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.

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.

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.

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.

Histone-like proteins in the purified Escherichia coli deoxyribonucleoprotein.

Analysis of E.coli chromosomes isolated under conditions similar to those used for isolation of eukaryotic chromatin has shown that: 1) The proteins of highly purified E.coli deoxyribonucleoprotein are mainly in addition to RNA polymerase two specific histone-like proteins of apparent molecular weight of 17,000 and 9,000 (proteins 1 and 2, respectively). 2) Proteins 1 and 2 occur in approximately equal molar amounts in the isolated E.coli chromosome, and their relative content corresponds to one molecule of protein 1 plus one molecule of protein 2 per 150-200 base pairs of DNA. 3) There are no long stretches of naked DNA in the purified E.coli deoxyribonucleoprotein suggesting a fairly uniform distribution of the proteins 1 and 2 along DNA. 4) The protein 2 is apparently identical to the DNA-binding protein HU which was isolated previously /1/ from extracts of E.coli cells. 5) Digestion of the isolated E.coli chromosomes with staphylococcal nuclease proceeds through discrete deoxyribonucleoprotein intermediates (in particular, at approximately 120 base pairs) which contain both proteins 1 and 2. However, since no repeating multimer structure was observed so far in nuclease digests of the E.coli chromosome, it seems premature to draw definite conclusions about possible similarities between the nucleosomal organization of the eukaryotic chromatin and the E.coli chromatin structure.Images