Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by attacks of fever and serositis. In this paper, we define a minimal co-segregating region of 60 kb containing the FMF gene (MEFV) and identify four different transcript units within this region. One of these transcripts encodes a new protein (marenostrin) related to the ret-finger protein and to butyrophilin. Four conservative missense variations co-segregating with FMF have been found within the MEFV candidate gene in 85% of the carrier chromosomes. These variations, which cluster at the carboxy terminal domain of the protein, were not present in 308 control chromosomes, including 162 validated non-carriers. We therefore propose that the sequence alterations in the marenostrin protein are responsible for the FMF disease.

A candidate gene for familial Mediterranean fever

One stop mycology

Gene amplification plays a critical role in tumor progression. Hence, understanding the factors triggering this process in human cancers is an important concern. Unfortunately, the structures formed at early stages are usually unavailable for study, hampering the identification of the initiating events in tumors. Here, we show that the region containing the PIP gene, which is overexpressed in 80% of primary and metastatic breast cancers, is duplicated in the breast carcinoma cell line T47D. The two copies are organized as a large palindrome, lying 'in loco' on one chromosome 7. Such features constitute the landmark of the breakage-fusion-bridge (BFB) cycle mechanism. In hamster cells selected in vitro to resist cytotoxic drugs, common fragile site (CFS) activation has been shown to trigger this mechanism. Here, we characterize FRA7I at the molecular level and demonstrate that it lies 2 Mb telomeric to the PIP gene and sets the distal end of the repeated sequence. Moreover, our results suggest that the BFB process was frozen within the first cycle by healing of the broken chromosome. T47D cells thus offer a unique opportunity to observe the earliest products of the BFB cycle mechanism. Our findings constitute the first evidence that this amplification mechanism can be initiated in vivo by fragile site activation.

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Initiation of the breakage–fusion-bridge mechanism through common fragile site activation in human breast cancer cells: the model of PIP gene duplication from a break at FRA7I

The Le(x) oligosaccharide is expressed in organ buds progressing in mesenchyma, during human embryogenesis. Myeloid-like alpha3-fucosyltransferases are good candidates to synthesize this oligosaccharide. We investigated by Northern analysis all the alpha3-fucosyltransferase gene transcripts and only FUT4 and FUT9 were detected. The enzymes encoded by the FUT4 and FUT9 genes are the first alpha3-fucosyltransferases strongly expressed during the first two months of embryogenesis. The Northern profile of expression of the embryo FUT4 transcripts is similar in size and sequence to the known FUT4 transcripts of 6 kb, 3 kb, and 2.3 kb, but a new FUT9 transcript of 2501 bp, different from the known mouse (2170 bp) and human (3019 bp) transcripts was cloned. FUT3, FUT5, FUT6, and FUT7 were not detected by Northern blot. The FUT3 and FUT6 transcripts start to appear at this stage, but are only detected by reverse transcriptase-PCR analysis. The expression of FUT5 is weaker than FUT3 and FUT6 and the RT-PCR signal is faint and irregular. FUT7 is not detected at all. Using mRNA from 40- to 65-day-old embryos, we have prepared different hexamer and oligo-dT cDNA libraries and cloned, by rapid amplification cDNA ends-PCR, FUT4 and FUT9 alpha3-fucosyltransferase transcripts. The tissue expression of the embryonic FUT9 transcript is closer to that observed for the mouse (brain), than to the known human (stomach) transcripts. The acceptor specificity and the kinetics of the alpha3-fucosyltransferase encoded by this FUT9 transcript are similar to the FUT4 enzyme, except for the utilization of the lac-di-NAc acceptor which is not efficiently transformed by the FUT9 enzyme. Like FUT4, this embryonic FUT9 is N-ethylmaleimide and heat resistant and the corresponding gene was confirmed to be localized in the chromosome band 6q16. Finally, this FUT9 transcript has a single expressed exon as has been observed for most of the other vertebrate alpha2- and alpha3-fucosyltransferases.

FUT4 and FUT9 genes are expressed early in human embryogenesis.

Molecular cytogenetic analysis of several sporadic papillary renal cell carcinomas and of their xenografts in immunodeficient mice had previously allowed us to delimit a minimal overrepresented region of chromosome 7 shared by all of them to band 7q31. We have refined the location of the overlapping region to the junction of the subbands 7q31.2 and 7q31.3 by reverse painting with two differently labelled probes prepared from the small chromosome 7 derivatives microdissected from the cells of two distinct tumours. This small region was shown to contain the MET proto-oncogene, present at three to four copies per cell as determined by Southern blot analysis. The increased copy number of the MET gene was found to be associated with its overexpression at the mRNA level. However, no change in MET copy number or expression level was observed in the cells from two xenografted tumours serially transplanted into immunodeficient mice, as compared to those from the corresponding initial tumours. Our results indicate that expression of the MET proto-oncogene above a critical threshold is required for the maintenance of the tumorigenic phenotype of at least some papillary renal cell carcinomas, but does not further increase during tumour progression.

Mapping of the 7q31 subregion common to the small chromosome 7 derivatives from two sporadic papillary renal cell carcinomas: increased copy number and overexpression of the MET proto-oncogene.

La localisation chromosomique et l'analyse du chimerisme de 22 YACs ont ete faites par hybridation in situ fluorescente. Les sondes sont obtenues en amplifiant par PCR la partie humaine du YAC grâce a des amorces Alu. Le maximum d'amplification des differents elements inter-Alu est obtenu pour une temperature d'hybridation des amorces inferieure a la temperature optimale utilisee pour une specificite stricte. Dans ces conditions, l'ADN de levure contribue aussi a la formation de produits d'amplification et, puisqu'une forte competition est necessaire pour la suppression de toutes les sequences Alu, les produits de PCR obtenus a partir de la levure sans YAC se sont averes d'efficaces competiteurs.

F. Pulcini

Papers

Mapping of 22 YACs on human chromosomes by fish using yeast DNA Alu-PCR products for competition.