Isolation and molecular characterization of the first genomic clone of a major peanut allergen, Ara h 2.

BACKGROUND: Peanuts have been identified as potent food allergens responsible for life-threatening IgE reactions among hypersensitive individuals. With the current increase of peanut allergies, there is an urgent need to molecularly characterize the genes encoding the target proteins and to understand the nature of their regulation. OBJECTIVES: The objectives of this study were to isolate, sequence, and characterize at least one full-length genomic clone encoding the major peanut allergen Ara h 2. METHODS: A peanut genomic library, constructed in a Lambda Fix II vector, was screened with an 80-bp oligonucleotide probe constructed on the basis of the 5' end of a published Ara h 2 cDNA partial sequence. One putative positive lambda clone was isolated, digested with Bam HI to release its 16-kb insert, and confirmed by means of dot blot and Southern hybridization. The positive clone was subcloned in pBluescript SK+ vector, sequenced, and characterized. RESULTS: Sequence analysis revealed a full-length genomic clone with an open reading frame starting with an initiation codon (ATG) at position 1 and ending with a termination codon (TGA) at position 622. One putative polyadenylation signal (AATAAA) is identified at positions 951 in the 3' untranslated region, and 6 additional stop codons are located at positions 628, 769, 901, 946, 967, and 982 downstream from the start codon. In the 5' promoter region, a putative TATA box (TATTATTA) is located at position -72 upstream from the start codon. The deduced amino acid sequence has 207 residues and includes a putative signal peptide of 21 residues. CONCLUSIONS: The results reveal for the first time information on the structure of a major peanut allergen, Ara h 2. Comparison of the cDNA and genomic sequences revealed the absence of an intron but the presence of 2 isoforms of Ara h 2 or different members of the same gene family.

Development of a 1.4-Mb BAC/PAC contig and physical map within the critical region for complete X-linked congenital stationary night blindness in Xp11.4.

A physical map internal to the markers DXS1368 and DXS228 was developed for the p11.4 region of the human X chromosome. Twenty-four BACs and 10 PACs with an average insert size of 149 kb were aligned to form a contig across an estimated 1.4 Mb of DNA. This contig, which has on average fourfold clone coverage, was assembled by STS and EST content analysis using 46 markers, including 8 ESTs, two retinally expressed genes, and 22 new STSs developed from BAC- and PAC-derived DNA sequence. The average intermarker distance was 30 kb. This physical map provides resources for high-resolution mapping as well as suitable clones for large-scale sequencing efforts in Xp11.4, a region known to contain the gene for complete X-linked congenital stationary night blindness.

Construction of a 1.5-Mb bacterial artificial chromosome contig in Xp11.23, a region of high gene content.

To generate sequence-ready templates for the gene-rich Xp11.23 region, we have constructed a 1.5-Mb bacterial artificial chromosome (BAC) contig spanning the interval between the DNA markers OATL1 and DXS255. The contig includes 28 BACs, ranging in size from 58 to 258 kb with an average size of 135 kb, which provide 2.5-fold coverage of the region. The BAC contig was constructed based entirely on the content of 40 DNA markers from a previously established YAC contig and 11 new markers developed from BAC-end DNA sequences, 4 of which were required to close gaps in the map. There was no evidence of rearrangement, instability, or chimerism in any of the BAC clones. The BAC cloning system appears to provide robust and total physical coverage of this gene-rich region with clones that are suitable for DNA sequencing.