Functional analysis of the DXPas34 locus, a 3' regulator of Xist expression.

X inactivation in female mammals is controlled by a key locus on the X chromosome, the X-inactivation center (Xic). The Xic controls the initiation and propagation of inactivation in cis. It also ensures that the correct number of X chromosomes undergo inactivation (counting) and determines which X chromosome becomes inactivated (choice). The Xist gene maps to the Xic region and is essential for the initiation of X inactivation in cis. Regulatory elements of X inactivation have been proposed to lie 3' to Xist. One such element, lying 15 kb downstream of Xist, is the DXPas34 locus, which was first identified as a result of its hypermethylation on the active X chromosome and the correlation of its methylation level with allelism at the X-controlling element (Xce), a locus known to affect choice. In this study, we have tested the potential function of the DXPas34 locus in Xist regulation and X-inactivation initiation by deleting it in the context of large Xist-containing yeast artificial chromosome transgenes. Deletion of DXPas34 eliminates both Xist expression and antisense transcription present in this region in undifferentiated ES cells. It also leads to nonrandom inactivation of the deleted transgene upon differentiation. DXPas34 thus appears to be a critical regulator of Xist activity and X inactivation. The expression pattern of DXPas34 during early embryonic development, which we report here, further suggests that it could be implicated in the regulation of imprinted Xist expression.

Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells.

Initiation of X chromosome inactivation requires the presence, in cis, of the X inactivation center (XIC). The Xist gene, which lies within the XIC region in both human and mouse and has the unique property of being expressed only from the inactive X chromosome in female somatic cells, is known to be essential for X inactivation based on targeted deletions in the mouse. Although our understanding of the developmental regulation and function of the mouse Xist gene has progressed rapidly, less is known about its human homolog. To address this and to assess the cross-species conservation of X inactivation, a 480-kb yeast artificial chromosome containing the human XIST gene was introduced into mouse embryonic stem (ES) cells. The human XIST transcript was expressed and could coat the mouse autosome from which it was transcribed, indicating that the factors required for cis association are conserved in mouse ES cells. Cis inactivation as a result of human XIST expression was found in only a proportion of differentiated cells, suggesting that the events downstream of XIST RNA coating that culminate in stable inactivation may require species-specific factors. Human XIST RNA appears to coat mouse autosomes in ES cells before in vitro differentiation, in contrast to the behavior of the mouse Xist gene in undifferentiated ES cells, where an unstable transcript and no chromosome coating are found. This may not only reflect important species differences in Xist regulation but also provides evidence that factors implicated in Xist RNA chromosome coating may already be present in undifferentiated ES cells.

Use of polymerase chain reaction for improved diagnosis of tuberculosis in children.

OBJECTIVE: To study the value of a rapid diagnostic method based on the amplification by polymerase chain reaction (PCR) of a fragment of the IS6110 insertion element for the detection of Mycobacterium tuberculosis in children. DESIGN: We tested 199 specimens obtained from 68 children referred for evaluation of suspected tuberculosis. RESULTS: In 83.3% of children with active disease and 38.9% with tuberculous infection but no evidence of disease, at least one positive PCR result was observed. No child without tuberculosis had positive PCR results (100% specificity). The sensitivity of the PCR was increased by testing of multiple samples from the same child and use of Chelex particles (Bio-Rad Laboratories, Ivry, France) rather than guanidine isothiocyanate-silica particles for DNA extraction. Bronchoalveolar lavage samples were no more useful than gastric aspirates. CONCLUSIONS: If appropriate laboratory methods are used, DNA amplification is a reliable method for the early diagnosis of tuberculosis in children and appears to be very helpful in clinical pediatric practice when the diagnosis of active tuberculosis is difficult or needs to be rapidly confirmed.

The detection of Mycobacterium tuberculosis in uncultured clinical specimens using the polymerase chain reaction and a non-radioactive DNA probe.

A Sal I-Hin dIII restriction fragment from Mycobacterium tuberculosis was found to hybridize specifically with genomic DNA from M. tuberculosis. Primers were designed from the sequence of this fragment and used to amplify uniquely M. tuberculosis-group DNA in a polymerase chain reaction. It is suggested that a combination of these primers and an acetylaminofluorene-labelled probe will prove to be a useful tool for the early diagnosis of tuberculous infections.

Diagnosis of tuberculosis by DNA amplification in clinical practice evaluation.

Various polymerase chain reaction (PCR) assays have been devised for the rapid identification of mycobacteria in clinical specimens. To assess the value of such assays in routine laboratory work the results obtained by PCR were compared with those obtained by standard microbiological methods for 514 specimens collected for investigation of mycobacterial infection. Specimens were tested for the presence of Mycobacterium tuberculosis complex and atypical mycobacteria in two assays, one based on amplification of the 65 kDa gene and the other on the IS6110 insertion sequence. For the 489 samples that did not contain inhibitors of the amplification reaction PCR findings correlated well with bacteriological and/or clinical data in 476 (97.4%). 6 PCR results turned out to be false negatives, 3 to be false positives and 4 to be mis-identification of strains. Pre-treatment of samples with guanidium thiocyanate reduced the proportion of false-negative results and of samples that contained inhibitors. This study confirms the potential of DNA amplification for early diagnosis of mycobacterial infections.