Isolation, characterization, and mapping to chromosome 19 of the human apolipoprotein E gene

The human apo-E gene has been isolated from a lambda phage library using as a probe the previously reported apo-E cDNA clone pE-301. Lambda apo-E was mapped and subcloned, and the apo-E gene was completely sequenced. The DNA sequence was compared with that of a near full length cDNA clone pE-368 and revealed three introns. The first intron was in the region that corresponds to the 5' untranslated region of apo-E mRNA. The second intron interrupted the codon specifying amino acid -4 of the apo-E signal peptide. The third intron interrupted the codon specifying amino acid 61 of the mature protein. Analysis of the DNA sequence revealed four Alu sequences. Two were in opposite orientations in the second intron, and one each occurred in the regions 5' and 3' to the apo-E gene. There were two base differences between the apo-E gene sequence and the sequence derived from the cDNA clones. At the codon for amino acid residue 112, the apo-E gene contained CGC, specifying Arg, whereas the cDNA contained TGC, specifying Cys. The other base difference was in the area corresponding to the 5' untranslated region of apo-E mRNA. Apo-E is commonly polymorphic in the population and the data suggest that the genomic clone was derived from the epsilon 4 apo-E allele, whereas the cDNA clones were derived from the epsilon 3 apo-E allele. S1 nuclease protection and primer extension experiments allowed the tentative assignment of the cap site of apo-E mRNA to the A approximately 44 base pairs upstream of the GT that begins the first intron. The sequence TATAATT was identified beginning 33 base pairs upstream of the proposed cap site and is presumably one element of the apo-E promoter. Finally, the apo-E gene was mapped in the human genome to chromosome 19 through the use of DNA probes and human-rodent somatic cell hybrids.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

Genomic structure and chromosomal mapping of the murine CD40 gene.

The B cell-associated surface molecule, CD40, is likely to play a central role in the expansion of Ag-stimulated B cells, and their interaction with activated Th cells. In our study we have isolated genomic clones of murine CD40 from a mouse liver genomic DNA library. Comparison with the murine CD40 cDNA sequence revealed the presence of nine exons that together contain the entire murine CD40 coding region, and span approximately 16.3 kb of genomic DNA. The intron/exon structure of the CD40 gene resembles that of the low affinity nerve growth factor receptor gene, a close homolog of both human and murine CD40. In both cases the functional domains of the receptor molecules are separated onto different exons throughout the genes. Southern blot analysis demonstrated that murine CD40 is a single copy gene that maps in the distal region of mouse chromosome 2.     

Molecular cloning and characterization of a novel peptidylprolyl isomerase (cyclophilin)-like gene (PPIL3) from human fetal brain.

During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated two cDNA clones encoding two novel proteins, which show 52% and 72% identity to the cyclophilin isoform 10 of C. elegans, respectively. Sequence analysis revealed these two cDNA clones are two different splicing variants of a novel cyclophilin-like gene (PPIL3). The PPIL3 gene was identified on a completely sequenced BAC (GenBank accession AC005037) from chromosome 2q33 between STS markers stSG2762 (proximal) and SHGC-3074 (distal), oriented toward the telomere. The PPIL3 gene consisted of eight exons spanning more than 18 kb of genomic DNA. RT-PCR analysis indicated that PPIL3 was ubiquitously expressed in adult human tissues.     

Mouse ferritin H multigene family is polymorphic and contains a single multiallelic functional gene located on chromosome 19.

Multiple ferritin H subunit sequences are present in the genome of higher vertebrates, but it is not yet known with certainty if more than one is expressed. In this paper, we provide evidence that there is only one functional ferritin H gene in the mouse. We screened a mouse genomic library using a mouse ferritin H cDNA as a probe and characterized five clones. These genomic clones proved to contain three pseudogenes and two allelic forms of a unique functional gene. These two alleles differed by only two point mutations in the promoter and three in the first intron and by a 31-bp insertion in the first intron. They were equally expressed when transiently transfected in HeLa cells. These five genomic clones account for all the bands observed on a Southern blot of mouse genomic DNA hybridized with a ferritin H cDNA, and these bands present a restriction fragment length polymorphism between various representatives of the genus Mus. Using a DNA panel prepared from the backcross progeny (C57BL/6 X Mus spretus)F1 X C57BL/6, we localized the functional ferritin H gene (Fth) in region B of mouse chromosome 19 and established cen-Ly-1-Fth-Pax-2 as the most likely gene order, thus defining a conserved syntenic fragment with human chromosome 11q.     

Isolation and characterization of the human ornithine transcarbamylase gene: structure of the 5'-end region

We isolated over 20 phage clones carrying the ornithine transcarbamylase (OTC) [EC 2.1.3.3] gene, from two independently constructed human genomic DNA libraries, using as probes either a rat OTC cDNA or several nuclear DNA fragments derived from some of these isolated clones. These clones, classified into 10 different groups, overlapped and spanned a region of more than 85 kilobase pairs of the human genomic DNA. Restriction mapping and Southern blot analyses demonstrated that one of the clones covers the 5'-end region of the OTC gene. We sequenced the 5'-end region of the OTC gene and found that it covered 665 base pairs of the 5'-flanking region, the complete first exon and a part of the first intron (150 base pairs). In the 5'-flanking region, there were two pairs of putative CAAT and TATA boxes and one enhancer core-like sequence, GTGGAAAG. The first exon contained a coding region for most of the OTC presequence, i.e. 26 out of 32 amino acid residues of the presequence, including the initiation methionine.     

Sequence of the Human Iduronate 2-Sulfatase (IDS) Gene

Deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS; EC 3.1.6.13) results in the storage of the glycosaminoglycans heparan sulfate and dermatan sulfate, which leads to the lysosomal storage disorder mucopolysaccharidosis type II. Three overlapping genomic clones derived from an X-chromosome-specific library containing the entire IDS gene were isolated and the sequences of the intron boundaries and the 5′ promoter region were determined. The IDS gene is split into nine exons spanning approximately 24 kb. The potential promoter for IDS lacks a TATA box but contains GC box consensus sequences, consistent with its role as a housekeeping gene. A polypyrimidine-like repeat is found in intron 1.     

Human urate oxidase gene: cloning and partial sequence analysis reveal a stop codon within the fifth exon

Using the cDNA and selected genomic probes of rat urate oxidase, we have screened the human genomic library and isolated seven clones; one clone (clone 13) contained exonic regions which correspond to the exons 5, 6, and 7 of rat urate oxidase gene. The nucleotide sequence was determined for these three exons and exon/intron junctions, and compared with the sequence from the rat gene. A mutation resulting in a stop codon TGA was found in the fifth exon of the human urate oxidase gene. Sequence analysis of the polymerase chain reaction amplified DNA, corresponding to the fifth exon of urate oxidase from DNA samples from four different individuals, confirmed the same TGA stop codon in all. This single stop codon mutation and/or other mutation(s) in this gene may be responsible for the lack of urate oxidase activity in the human.