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Homocysteine metabolism in children with Down syndrome: in vitro modulation.

The gene for cystathionine beta-synthase (CBS) is located on chromosome 21 and is overexpressed in children with Down syndrome (DS), or trisomy 21. The dual purpose of the present study was to evaluate the impact of overexpression of the CBS gene on homocysteine metabolism in children with DS and to determine whether the supplementation of trisomy 21 lymphoblasts in vitro with selected nutrients would shift the genetically induced metabolic imbalance. Plasma samples were obtained from 42 children with karyotypically confirmed full trisomy 21 and from 36 normal siblings (mean age 7.4 years). Metabolites involved in homocysteine metabolism were measured and compared to those of normal siblings used as controls. Lymphocyte DNA methylation status was determined as a functional endpoint. The results indicated that plasma levels of homocysteine, methionine, S-adenosylhomocysteine, and S-adenosylmethionine were all significantly decreased in children with DS and that their lymphocyte DNA was hypermethylated relative to that in normal siblings. Plasma levels of cystathionine and cysteine were significantly increased, consistent with an increase in CBS activity. Plasma glutathione levels were significantly reduced in the children with DS and may reflect an increase in oxidative stress due to the overexpression of the superoxide dismutase gene, also located on chromosome 21. The addition of methionine, folinic acid, methyl-B(12), thymidine, or dimethylglycine to the cultured trisomy 21 lymphoblastoid cells improved the metabolic profile in vitro. The increased activity of CBS in children with DS significantly alters homocysteine metabolism such that the folate-dependent resynthesis of methionine is compromised. The decreased availability of homocysteine promotes the well-established "folate trap," creating a functional folate deficiency that may contribute to the metabolic pathology of this complex genetic disorder.

Adenosine↗

Indistinguishable physical and catalytic properties of DNA methyltransferase from normal rat liver and a transplantable rat hepatocellular carcinoma.

DNA methyltransferase (DMase) was purified 700- and 1002-fold from normal rat liver and transplantable hepatocellular carcinoma 252 (THC 252) nuclei, respectively, using a four-step procedure that included chromatography on phosphocellulose, hydroxylapatite, DEAE-Sephacel and gel filtration on AcA 34. The enzymes had identical characteristics: pI = 7.4-7.6; Mr = 280 000 by gel filtration; preference for methylating double-stranded over single-stranded DNA and hemimethylated over unmethylated DNA templates; and apparent km of 10 microM for dinucleotide units in poly(dC-dG) and 0.5 microM for S-adenosylmethionine (SAM). Thermal inactivation profiles and sulfhydryl group alkylation inhibition curves for fraction III produced very similar single-transition curves, suggesting the presence of a single-functional enzyme species that is indistinguishable between normal and tumor tissue. Single-value Michaelis-Menten kinetics were obtained for fraction IV enzymes with respect to the concentration of SAM and dinucleotide units in poly(dC-dG), suggesting the absence of isozymic or multiple forms of DMase in normal and malignant liver tissues.

Animals↗

Cryobacterium psychrophilum gen. nov., sp. nov., nom. rev., comb. nov., an obligately psychrophilic actinomycete to accommodate "Curtobacterium psychrophilum" Inoue and Komagata 1976.

"Curtobacterium psychrophilum," proposed by Inoue and Komagata in 1976, is a psychrophilic gram-positive irregular rod isolated from Antarctic soil. This organism grew optimally at 9 to 12 degrees C and did not grow at higher than 18 degrees C. Chemotaxonomic characteristics of this organism were the presence of 2,4-diaminobutyric acid in the cell wall and menaquinone-10 as the predominant respiratory quinone. The cellular fatty acid profile, which contained a significant amount of an anteiso-branched monounsaturated acid, 12-methyl tetradecenoic acid, was a distinctive characteristic of this organism and was reasonable for adaptation to low temperature. Phylogenetic analysis based on 16S ribosomal DNA sequences revealed that this organism was positioned at a separate branch in the family Microbacteriaceae, actinomycetes with group B peptidoglycan. We propose the name Cryobacterium psychrophilum gen. nov., sp. nov. for this organism. The type strain is JCM 1463 (= IAM 12024 = ATCC 43563 = IFO 15735 = NCIMB 2068).

Actinomycetales↗

3-Methyladenine DNA glycosylase activity in a glial cell line sensitive to the haloethylnitrosoureas in comparison with a resistant cell line.

Extracts of a glial cell line (SF-126) which is sensitive to the cytotoxic effect of the haloethylnitrosoureas and of a cell line (SF-188) which is resistant to these agents have been tested for their ability to release methylated bases from a DNA substrate which has been modified with [3H]dimethyl sulfate. In comparison with the sensitive cell line, extracts from the resistant cell line have 2-3-fold higher enzymatic activity. High performance liquid chromatography profiles of the bases which are released by these extracts show that the activity is specific for 3-methyladenine, suggesting that the resistant cells contain elevated levels of 3-methyladenine DNA glycosylase. Previous studies have shown that these cells also contain elevated levels of O6-alkylguanine-DNA alkyl-transferase, suggesting that both enzyme activities may be involved in the resistance of this cell line to the haloethylnitrosoureas.

DNA↗

Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.

Xist gene expression begins at the late 2-cell stage in female mouse embryos and by the third division results in the accumulation of an average 100 copies of Xist RNA per cell, as measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). In the blastocyst, the trophectoderm maintains the paternally imprinted pattern of Xist expression present during early development, while either the maternal or the paternal X chromosome can express Xist among cells of the inner mass. Fluorescent in situ hybridization (FISH) has previously established that Xist transcripts are localized on the silenced X chromosome, forming aggregates of variable dimensions in blastomeres of 8-cell embryos. This observation and the fact that Xist RNA accumulation per cell sharply decreases after morula stage raise the possibility that cells of cleaving embryos contain different levels of Xist RNA, perhaps linked to their subsequent developmental fates. We show here that Xist RNA is efficiently recovered from single blastomeres isolated from 8-cell embryos following laser zona drilling. Sexing of the samples and simultaneous quantification of Xist RNA in individual cells is achieved with a multiplex Xist/Sry real-time RT-PCR assay sensitive to the single-copy level. This analysis reveals that Xist RNA is indeed accumulated to substantially different levels in individual blastomeres of the same 8-cell embryo and that two blastomeres contain most of the molecules per embryo. These results support the conclusion that cells of the early mammalian embryo are not all functionally equivalent. Differential Xist gene expression could arise from differences in DNA methylation, or the order in which cells divide.

Animals↗

Single-step quantitation of DNA in microchip electrophoresis with linear imaging UV detection and fluorescence detection through comigration with a digest.

We demonstrate a convenient single-step quantitation technique for double-stranded DNA (dsDNA) fragments in polymerase chain reaction (PCR) products based on microchip capillary electrophoresis (micro-CE)/UV or fluorescence detection. PCR products of polymorphisms on the human Y-chromosome related to spermatogenic failure did not need purification. They were premixed and comigrated with a DNA digest whose concentration was known. Hydroxyethyl cellulose (HEC) dissolved in 5x Tris-borate-EDTA (5x TBE, pH 8.3) was used as a separation matrix in a linear polyacrylamide-coated quartz microchip, while mixed poly(ethyl oxides) (PEOs) of different molar-masses dissolved in 1 x TBE (pH 8.3) containing 1 ng/microl ethidium bromide was used as a separation matrix in an uncoated poly(methyl methacrylate) (PMMA) microchip. Elution profiles were monitored under either real-time linear imaging UV detection in the snapshot mode where the total separation time is fixed, or light-emitting diode (LED) confocal fluorescence detection in the finishline mode where solutes migrate over the same separation length. It is found that, in both modes, a linear relation exists between the peak areas (A) and the multiplication of the digest concentrations (C) and the fragment sizes (L) in a DNA restrictive digest. Using the comigration electropherogram of a single-step experiment, the concentrations of PCR products were directly determined using the A versus LC linear relationship. The sole condition to obey is that the chosen digest has different fragment sizes with the PCR products of interest. This condition is easy to obey, because micro-CE owns high separation ability, and many digests are commercially available. The recovery of the technique was between 98 and 105%. The R.S.D. for chip-to-chip concentration measurements was less than 6.0% (n = 6). Hence, the technique was accurate and reliable for DNA assays.

DNA↗

A thermodynamic investigation of the melting of B-Z junction forming DNA oligomers.

Ultraviolet absorbance methods were used to characterize the thermodynamics of melting of a series of 16 bp deoxyoligonucleotides over a wide range of NaCl concentrations (0-4.5 M) and to obtain complete thermodynamic profiles for their melting at 0.115 and 4.5 M NaCl. The sequence of the series (one strand of duplex) was: 5'-CGCGCGCGAMNGACTG-3', where C indicates m5dC and -MN- was varied to include all combinations of Py:Py stacks (CC, TT, CT, TC). The unmethylated deoxyoligonucleotide 5'-CGCGCGCGACTGACTG-3' was used as a control sequence. All of the methylated oligonucleotides studied undergo a NaCl-induced transition to a hybrid form containing a left-handed, Z-DNA, region joined to a right-handed region by a B-Z junction. Our experiments allowed us to quantitatively evaluate the effects of NaCl, sequence, and methylation and the transition to the hybrid BZ structure on DNA thermal stability. We found that alteration of a single dinucleotide step has profound effects on the thermal stabilities of the 16 bp fragments studied. Methylation was found to destabilize the double helix, resulting in a decrease in Tm. Transition to the hybrid BZ structure, somewhat surprisingly, was found to only slightly destabilize DNA, with an observed decrease in free energy of melting of approximately 0.5 kcal/mol relative to the control, right-handed, sequence in high salt. Transition melting temperatures (Tm) were found, in agreement with previous studies on polymeric DNA, to depend upon NaCl concentration in a complicated, nonlinear fashion. m values increase to maximal values at circa 1.0 M NaCl, but decrease thereafter with further addition of salt.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

The ability of atypical antipsychotic drugs vs. haloperidol to protect PC12 cells against MPP+-induced apoptosis.

The present study examined the effects of the atypical antipsychotic drugs clozapine, olanzapine, quetiapine and risperidone, on N-methyl-4-phenylpyridinium ion-induced apoptosis and DNA damage in PC12 cells, and explored the molecular mechanisms underlying these effects. Haloperidol, a typical antipsychotic drug, was used for comparison. Exposure of PC12 cells to 50 micro m N-methyl-4-phenylpyridinium ion for 24 h resulted in a 35-45% loss of cells in culture. Pretreatment with the aforementioned atypical antipsychotic drugs significantly reduced the N-methyl-4-phenylpyridinium ion-induced cell loss, whereas haloperidol (10-100 micro m) did not have this protective effect. Hoechst 33258 staining revealed the apoptotic nuclear features of the N-methyl-4-phenylpyridinium ion-induced cell death, and showed that the atypical antipsychotic drugs, but not haloperidol, effectively prevented PC12 cells from this N-methyl-4-phenylpyridinium ion-induced apoptosis. DNA fragmentation assays further confirmed the N-methyl-4-phenylpyridinium ion-induced nuclear fragmentation. Pretreatment with the atypical antipsychotic drugs completely prevented this nuclear fragmentation, whereas haloperidol only partially prevented it. In vitro oligonucleotide assays indicated an activation of a specific glycosylase that recognizes and cleaves bases (at the 8-hydroxyl-2-deoxyguanine site) that were damaged by N-methyl-4-phenylpyridinium ion. Pretreatment with the atypical antipsychotic drugs more effectively attenuated this N-methyl-4-phenylpyridinium ion-induced activation than did haloperidol. Northern blot analyses showed that the atypical antipsychotic drugs, but not haloperidol, blocked the N-methyl-4-phenylpyridinium ion-induced substantial increase of copper/zinc superoxide dismutase mRNA in PC12 cells. Atypical antipsychotic drugs slightly up-regulated the expression of copper/zinc superoxide dismutase mRNA, whereas haloperidol strongly increased the expression of copper/zinc superoxide dismutase mRNA. These data may account for the different therapeutic effects and side-effect profiles of typical and atypical antipsychotic drugs in schizophrenia.

1-Methyl-4-phenylpyridinium↗

Biphasic kinetics of the human DNA repair protein MED1 (MBD4), a mismatch-specific DNA N-glycosylase.

The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.

Base Pair Mismatch↗

Set1 is required for meiotic S-phase onset, double-strand break formation and middle gene expression.

The Set1 protein of Saccharomyces cerevisiae is a histone methyltransferase (HMTase) acting on lysine 4 of histone H3. Inactivation of the SET1 gene in a diploid leads to a sporulation defect. We have studied various processes that take place during meiotic differentiation in set1delta diploid cells. The absence of Set1 leads to a delay of meiotic S-phase onset, which reflects a defect in DNA replication initiation. The timely induction of meiotic DNA replication does not require the Set1 HMTase activity, but depends on the SET domain. In addition, set1delta displays a severe impairment of the DNA double-strand break formation, which is not only the consequence of the replication delay. Transcriptional profiling experiments show that the induction of middle meiotic genes, but not of early meiotic genes, is affected by the loss of Set1. In contrast to meiotic replication, the transcriptional induction of the middle meiotic genes appears to depend on the methylation of H3-K4. Our results unveil multiple roles of Set1 in meiotic differentiation and distinguish between HMTase-dependent and -independent Set1 functions.

DNA Damage↗

Defective expression of HRK is associated with promoter methylation in primary central nervous system lymphomas.

OBJECTIVES: Recently, it has been reported that expression of the HRK gene was significantly reduced by hypermethylation in astrocytic tumors. Our aim is to verify the alterations in the HRK gene in primary central nervous system lymphomas (PCNSLs). METHODS: We analyzed the hypermethylation status and expression of the gene and 12q13.1 loss of heterozygosity in 31 PCNSLs. RESULTS: A total of 13 PCNSLs (31%) demonstrated hypermethylation in either the promoter or exon 1; loss of HRK expression was immunohistochemically observed in 9 tumors and was significantly associated with promoter methylation. In addition, higher apoptotic counts were associated with HRK positivity. PCNSLs with HRK methylation also showed methylation of multiple genes, such as p14ARF, p16INK4a, RB1, p27Kip1 and O6-MGMT. Patients with tumors demonstrating concurrent methylation of more than half of their genes demonstrated significantly poorer survival and earlier recurrence. Hypermethylation of the HRK promoter alone was not associated with overall outcome, but relapse-free survival was significantly shorter. CONCLUSIONS: Our findings suggest that transcriptional repression of HRK is caused by promoter hypermethylation in PCNSL, and that the loss of HRK associated with the methylation profile of other genes is a potential step in the modulation of cellular death by apoptosis during PCNSL tumorigenesis.

Apoptosis↗

Chromosomal assignment of human genomic NotI restriction fragments in a two-dimensional electrophoresis profile.

Using DNA from sorted human chromosomes and two-dimensional gel electrophoresis, we assigned 2295 NotI sites, 43% of the total, to specific chromosomes and designated the procedure CA-RLGS (chromosome-assigned restriction landmark genomic scanning). Although the NotI enzyme is sensitive to DNA methylation, our results suggested that the majority of the spots did not seem to be affected by this modification. The NotI sites were distributed at higher levels in chromosomes 17, 19, and 22, suggesting higher gene content in these chromosomes. Most spots were assigned to unique chromosomes, but some spots were found on two or more chromosomes. Quantitative analysis revealed the intensity of the DNA spots on the sex chromosomes to be haploid and that of the chromosome 21 spots in DNA from a male with Down syndrome to be trisomic, although there were exceptions. We report here the first-generation CA-RLGS map of the human genome.

Chromosome Aberrations↗

Mutations and aberrant DNA methylation of the PROX1 gene in hematologic malignancies.

The homeobox gene PROX1 is related to the Drosophila prospero gene, which is expressed in the developing central nervous system and lens-secreting cone cells. We found that the PROX1 gene had missense and nonsense mutations in 4 of 29 hematologic cell lines analyzed. Decreased mRNA expression was also observed in half of these cell lines by RT-PCR. The restoration of PROX1 gene expression after treatment with the demethylating agent 5-aza-2'-deoxycytidine, as well as bisulfite sequencing analysis, indicated that gene silencing is caused by DNA hypermethylation at intron 1. Such hypermethylation was also seen in primary lymphomas (56.3%, 18/32) in a tumor-specific manner. These findings indicate that the profile of the PROX1 gene corresponds to that of a candidate tumor-suppressor gene.

Azacitidine↗

Mutational analysis of the function of Gln115 in the EcoRI restriction endonuclease, a critical amino acid for recognition of the inner thymidine residue in the sequence -GAATTC- and for coupling specific DNA binding to catalysis.

The Gln115 residue of the EcoRI restriction endonuclease has been proposed to form a hydrophobic contact to the methyl group of the inner thymidine of the EcoRI recognition sequence -GAATTC- and to be involved in intramolecular hydrogen bonds to the mainchain at positions 140 and 143 as well as to the side-chain of Asn173. We have exchanged Gln115 for Ala and Glu by site-directed mutagenesis and analysed the purified mutant proteins (Q115A and Q115E) biochemically and physico-chemically. Q115A and Q115E have the same secondary structure composition as wild-type EcoRI but are less stable towards thermal denaturation than the wild-type enzyme. In contrast to wild-type EcoRI the mutant proteins show a biphasic denaturation profile under alkaline pH, presumably because the amino acid exchange labilizes one part of the molecule, which unfolds before the rest of the protein is denatured. Q115A is catalytically inactive under normal buffer conditions, in part due to a diminished affinity towards DNA. At low ionic strength and alkaline pH, as well as in the presence of Mn2+, i.e. under conditions where wild-type EcoRI shows a relaxed specificity, Q115A is active, however not as much as wild-type EcoRI. Under these conditions it cleaves the canonical sequence -GAATTC- with the same kcat/Km value as the sequence -GAAUTC-, which differs from the former sequence by a single methyl group, while wild-type EcoRI shows a tenfold lower kcat/Km for cleavage of -GAAUTC- than for -GAATTC-. Binding experiments, carried out in the absence of Mg2+, demonstrate that Q115A has a similar affinity towards -GAATTC- as to -GAAUTC-, while wild-type EcoRI binds to -GAATTC- with a tenfold preference over -GAAUTC-. On the basis of these thermodynamic and kinetic results it can be concluded that the hydrophobic contact between the gamma-methylene group of Gln115 and the methyl group of the inner thymidine contributes about 3 kJ/mol (0.7 kcal/mol) to the energy of interaction, both in the ground and the transition state. Q115E is catalytically inactive under normal buffer conditions, but becomes active at low ionic strength or in the presence of Mn2+. Different from Q115A, Q115E is inactive at alkaline pH and its DNA binding affinity is highest at acidic pH.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

Construction and characteristics of 3-end enriched cDNA library from individual embryos of cattle.

To analyze stage-specific gene expression profiles of pre-implantation embryos and evaluate potential viability, techniques were adapted to generate 3-end enriched cDNA libraries from individual embryos of cattle based on RT-PCR methodology. The reproducibility of constructing a cDNA library was tested by five independent PCR experiments with specific primers for the presence of several rare genes such as DNMT1 (DNA methylation transferase 1), DNMT2, DNMT3A, Oct-4/3 (octmer-binding transcription factor), IFN-iota, IGF-2r (insulin like growth factor 2 receptor), and the housekeeping genes, H2A and beta-actin. Results indicated repeatability and that a proportion of expressed genes in the cDNA library from an individual embryo was not affected by limited PCR amplification. From the cDNA library, 134 clones were randomly selected for sequencing and showed that structure related elements accounted for 33.5% of transcripts and the energy- and metabolism-related genes were also an important component being 11.9% in the cDNA library. Approximately 14% of genes in the library were functionally unknown including greater than 5% of genes that were likely novel because there was no identity in Genbank. The frequency of structure-related genes such as beta-actin and ribosomal proteins in the cDNA library corresponded to other reports and suggested that the cDNA library constructed by RT-PCR might be proportional to the mRNA populations. The cDNA libraries constructed from different stage embryos will provide a powerful tool to explore novel genes relevant to embryogenesis, determine the profiling of stage-specific gene expression, and evaluate the potential viability of embryos.

Animals↗

Genome-wide epigenomic atlas and multi-omics responses of Eriocheir sinensis to natural extreme heat.

BACKGROUND: Global climate warming has led to increasingly frequent and prolonged extreme summer heat events, posing severe environmental challenges to aquaculture systems. Extreme summer heat can disrupt the performance of pond-cultured ectotherms. The Chinese mitten crab (Eriocheir sinensis) is an economically important freshwater crustacean, but coordinated molecular differences following contrasting natural summers remain incompletely characterized. RESULTS: We performed a comprehensive multi-omics analysis integrating meteorological monitoring, mRNA/lncRNA transcriptomics, small-RNA profiling of miRNAs, DNA methylomics, and LC-MS metabolomics in E. sinensis populations collected from Yancheng, China, between 2020 and 2024. Across the ten farms, survival was significantly lower in 2024, whereas yield and the proportion of large individuals showed nonsignificant downward trends. Gene-set analyses showed negative enrichment of cellular heat-response, protein-folding, oxidative-phosphorylation, and mitochondrial ATP-production terms in the 2024 cohort at the time of sampling. The integrated transcript annotation contained 72,240 lncRNAs and 63,833 mRNAs, and CpG was the predominant methylation context. Differential methylation analysis identified 73 regions and 185 cytosines, with hypomethylated events predominating within the significant subset. Metabolomic profiles differed between annual cohorts and mapped to carbohydrate, lipid, and amino-acid pathways. Cross-omics integration prioritized eight candidate genes-ADCY9, UNC79, UBN1, IFT52, ACO2, LOC126986070, LOC127001126, and LOC126997895-and qPCR reproduced the reported directions of expression for selected RNAs. CONCLUSION: This study provides the first integrative multi-omics framework for understanding chronic heat adaptation in E. sinensis. By linking transcriptomic, epigenomic, and metabolic remodeling, we elucidate the molecular mechanisms underlying energy imbalance, epigenetic reprogramming, and immune dysregulation during prolonged thermal stress. These findings offer valuable insights and genomic resources for breeding heat-tolerant crab strains and improving aquaculture resilience under ongoing climate change.

DNA methylation↗

ECHO: a nanopore sequencing-based workflow for (epi)genetic profiling of the human repeatome.

SUMMARY: The human genome is dominated by repetitive DNA, whose genetic and epigenetic variation plays a key role in gene regulation, genome stability, and disease. Recent advances in long-read sequencing now enable large-scale, haplotype-resolved, and DNA methylation-informative analysis of the human genome, including on previously inaccessible complex and repetitive regions. However, the comprehensive, simultaneous characterisation of the "human repeatome" remains challenging, largely due to the lack of comprehensive tools integrated in a single pipeline that can capture the full spectrum of variation across diverse types of DNA repeats. Here, we present ECHO, a user-friendly, Snakemake-based pipeline for the "(Epi)genomic Characterisation of Human Repetitive Elements using Oxford Nanopore Sequencing." ECHO provides a reproducible and scalable framework for end-to-end analysis of whole-genome nanopore sequencing data, enabling integrative but also tailored (epi)genetic analyses of the human repeatome. AVAILABILITY AND IMPLEMENTATION: ECHO is freely available at Github: https://github.com/leenput/ECHO-pipeline, with the archived version at Zenodo: https://zenodo.org/records/19068468.

Humans↗

Restriction endonuclease analysis of total deoxyribonucleic acid of Mycobacterium tuberculosis H37RV (ATCC 27294) and of M. bovis (ATCC 19210).

Total DNA from two slowly-growing pathogenic mycobacterial species propagated in vitro was isolated, digested with each of 34 restriction endonucleases and analyzed by agarose gel electrophoresis. The most distinct profiles for M. tuberculosis (ATCC 27294) and for M. bovis (ATCC 19210) were obtained respectively using (BamHI, DraI, ClaI, EcoRI, EcoRV, HindIII, HpaI, SalI, SmaI, XbaI, and XmaI). The patterns produced for these strains were reproducible and distinguishable from each other. However, with several enzymes the patterns for M. tuberculosis and M. bovis were similar. Evidence was obtained for the presence of dam and dcmI methylations in the DNA of each mycobacterial species.

DNA Restriction Enzymes↗