Chemical cleavage of mismatches in heteroduplexes of the rpoB gene for detection of mutations associated with resistance of Mycobacterium tuberculosis to rifampin.

In order to detect mutations in the core region of the RNA polymerase B (rpoB) subunit gene of Mycobacterium tuberculosis that are known to be associated with resistance to rifampin, we applied rapid chemical cleavage of mismatches (CCM) to heteroduplexes formed between the DNA of M. tuberculosis H37Rv and strains resistant to rifampin. DNA fragments amplified from normal and mutant rpoB genes by polymerase chain reaction were mixed, denatured and re-annealed to create heteroduplexes containing mispaired bases reactive to modification by hydroxylamine (cytosine mismatches) or osmium tetroxide (thymine mismatches) and cleavage of DNA by piperidine at the position of modified base. The cleaved products and the heteroduplexes were separated by polyacrylamide-urea gel electrophoresis and detected by autoradiography. The position of mutations was confirmed by DNA sequencing of the amplified DNA fragments. The results suggest further applicability of the CCM method as a means to screen M. tuberculosis isolates for mutations associated with drug resistance.

Treatment monitoring and prevalence of drug resistance in tuberculosis patients in Tehran.

SETTING: Health care clinics and private practitioners in Tehran. OBJECTIVE: To analyse rates of drug resistance and response to treatment in tuberculosis patients. DESIGN: A prospective study of 257 patients undergoing treatment for whom data were collected on drug susceptibility testing and outcome as well as age, sex and history of treatment. RESULTS: Of 774 initially diagnosed patients, 380 were female and 394 were male; 520 (67%) of the cases had pulmonary disease. The overall rate of primary drug resistance among Mycobacterium tuberculosis isolates resistant to at least one drug was 87/563 (15.5%). Twenty-three patients were multidrug-resistant. Among 215 patients with drug-susceptible cultures recruited for follow-up, rapid response to short-course chemotherapy was observed in 190 (88%) who were successively both smear and culture negative after 2 and 4 months of treatment. After 6 months of treatment, 12 of the 25 patients with slow response to treatment had positive cultures; one was smear-positive. Of the 42 patients with drug-resistant isolates, satisfactory bacteriological response was observed after 6 months of treatment in 30 (71%). CONCLUSIONS: These observations support regional recommendations for short-course treatment regimens. Culture rather than smear result could be a key parameter for individually guiding the duration of treatment in patients with poor response to treatment.

Use of restriction enzyme analysis of amplified DNA coding for the hsp65 gene and polymerase chain reaction with universal primer for rapid differentiation of mycobacterium species in the clinical laboratory.

Two rapid procedures, restriction enzyme analysis of the amplified segment of the gene encoding for the 65000 mol. wt heat shock protein and a polymerase chain reaction with single universal primer (UP-PCR), were used for the identification of Mycobacterium tuberculosis complex (n = 47) and proving the species identity of non-tuberculous mycobacteria (NTM, n=36) cultured from clinical samples by comparing the resulting DNA banding pattern with patterns derived from mycobacterial type strains (n = 24). UP-PCR assay provided a rather wide limit of tolerance for variations in procedure. Although mycobacterial strains were found to generate species-specific banding patterns in both assays, M. tuberculosis and M. bovis strains and isolates produced nearly the same DNA patterns, which were very distinctive from that of all NTM tested. Investigation of the majority of M. fortuitum (n = 14) and M. kansasii (n = 7), mycobacteria most frequently causing mycobacterioses in the region, as well as other NTM isolates, showed reproducible patterns characteristic of corresponding type strains. Both methods combine the advantages of ordinary PCR and PCR 'fingerprinting', namely, the species-specific DNA pattern and primers applicable to different species. They may be applied as rapid tests for proving the identity of Mycobacterium species in a clinical laboratory.

Polymerase chain reaction of bacterial genomes with single universal primer: application to distinguishing mycobacteria species.

The polymerase chain reaction with a single universal primer (UP-PCR) was applied to bacterial strains and mycobacteria isolates alongside conventional methods. A universal protocol of preparation of PCR samples from cultures representing Escherichia coli, Enterobacter aerogenes, Serratia marcescens, Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Mycobacterium tuberculosis, Mycobacterium bovis, and several non-tuberculous mycobacteria was found to be reproducible and efficient with these organisms. The bands of UP-PCR products observed in an agarose gel after electrophoresis were species-specific and provided an efficient means of distinguishing bacterial species. The applicability of this approach to mycobacteria identification was assessed by comparing the DNA bands obtained for different strains. Three reference strains and 22 clinical isolates of M. tuberculosis and M. bovis produced very similar DNA banding patterns. They comprised a triplet of prominent and several minor fragments within the 200-500 base pair (bp) size range and were the easiest to interpret. The DNA profiles of unrelated mycobacteria clearly differed from each other when subjected to electrophoretic analysis and correlated well with results of culture method. The method provides a real promise of its application in clinical studies as a simple assay for distinguishing between tubercle bacilli.

Detection and identification of non-tuberculous mycobacterial infections in 6,472 tuberculosis suspected patients.

Between March 1993 and March 1994, 82 patients with infection by non-tuberculous mycobacteria (NTM) and 443 patients with tuberculosis (TB) were registered among 6,472 patients with suspected tuberculosis. Skin-test reactivity to purified protein derivative (PPD) in patients demonstrated indurations of 10-14 mm or more for the majority of patients in both groups. Most patients with NTM infection had abnormal chest roentgenograms showing sporadic infiltrations, nodular abscesses, and cavities resembling TB radiological evidence. The similarity in age range, PPD skin reaction, and radiological evidence in patients infected with NTM or Mycobacterium tuberculosis (MTB) can mislead the physician. Some NTM species were recovered more often than others. Mycobacterium fortuitum from 22 clinical specimens (26.8%); Mycobacterium gastric 19 (23.1%); and Mycobacterium terrae complex 15 (18.3%). The antimicrobial drug susceptibility tests of the isolated organisms showed that 42 (9.5%) isolates of MTB were resistant to isoniazid and 31 (7.0%) to streptomycin. a few strains (1.3%) were identified as being resistant to a combination of 3 primary drugs. These findings suggest that drug-resistant mycobacterial infections are becoming an important problem in the region.

Use of polymerase chain reaction for primary diagnosis of pulmonary tuberculosis in the clinical laboratory.

A nested polymerase chain reaction (PCR) has been used for the rapid detection of tubercule bacilli in respiratory specimens from 287 patients suspected of tuberculosis. The results of PCR testing were compared with isolation methods (conventional culture and Bactec system) in 110 smear-positive and 177 smear-negative patients. There were only 4 false negative results by PCR in the 171 specimens that were M. tuberculosis complex culture-positive. Of 92 PCR-positive samples prepared from the smear-positive specimens 90 (97.8%) were confirmed by culture. However, a poor correlation was obtained between initial 122 PCR-positive results and combined 81 culture recovered organisms in smear-negative patients. After verification of the efficacy of isolation method, retesting PCR-positive culture-negative samples, and studies of patients' clinical histories, only 18 of the cases were found to be associated with the disease. The other 29 results out of the original 47 discrepants were considered PCR false positives, possibly due to contamination. In conclusion, the PCR assay described is suitable for implementation in daily routine work with respiratory specimens, however it should be validated with culture, especially for the smear-negative patients.

Topoisomerase I is associated with the regulatory region of transcriptionally active SV 40 minichromosomes.

Proteins bound to SV 40 DNA in sarkosyl-treated nuclei have been studied. The major component is topoisomerase I, a 60-70 kDa protein which possesses a strong DNA-nicking activity in the presence of sarkosyl and camptothecin. An SV 40 DNA fraction containing sarkosyl-resistant proteins constitutes 2 to 3% of the total nuclear SV 40 DNA and is enriched in transcriptionally active DNA (as monitored by distribution of RNase-resistant in vivo pulse-labeled RNA). An SV 40 DNA fraction, which is nicked due to covalent binding of topoisomerase I upon sarkosyl treatment is also enriched in transcriptionally active DNA. Topoisomerase I cleavage sites on SV 40 DNA which arise following sarkosyl treatment of nuclei or camptothecin treatment of infected cells have been mapped. The strongest site is located at nucleotide 381 on the non-coding strand and is framed by nuclease hypersensitive sites.

Effect of X-ray induced DNA damage on DNAase I hypersensitivity of SV40 chromatin: relation to elastic torsional strain in DNA.

The effect of X-irradiation on DNAase I hypersensitivity of SV40 minichromosomes within nuclei or free in solution was investigated. The susceptibility of the specific DNA sites in the control region of minichromosomes to DNAase I decreased in a dose dependent manner after irradiation of isolated nuclei. On the other hand, the irradiation of minichromosomes extracted from nuclei in 0.1 M NaCl-containing buffer almost did not affect the level of their hypersensitivity to DNAase I. This suggests that DNAase I hypersensitivity may be determined by two different mechanisms. One of them may be connected with elastic torsional strain within a fraction of minichromosomes and another seems to be determined by nucleosome free region. The first mechanism may be primarily responsible for the hypersensitivity of minichromosomes within nuclei. After irradiation of the intact cells, DNAase I hypersensitivity tested in nuclei substantially increased. This was connected with activation of endogeneous nucleases by X-irradiation which led to accumulation of single- and double-strand breaks superimposed to DNAase I induced breaks in the control region of SV40 DNA.

DNAaseI-hypersensitive minichromosomes of SV40 possess an elastic torsional strain in DNA.

Previously, we have shown that DNA in a small fraction (2-5%) of SV40 minichromosomes was torsionally strained and could be relaxed by treating minichromosomes with topoisomerase I. This fraction was enriched with endogeneous RNA polymerase II (Luchnik et al., 1982, EMBO J., 1, 1353). Here we show that one and the same fraction of SV40 minichromosomes is hypersensitive to DNAase I and is relaxable by topoisomerase I. Moreover, this fraction completely loses its hypersensitivity to DNAase I upon relaxation. The possibility that this fraction of minichromosomes can be represented by naked DNA is ruled out by the results of studying the kinetics of minichromosome digestion by DNAase I in comparison to digestion of pure SV40 DNA and by measuring the buoyant density of SV40 chromatin in equilibrium CsCl gradient. Our data obtained with SV40 minichromosomes may be relevant to the mechanism responsible for DNAase I hypersensitivity in the loops or domains of cellular chromatin.

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

After treatment of SV40 minichromosomes with DNA topoisomerase I, the superhelicity in the bulk of the DNA extracted from minichromosomes is known to remain unchanged. However, we found that the DNA extracted from a small fraction of SV40 minichromosomes (2-5%), was almost completely relaxed, and covalently closed as shown by agarose gel electrophoresis. Thus, the DNA in these 2-5% of SV40 minichromosomes was probably torsionally strained (TS). The proportion of such TS minichromosomes is close to the estimated proportion of transcriptionally active minichromosomes. The distribution of the TS minichromosomes in sucrose gradient coincided with the distribution of transcriptionally active complexes. Both sedimented faster than the majority of minichromosomes. Furthermore, after treatment with topoisomerase I the relaxed minichromosomes could be quantitatively separated from the bulk of material by recentrifugation in a sucrose gradient. A major part of the endogenous RNA polymerase activity was recovered in the relaxed fraction. These data suggest that TS-minichromosomes correspond to transcriptionally active chromatin. After relaxation with topoisomerase I the TS minichromosomes lacked histones.

Studies on structure and function of chromatin.

This article covers research work in this laboratory on the structure and function of chromatin. Early studies have led to discovery of skeletal fibrils (nucleonemas) within the nuclei and showed the specific role of histone H1 in chromatin condensation and in restriction of transcription. More recently with the aid of a novel DNP electrophoresis technique the relation of histone H1 and non-histone proteins to nucleosomes was studied. Three types of mononucleosomes and a number of subnucleosomes were identified in chromatin digests. The complexes of certain HMG proteins with short DNA segments were isolated and found to originate frm transcriptionally active chromatin. Different forms of SV40 minichromosome were characterized. A method for the analysis of nucleosome distribution along the DNA sequence was elaborated and used to show non-random (phased) location of nucleosomes on SV40 DNA. The attachment of DNA to skeletal elements of interphase nuclei and metaphase chromosomes was shown to be a non-random, probably sequence-specific process.

Studies on protein organization of nucleosomes using cross-linking.

When chromosomal proteins in chromatin or in mononucleosomes were extensively cross-linked with an imido ester, the H1-containing nonameric histone complex was revealed. In this complex, histone H1 is connected with the octamer of core histones. The cross-linking of H1 of the octamer is realized preferentially through H2a and H3 histones. Some HMG (high mortality group) proteins located presumably in the linker regions of a nucleosome fiber also take part in the formation of dimers, possibly with the histones of a nucleosomal core. The results suggest mutant interactions between some linker-associated proteins and intranucleosomal histones. Experiments involving extensive cross-linking of proteins in the purified mononucleosome subfractions demonstrated differences in the organization of core histones between 'complete' nucleosomes and nucleosomes lacking H1.

Subnucleosome particles containing high mobility group proteins HMG-E and HMG-G originate from transcriptionally active chromatin.

Subnucleosome particles SN2 and SN3 containing short DNA fragments and non-histone proteins of the high mobility group, HMG-G and HMG-E respectively, were purified from the chromatin preparations of mouse L cells partially digested with staphylococcal nuclease. Labeled DNAs prepared from these particles were hybridized to an excess of nuclear RNA. The binding of subnucleosomal DNA was about 3-fold higher comparing to total cellular DNA fragmented to the same size. Special control experiments showed that DNA.protein complexes present in subnucleosomes SN2 and SN3 preexisted in nontreated nuclei. The conclusion has been drawn that non-histone proteins HMG-G and HMG-E are associated with the DNA of transcriptionally active chromatin and are released by nuclease as subnucleosomes.

Non-histone proteins in mononucleosomes and subnucleosomes.

Nucleosomes and subnucleosomes separated either by sucrose gradient ultracentrifugation or by polyacrylamide gel electrophoresis contain proteins incorporating [3H]tryptophan, i.e. non-histone proteins. The fractions of mononucleosomes MN3 and MN2 are enriched in these proteins as compared to the MN1 fraction. Two-dimensional gel electrophoresis of chromatin digests reveals a number of non-histone proteins comigrating with deoxyribonucleoprotein particles in the first direction (in non-dissociating conditions). A significant fraction of these proteins corresponds to basic non-histone proteins, so-called HMG (high-mobility-group) proteins. Two HMG proteins are present in mononucleosomes MN3 exclusively and three others in mononucleosomes MN3 and MN2. One of them is recovered also in subnucleosomes SN2, and another in SN3 subnucleosome fraction, At least three HMG proteins are rapidly released from the oligonucleosome fractions as well as from the insoluble DNA . protein residue. Thus, they are located in chromatin readily available to DNAase action. Apart from HMG proteins, a number of other non-histone proteins are present in mononucleosomes but their relative content in the oligonucleosome fraction is much higher. The conclusion has been drawn that many non-histone proteins, in particular HMG proteins, interact with linker DNA in chromatin.

Separation of nucleosomes containing histones H1 and H5.

Hen erythrocyte chromatin was digested with staphylococcal nuclease and fractionated by electrophoresis in polyacrylamide gels. Instead of the three bands described for mouse carcinoma chromatin, four main discrete components (MN1, MN2, MN2E and MN3) were resolved in the mononucleosome fraction of erythrocyte chromatin. MN2 contained all five histones and a DNA fragment of 165--180 base pairs. MN2E comprised four nucleosomal histones plus histone H5 (but not H1) and a DNA fragment of 170--190 base pairs. The relatively nuclease resistant MN3 fraction of erythrocyte nucleosomes contained H1 but no H5 histone. A more accurate analysis of the MN2 fraction in mouse carcinoma nucleosomes revealed some additional microheterogeneity depending on the presence of two different subfractions of H1.

Chromosome of the mature virion of simian virus 40 contains H1 histone.

In addition to free SV40 minichromosomes in the compact form, complete virions were obtained from the nuclear extract of productively infected cells. Capsid proteins VP1, VP2, and VP3, as well as histones, were observed on electrophoregrams of proteins prepared from virions. In contrast to the widely accepted view, histone H1 was found in virions in stoichiometric amounts with respect to other histones. The same is true for virions isolated by a conventional method. Free minichromosomes present in infected cells contain all histones and practically no viral proteins.