Identification of sequences of chromosome 7 that are expressed in sweat gland epithelial cells.

This paper describes an approach that can be used to identify specifically expressed coding sequences in defined regions of genomic DNA. We developed this method to identify expressed sequences from chromosome 7 located at or near the cystic fibrosis (CF) locus. Radioactively labelled single-stranded cDNAs derived from sweat gland epithelial cells and from fibroblasts were used to screen a genomic library constructed from flow-sorted chromosomes. Differential screening of phage lifts with these two probes yielded 36 different DNA segments. By using somatic cell hybrids containing different portions of chromosome 7, four of the clones were mapped to the 7q31 region in which the CF locus is located. These four clones and two others that gave strong differential epithelial signals but that were not within 7q31 were studied further. Restriction fragment length polymorphisms (RFLPs) were identified for two of the DNA segments within 7q31 and used for linkage analysis using a panel of CF families. One DNA segment was assigned to a location centromeric to the met locus. The other marker did not show recombination with CF but was subsequently excluded from the CF region by physical mapping. Three of the six DNA segments were found to hybridize to various RNAs using the Northern technique and therefore contain portions of genes. One of the clones showed strong differential expression when epithelial tissues were compared to fibroblasts and may represent an epithelium-specific gene.

Microdissection and microcloning of human chromosome 7q22-32 region.

Genetic information contributing to cystic fibrosis in addition to the CF gene is suggested to reside on the long arm of the human chromosome 7. In our attempt to analyze this genomic region in detail, we generated a region-specific DNA probe library by microdissection and microcloning of the midpiece of the chromosome 7q arm. Microdissection was performed in unstained metaphase spreads from a human x mouse hybrid cell line containing chromosome 7 as the only human chromosome. We obtained 593 clones from 75 dissected chromosomal fragments. At least 88% of the microclones were true recombinants; 40% of the clones contained repetitive sequences as determined by plaque hybridization with genomic DNA as probe. The overall mean fragment size of insert fragments was 3.2 kb, the median size was 3.5 kb. Regional mapping of 30 DNA fragments was performed by the aid of hybrid cell lines containing different segments of human chromosome 7; 50% of the microcloned inserts were found to map to 7q22-32.

[Use of the polymerase chain reaction technic in the genetic analysis of cystic fibrosis]. / Aplicación del método de la reacción en cadena de la polimerasa al análisis genético de la fibrosis quística.

The development of polymerase chain reaction (PCR) which allows the specific amplification of DNA sequences has improved considerably the genetic analysis of hereditary diseases. We present here the application of this new technique to the genetic analysis of cystic fibrosis (CF), the most frequent severe genetic disease in caucasians. We have amplified four sequences containing polymorphisms linked to the CF gene (CS.7, KM.19, MP6d-9 and J3.11), and analysed the amplified products with restriction enzymes. Complete concordance was found with classical Southern methods, allowing the application of PCR to routine CF family studies.

A new polymorphic locus, D7S411, isolated by cloning from preparative pulse-field gels is close to the mutation causing cystic fibrosis.

The mutation causing cystic fibrosis (CF) has been localized to the DNA sequence of 700 kb bounded by the loci identified by the markers pMP6d-9 (D7S399) and pJ3.11 (D7S8). A 560-kb fragment obtained after SacII digestion of DNA from a cell line containing this region of human chromosome 7 in a mouse background was separated using pulse-field gel electrophoresis and isolated from the gel. The DNA was digested with BamHI prior to cloning into lambda EMBL3. Approximately 0.1% of the resulting clones contained human repetitive sequences, and 24 such recombinants were studied. Of these, 23 are on chromosome 7; 8 clones were duplicated, and of the 15 different recombinants, 7 are between MET and INT1L1, and a further 7 are between INT1L1 and pMP6d-9, leaving a single marker, pG2, which is between pMP6d-9 and pJ3.11. pG2 recognizes an RFLP with XbaI. A cosmid walk from pG2 has generated a further marker, H80, which recognizes an RFLP with PstI. This new locus (D7S411) divides the remaining region between the CF flanking markers, thereby making it more accessible to fine pulse-field mapping and allowing the precise localization of further clones to this region. Although it is not possible to position the CF locus unequivocally with respect to D7S411, both polymorphic markers at this locus exhibit low but significant linkage disequilibrium with CF, placing the emphasis for the search for the gene on the D7S399 to D7S411 interval of 250 kb.

[Mucoviscidosis: current diagnostic possibilities. Applications in perinatology]. / Mucoviscidose: de nouvelles possibilités diagnostiques. Applications en anténatalogie.

The gene of cystic fibrosis is localised on the long arm of chromosome 7. DNA probes placed close to the gene enable a study of restriction polymorphism to follow the transmission of the gene in index families. It is now possible to counsel those families, who already have an affected child, with an early antenatal diagnosis at ten weeks after the last period. In our personal experience, based on a study of the genotype of 48 families, 70% were informative when they were studied by two probes corresponding to the local pJ3.11 and met. When the latter probes Km19-XV2c were studied concurrently useful information was achieved in 96%. DNA analysis non enables the detection of the chromosome carrying the deleterious gene in practically every family where there is a child suffering from the disease.