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PubMed1996

Tad, a Neurospora LINE-like retrotransposon exhibits a complex pattern of transcription.

Tad is a LINE-like element from Neurospora. Inactive Tad elements have been found in all strains of Neurospora examined; however, active copies of Tad were initially found in a single strain (Adiopodoumé) collected from nature. Active Tad elements can be transferred to laboratory strains by means of crosses, heterokaryons or by transformation with cloned active elements. We have analyzed the pattern of transcripts from Tad elements present in strains derived from Adiopodoumé containing either high or low copy numbers of active Tad. In both kinds of strains we found a complex set of discrete polyadenylated transcripts. As expected, a full-length transcript was detected with 5' and 3' ends that coincide with the consensus 5' and 3' ends of the Tad element. Two additional transcripts with the same polarity as the full-length transcript were identified. One of these plus-strand transcripts is 2.0 kb in length with a 5' end identical to that of the full-length transcript and an internal polyadenylation site. The other (4.2 kb) has an internal start site with termination occurring at the 3' consensus end of the element. The 2.0-kb transcript encodes all of ORF1. The 4.2 kb transcript could encode a 5' truncated version of ORF2. Two minusstrand transcripts were also consistently identified. These represent overlapping transcripts from the 3' tail of Tad. These transcripts share a small intron and 3' polyadenylation site. Their 5' start sites are both internal to the consensus 3' end of Tad. Possible functions of this complex array of transcripts are discussed.

Base Sequence↗
PubMed2000

Comparative genomic hybridization reveals complex genetic changes in primary breast cancer tumors and their cell lines.

DNA copy number changes were characterized by comparative genomic hybridization (CGH) in 18 breast cancer cell lines. In 5 of these, the results were comparable with those from the primary tumors of which the cell lines were established. All of the cell lines showed extensive DNA copy number changes, with a mean of 16.3 +/- 1.1 aberrations per sample (range 7-26). All of the cell lines had a gain at 8q22-qter. Other common gains of DNA sequences occurred at 1q31-32 (89%), 20q12-q13.2 (83%), 8q13 (72%), 3q26.1-qter (67%), 17q21-qter (67%) 5p14 (61%), 6p22 (56%), and 22pter-qter (50%). High-level amplifications were observed in all cell lines; the most frequent minimal common regions were 8q24.1 (89%), 20q12 (61%), 1q41 (39%), and 20p11.2 (28%). Losses were observed less frequently than gains and the minimal common regions of the most frequent losses were Xq11-q12 (56%), Xp11.2-pter (50%), 13q21 (50%), 8p12-pter (44%), 4p13-p14 (39%), 6q15-q22 (39%), and 18q11.2-qter (33%). Although the cell lines showed more DNA copy number changes than the primary tumors, all aberrations, except one found in a primary tumor, were always present in the corresponding cell line. High-level amplifications found both in primary tumors and cell lines were at 1q, 8q, 17q, and 20q. The DNA copy number changes detected in these cell lines can be valuable in investigation of tumor progression in vitro and for a more detailed mapping and isolation of genes implicated in breast cancer.

Breast Neoplasms↗
PubMed2004

Detection of BK virus and JC virus DNA in urine samples from immunocompromised (HIV-infected) and immunocompetent (HIV-non-infected) patients using polymerase chain reaction and microplate hybridisation.

BACKGROUND: The majority of the human population is infected with two human polyomaviruses BK virus (BKV) and JC virus (JCV) during childhood. After initial infection both viruses persist within renal system. Reactivation of both viruses may be linked with immunodeficiency or immunosuppressive therapy. OBJECTIVE: To evaluate the relationship between immunodeficiency and viruria, prevalence of BK and JC viruria over time was investigated in a cohort of HIV seropositive individuals at different stages of disease. The excretion in this group was compared with virus excretion in their HIV seronegative partners and in an unselected cohort of patients attending a Genito-Urinary Medicine (GUM) clinic. STUDY DESIGN: The excretion of BKV and JCV DNA in multiple urine samples from HIV-infected patients at different stages of disease and their HIV-negative partners, and in single samples from a cohort of patients at a GUM clinic was investigated. A microplate hybridisation method was developed to increase both the sensitivity and specificity of detection of the PCR product. The method was also applied to estimate the DNA copy numbers of BKV and JCV in urine samples. RESULTS: Within the HIV group, the level of immunosuppression (CD4+ category) was not associated with JCV viruria. By contrast, there was a modest correlation between immunodeficiency as indicated by a decline in CD4+ count and BKV viruria. Shedding of both BKV and JCV DNA together in urine samples of HIV-infected patients was much higher than in control groups (P = 0.02), indicating that HIV infection may associate with polyomavirus reactivation. The incidence of flu-like syndrome was much higher in HIV-infected asymptomatic individuals than acquired immunodeficiency syndrome (AIDS)-related complex (ARC)/AIDS patients. In general, the concentration of BKV DNA viruria (DNA copy number) was dependent to CD4+ counts (P = 0.008) while concentration of JCV DNA was independent to CD4+ cell count (P = 0.54). The prevalence of BKV and JCV DNA in patients who were infected with C. trachomatis was 9/50 (18%) and 11/50 (22%), respectively. BKV and JCV DNA was detected in 3/19 (15%) and 2/19 (10%) of patients who were infected with N. gonorrhoea. Results suggested that persons infected with C. trachomatis were more likely to show BKV and JCV viruria. CONCLUSION: These results confirm that shedding of BK and JC viruses in urine is not exclusively found in immunosupression, it may also occur in healthy individuals. The frequency of virus excretion is however, apparently increased in HIV-infected patients, although no firm statistical difference could be established. One of the interesting aspects of these findings was the relatively high incidence of BKV and JCV viruria in both control groups, i.e. HIV-negative partners of HIV-infected patients and patients attending a GUM clinic.

Adolescent↗
PubMed2006

DNA copy-number analysis in bipolar disorder and schizophrenia reveals aberrations in genes involved in glutamate signaling.

Using bacterial artificial chromosome (BAC) array comparative genome hybridization (aCGH) at approximately 1.4 Mbp resolution, we screened post-mortem brain DNA from bipolar disorder cases, schizophrenia cases and control individuals (n=35 each) for DNA copy-number aberrations. DNA copy number is a largely unexplored source of human genetic variation that may contribute risk for complex disease. We report aberrations at four loci which were seen in affected but not control individuals, and which were verified by quantitative real-time PCR. These aberrant loci contained the genes encoding EFNA5, GLUR7, CACNG2 and AKAP5; all brain-expressed proteins with known or postulated roles in neuronal function, and three of which (GLUR7, CACNG2 and AKAP5) are involved in glutamate signaling. A second cohort of psychiatric samples was also tested by quantitative PCR using the primer/probe sets for EFNA5, GLUR7, CACNG2 and AKAP5, and samples with aberrant copy number were found at three of the four loci (GLUR7, CACNG2 and AKAP5). Further scrutiny of these regions may reveal insights into the etiology and genetic risk factors for these complex psychiatric disorders.

A Kinase Anchor Proteins↗
PubMed2025

Optical genome mapping improves clinical interpretation of constitutional copy-number gains and reduces their VUS burden.

PURPOSE: Genomic structure of copy-number gains is critical for their clinical interpretation but cannot be determined by chromosomal microarray (CMA) analysis, which does not provide information about chromosomal location and orientation of multiplied regions. We thus hypothesized that in CMA testing gains have higher probability than losses to be classified as variants of uncertain significance (VUS) and that structural information from optical genome mapping (OGM) may improve their interpretation. METHODS: Using a χ2 test, we assessed the association between classification of copy-number variants as VUS and their type (gains vs losses) in a cohort of 4073 CMA cases. Thirty-three VUS gains involving disease-associated genes were characterized by OGM to evaluate if OGM data enable their more conclusive clinical interpretation. RESULTS: The proportion of variants reported as VUS compared with likely pathogenic/pathogenic was significantly higher for gains than losses, confirming their increased VUS burden. OGM successfully determined genomic structure for all 33 copy-number gains, showing that 26 of 33 were tandem duplications and 7 of 33 were complex rearrangements. Structural information facilitated clinical interpretation in majority of the cases; it supported benign nature for 27 of 33 gains and was inconclusive or supported pathogenic role for 6 of 33. An estimated 20% of reported VUS gains would not have been reportable if we had OGM data. CONCLUSION: We illustrate a specific advantage of OGM compared with CMA: in addition to detecting both copy-number variants and balanced rearrangements, OGM improves clinical interpretation of copy-number gains by providing structural information and is thus expected to significantly decrease their VUS burden.

Humans↗
PubMed1991

The Staphylococcus aureus mutation pcrA3 leads to the accumulation of pT181 replication initiation complexes.

In Staphylococcus aureus cells carrying the pcrA3 chromosomal mutation, plasmid pT181 and its derivatives were maintained at a reduced copy number. A significant proportion of their DNA migrated during agarose gel electrophoresis as nicked DNA. The results obtained in the characterization of this plasmid DNA species show that it represents replication initiation complexes. Such complexes could not be detected in a wild-type host. The replication initiation complexes present in pcrA3 cells could resume replication after a lag. It was concluded from these results that the pcrA3 host mutation affected a step in plasmid pT181 replication immediately following the formation of the replication initiation complex, and that in pcrA3 this step became rate-limiting for plasmid pT181 replication.

Bacterial Proteins↗
PubMed2003

Photosystem I trimers from Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits bind an IsiA ring of 17 units.

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of Photosystem I (PSI) and the chlorophyll-binding protein IsiA from a mutant of the cyanobacterium Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits. The circular complex consists of a central PSI trimer surrounded by a ring of 17 IsiA units, one less than in the wild-type supercomplex. We conclude that PsaF and PsaJ are not obligatory for the binding of the IsiA ring, and that the size of the PSI complex determines the number of IsiA units in the ring. The resulting number of 17 copies implies that each PSI monomer has a different association to the IsiA ring.

Bacterial Proteins↗
PubMed2004

FISH cytogenetics and prognosis in breast and non-small cell lung cancers.

BACKGROUND: Interphase cytogenetics by fluorescence in situ hybridization (FISH) has been demonstrated to be a valuable diagnostic tool in effusions from patients with solid tumors. As the next step, we investigated whether certain patterns of numeric aberrations in malignant effusion cells supply prognostic information. METHODS: From a large series of effusions from patients with solid tumors, 55 effusions from breast cancer and 39 effusions from non-small cell lung cancer (NSCLC) were classified as malignant by cytology or FISH. Tumor cells were classified as FISH aneuploid for chromosome 11 and/or 17 or as not aneuploid. Predominant cytogenetic anomalies and patterns of intratumor cytogenetic heterogeneity were brought in relation to overall survival rate. RESULTS: There was no difference with respect to overall survival rate when effusions with or without aneuploidy for chromosomes 11 and 17 were compared. Likewise, in effusions with aneuploidy, there was no difference in overall survival rate among patients with different modal chromosome copy numbers (e.g., trisomy vs. tetrasomy 11) or among patients with a low or high grade of intratumor complexity (defined by the intratumor heterogeneity of FISH aneuploidy). In breast cancer, aneuploidy with gain of chromosome 11 was associated with a significantly superior survival rate, suggesting that amplification of chromosome 11 DNA is associated with a less aggressive phenotype. CONCLUSIONS: Simple chromosomal changes as determined by FISH, such as gain of chromosome 11 copy numbers in breast cancer, may be prognostic. Prospective studies in primary tumors that classify distinct prognostic groups by FISH cytogenetics are warranted.

Aneuploidy↗
PubMed1994

Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors.

Comparative genomic hybridization (CGH) is a powerful new method for molecular cytogenetic analysis of cancer. In a single hybridization, CGH provides an overview of DNA sequence copy number changes (losses, deletions, gains, amplifications) in a tumor specimen and maps these changes on normal chromosomes. CGH is based on the in situ hybridization of differentially labeled total genomic tumor DNA and normal reference DNA to normal human metaphase chromosomes. After hybridization and fluorescent staining of the bound DNAs, copy number variations among the different sequences in the tumor DNA are detected by measuring the tumor/normal fluorescence intensity ratio for each locus in the target metaphase chromosomes. CGH is in particular useful for analysis of DNA sequence copy number changes in common solid tumors where high-quality metaphase preparations are often difficult to make, and where complex karyotypes with numerous markers, double minutes, and homogeneously stained chromosomal regions are common. CGH only detects changes that are present in a substantial proportion of tumor cells (i.e., clonal aberrations). It does not reveal translocations, inversions, and other aberrations that do not change copy number. At present, CGH is a research tool that complements previous methods for genetic analysis. CGH will advance our understanding of the genetic progression of cancer and highlight important genomic regions for further study. Direct clinical applications of CGH are possible, but will require further development and validation of the technique. We describe here our recent optimized procedures for CGH, including DNA labeling, hybridization, fluorescence microscopy, digital image analysis, data interpretation, and quality control, emphasizing those steps that are most critical. We will also assess sensitivity and resolution limits of CGH as well as discuss possible future technical improvements.

Chromosome Aberrations↗
PubMed2001

Clinical and molecular features of the Carney complex: diagnostic criteria and recommendations for patient evaluation.

Carney complex is a multiple neoplasia syndrome featuring cardiac, endocrine, cutaneous, and neural tumors, as well as a variety of pigmented lesions of the skin and mucosae. Carney complex is inherited as an autosomal dominant trait and may simultaneously involve multiple endocrine glands, as in the classic multiple endocrine neoplasia syndromes 1 and 2. Carney complex also has some similarities to McCuneAlbright syndrome, a sporadic condition that is also characterized by multiple endocrine and nonendocrine tumors. Carney complex shares skin abnormalities and some nonendocrine tumors with the lentiginoses and certain of the hamartomatoses, particularly Peutz-Jeghers syndrome, with which it shares mucosal lentiginosis and an unusual gonadal tumor, large-cell calcifying Sertoli cell tumor. Careful clinical analysis has enabled positional cloning efforts to identify two chromosomal loci harboring potential candidate genes for Carney complex. Most recently, at the 17q22-24 locus, the tumor suppressor gene PRKAR1A, coding for the type 1alpha regulatory subunit of PKA, was found to be mutated in approximately half of the known Carney complex kindreds. PRKAR1A acts a classic tumor suppressor gene as demonstrated by loss of heterozygosity at the 17q22-24 locus in tumors associated with the complex. The second locus, at chromosome 2p16, to which most (but not all) of the remaining kindreds map, is also involved in the molecular pathogenesis of Carney complex tumors, as demonstrated by multiple genetic changes at this locus, including loss of heterozygosity and copy number gain. Despite the known genetic heterogeneity in the disease, clinical analysis has not detected any corresponding phenotypic differences between patients with PRKAR1A mutations and those without. This article summarizes the clinical manifestations of Carney complex from a worldwide collection of affected patients and also presents revised diagnostic criteria for Carney complex. In light of the recent identification of mutations in the PRKAR1A gene, an estimate of penetrance and recommendations for genetic screening are provided.

Adrenal Cortex Neoplasms↗
PubMed2005

Rapid array-based genomic characterization of a subtle structural abnormality: a patient with psychosis and der(18)t(5;18)(p14.1;p11.23).

Array-based copy number analysis has recently emerged as a rapid means of mapping complex and/or subtle chromosomal abnormalities. We have compared two such techniques, using bacterial artificial chromosome (BAC) and single nucleotide polymorphism (SNP) arrays in the evaluation of a 45-year-old woman with dysmorphic features, mental retardation, psychosis, and an unbalanced derivative chromosome 18, (46,XX, der(18)t(18;?)(p12;?)). Both array-based methods demonstrated that the additional material on chromosome 18 was of 5p origin. The 5p duplication mapped telomeric to 25.320 Mb (BAC array) and 25.607 Mb (SNP array), corresponding to the band 5p14.1. Both BAC and SNP arrays also showed a deletion involving chromosome 18p extending telomeric from 8.437 Mb (BAC array) and 8.352 Mb (SNP array), corresponding to the band 18p11.23. Molecular cytogenetic mapping using fluorescence in situ hybridization (FISH) supported the array findings and further refined the breakpoint regions, confirming that the BAC and SNP chips were both useful in this regard. Both case reports and linkage analyses have implicated these chromosomal intervals in psychosis. The array-based experiments were completed over the course of several days. While these methods do not eliminate the requirement for traditional fine-mapping, they provide an efficient approach to identifying the origin and extent of deleted and duplicated material in chromosomal rearrangements.

Chromosome Banding↗
PubMed2005

High dose melphalan in the treatment of advanced neuroblastoma: results of a randomised trial (ENSG-1) by the European Neuroblastoma Study Group.

BACKGROUND: High dose myeloablative chemotherapy ("megatherapy"), with haematopoietic stem cell support, is now widely used to consolidate response to induction chemotherapy in patients with advanced neuroblastoma. PROCEDURE: In this study (European Neuroblastoma Study Group, ENSG1), the value of melphalan myeloablative "megatherapy" was evaluated in a randomised, multi-centre trial. Between 1982 and 1985, 167 children with stages IV and III neuroblastoma (123 stage IV > 1 year old at diagnosis and 44 stage III and stage IV from 6 to 12 months old at diagnosis) were treated with oncovin, cisplatin, epipodophyllotoxin, and cyclophosphamide (OPEC) induction chemotherapy every 3 weeks. After surgical excision of primary tumour, the 90 patients (69% of the total) who achieved complete response (CR) or good partial response (GPR) were eligible for randomisation either to high dose melphalan (180 mg per square meter) with autologous bone marrow support or to no further treatment. RESULTS: Sixty-five (72%) of eligible children were actually randomised and 21 of these patients were surviving at time of this analysis, with median follow-up from randomisation of 14.3 years. Five year event-free survival (EFS) was 38% (95% confidence interval (CI) 21-54%) in the melphalan-treated group and 27% (95% CI 12-42%) in the "no-melphalan" group. This difference was not statistically significant (P = 0.08, log rank test) but for the 48 randomised stage IV patients aged >1 year at diagnosis outcome was significantly better in the melphalan-treated group-5 year EFS 33% versus 17% (P = 0.01, log rank test). CONCLUSIONS: In this trial, high dose melphalan improved the length of EFS and overall survival of children with stage IV neuroblastoma >1 year of age who achieved CR or GPR after OPEC induction therapy and surgery. Multi-agent myeloablative regimens are now widely used as consolidation therapy for children with stage IV disease and in those with other disease stages when the MYCN gene copy number in tumour cells is amplified. Because they are more toxic, complex, and costly these combination megatherapy regimens should be compared with single agent melphalan in randomised clinical trials.

Antineoplastic Agents, Alkylating↗
PubMed2005

Individual differences in drug dependence in rats: the role of genetic factors and life events.

Drug dependence and addiction is a chronic mental illness that has far reaching consequences for society in terms of economic loss, health costs and judicial problems. A crucial question in drug addiction, is what factors are involved in its aetiology, and especially what mediates the shit from use to abuse. As in most other mental illnesses, addiction can best be described using the so-called three hit model, which states that a disease results from an interaction between genetic factors, early lie events and late environmental factors. However, the precise nature of these factors still remains to be elucidated. This present review discusses the results from an animal model in which these three different hit are currently being investigated. The apomorphine susceptible (APO-SUS) and apomorphine unsusceptible (APO-UNSUS) rats, originally selected on the basis of their behavioural response to the dopaminergic agonist apomorphine, were recently found to be genetically different in the number of gene copies of a component of the gamma-secretase complex called Aph-1b. Whereas APO-UNSUS rats have three copies of the gene, APO-SUS rats have either 1 or 2 copies. In addition we have shown that these rats show differences in cocaine and alcohol self-administration, and that both early life events and late environmental factors can alter this self-administration behaviour. Thus, the data so far support the hypothesis that the APO-SUS and APO-UNSUS rats offer an interesting animal model for drug dependence in which genes and environment interact. We finally propose a theoretical model which can explain this gene-environment interaction.

Amyloid Precursor Protein Secretases↗
PubMed2005

Common deletions and SNPs are in linkage disequilibrium in the human genome.

Humans show great variation in phenotypic traits such as height, eye color and susceptibility to disease. Genomic DNA sequence differences among individuals are responsible for the inherited components of these complex traits. Reports suggest that intermediate and large-scale DNA copy number and structural variations are prevalent enough to be an important source of genetic variation between individuals. Because association studies to identify genomic loci associated with particular phenotypic traits have focused primarily on genotyping SNPs, it is important to determine whether common structural polymorphisms are in linkage disequilibrium with common SNPs, and thus can be assessed indirectly in SNP-based studies. Here we examine 100 deletion polymorphisms ranging from 70 bp to 7 kb. We show that common deletions and SNPs ascertained with similar criteria have essentially the same distribution of linkage disequilibrium with surrounding SNPs, indicating that these polymorphisms may share evolutionary history and that most deletion polymorphisms are effectively assayed by proxy in SNP-based association studies.

Gene Frequency↗
PubMed2004

Comparative genomic hybridization (CGH)-arrays pave the way for identification of novel cancer-related genes.

Comparative genomic hybridization (CGH) has already made a significant impact on cancer cytogenetics. However, CGH to metaphase chromosomes can provide only limited resolution at the 5-10 Mb level. To circumvent this limitation, array-based CGH has been devised. Since spotted DNAs in a CGH-array contain sequence information directly connected with the genome database, we can easily note particular biological aspects of genes that lie within regions involved in copy-number aberrations. High-density, sub-megabase arrays can reveal nonrandom chromosome copy-number aberrations responsible for neoplastic transformation that have been masked under complex karyotypes in epithelial solid tumors. High-density CGH-array therefore paves the way for identification of disease-related genetic aberrations that have not yet been detected by existing technologies, and array-based CGH technology should soon be practical for diagnosis of cancer or genetic diseases in the clinical setting.

Chromosome Aberrations↗
PubMed2004

A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes.

BACKGROUND: Sequencing the genomes of multiple, taxonomically diverse eukaryotes enables in-depth comparative-genomic analysis which is expected to help in reconstructing ancestral eukaryotic genomes and major events in eukaryotic evolution and in making functional predictions for currently uncharacterized conserved genes. RESULTS: We examined functional and evolutionary patterns in the recently constructed set of 5,873 clusters of predicted orthologs (eukaryotic orthologous groups or KOGs) from seven eukaryotic genomes: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Encephalitozoon cuniculi. Conservation of KOGs through the phyletic range of eukaryotes strongly correlates with their functions and with the effect of gene knockout on the organism's viability. The approximately 40% of KOGs that are represented in six or seven species are enriched in proteins responsible for housekeeping functions, particularly translation and RNA processing. These conserved KOGs are often essential for survival and might approximate the minimal set of essential eukaryotic genes. The 131 single-member, pan-eukaryotic KOGs we identified were examined in detail. For around 20 that remained uncharacterized, functions were predicted by in-depth sequence analysis and examination of genomic context. Nearly all these proteins are subunits of known or predicted multiprotein complexes, in agreement with the balance hypothesis of evolution of gene copy number. Other KOGs show a variety of phyletic patterns, which points to major contributions of lineage-specific gene loss and the 'invention' of genes new to eukaryotic evolution. Examination of the sets of KOGs lost in individual lineages reveals co-elimination of functionally connected genes. Parsimonious scenarios of eukaryotic genome evolution and gene sets for ancestral eukaryotic forms were reconstructed. The gene set of the last common ancestor of the crown group consists of 3,413 KOGs and largely includes proteins involved in genome replication and expression, and central metabolism. Only 44% of the KOGs, mostly from the reconstructed gene set of the last common ancestor of the crown group, have detectable homologs in prokaryotes; the remainder apparently evolved via duplication with divergence and invention of new genes. CONCLUSIONS: The KOG analysis reveals a conserved core of largely essential eukaryotic genes as well as major diversification and innovation associated with evolution of eukaryotic genomes. The results provide quantitative support for major trends of eukaryotic evolution noticed previously at the qualitative level and a basis for detailed reconstruction of evolution of eukaryotic genomes and biology of ancestral forms.

Animals↗
PubMed2004

[Use of IS6110 polymorphism analysis in molecular epidemiological studies of tuberculosis (Belgrade study results)].

Tuberculosis (TB) remains a major health problem worldwide. One of the main approaches to tackling TB today is the control of transmission of the disease through monitoring the transmission of specific strains of Mycobacterium tuberculosis. In the past, efforts to type strains of M. tuberculosis were hampered by the lack of strain-specific phenotypic markers. In recent years, novel approaches to studying the epidemiology of TB have been provided by molecular biological techniques based on DNA fingerprinting. The most widely used polymorphic marker is the transposable element IS6110, which is an insertion sequence found throughout M. tuberculosis complex. It is a highly polymorphic marker which varies in both copy number and location in the M. tuberculosis genome. A standardized methodology for IS6110 DNA fingerpriniting of M. tuberculosis exploits restriction fragment length polymorphism analysis. To date, IS6110 fingerprinting has been successfully used to trace the TB transmission in different regions and populations, to quantify the relative contribution of recent infection to the TB population burden, to answer the question of endogenous reactivation versus exogenous reinfection, to trace small-scale outbreaks of TB, to analyze spread of drug-resistant and multidrug resistant strains, to confirm laboratory cross-contaminations, etc. The objective of this study was to illustrate the basic principles of molecular epidemilological studies of TB. Results of the study, which provided the first insight into the status of TB in Belgrade based on implementation of molecular methods, were used as an example.

DNA Fingerprinting↗
PubMed1983

Genetic mapping in the major histocompatibility complex by restriction enzyme site polymorphisms: most mouse class I genes map to the Tla complex.

From a genomic library constructed from sperm DNA of the inbred BALB/c mouse, we previously isolated 54 cosmid clones that contain 36 class I genes and can be divided by restriction map analyses into 13 gene clusters. We have isolated single- and low-copy DNA probes from each of these clusters to visualize restriction enzyme site polymorphisms in the DNAs from various congeneic and recombinant congeneic mice. These polymorphisms permit us to map each of the 13 cosmid clusters to a precise location in the major histocompatibility complex of the mouse. Thirty-one of 36 class I genes map into the Tla complex of the major histocompatibility complex whereas the remaining 5 genes map to the H-2 complex. Thus, all 36 class I genes are located in the major histocompatibility complex. Analysis of the number of restriction enzyme fragments visualized by the single- and low-copy DNA probes suggests that the class I genes in different inbred strains of mice probably undergo gene duplications and deletions, presumably by homologous but unequal crossing-over.

Animals↗