses is polyadenylated like bacterial mRNAs, pulse-labelled as well as the steady-state population of bacteriophage T7-specific transcripts were examined for the presence of poly(A) tracts by binding to oligo(dT) cellulose followed by hybridization with specific gene probes. Representatives of all classes of bacteriophage-specific mRNA--early, middle and late--were found to be polyadenylated. This conclusion was confirmed by screening the products of oligo(dT)-dependent cDNA synthesis. A cDNA library was prepared from RNA synthesized after bacteriophage T7 infection and the sequence of bacteriophage-specific clones was determined to define the sites of polyadenylation. About half of the clones were polyadenylated near the end of a protein-coding region, one of them at the site of post-transcriptional processing by RNase III. Other clones were polyadenylated within protein-coding regions. These observations suggest that polyadenylation occurs after the nucleolytic processing of primary transcripts and in some cases also after mRNA degradation has already begun.

In search of immunogenic Helicobacter pylori proteins by screening of expression library.

BACKGROUND: Prevention of Helicobacter pylori infection may help to control related gastritis, peptic ulcer and cancer. Of the possible preventive measures, immunization was successfully employed in various animal studies. However, no immunization protocol has been accepted for humans. A better characterization of the immune response against the pathogen may be required before a human vaccine is developed. AIM: To identify bacterial proteins which induce an immune response in infected humans or H. pylori-immunized rabbits. METHODS: An expression library of H. pylori genes was screened with sera from infected humans and from immunized rabbits. Positive clones were partially sequenced and identified on the basis of a homology search of a H. pylori genome database. Encoded proteins were expressed directly from positive clones and analyzed by SDS-PAGE/Western blot techniques. RESULTS: 114 positive clones were isolated: 79 by screening with human sera and 35 by screening with rabbit sera. Western blot analysis demonstrated that selected clones encoded one or more strongly immunoreactive proteins. 64 clones selected with human sera had no counterparts among clones from screening with rabbit serum. 13 of these clones encoded a total of 21 unknown H. pylori proteins. 17 clones selected with rabbit sera were not immunostained with human sera. They represent 2 various regions of the H. pylori genome which encoded 3 bacterial proteins of unknown function. CONCLUSIONS: Screening of H. pylori expression library identified immunogenic proteins - potential vaccine antigens.