Isolation and partial characterization of the structural gene for human acid alpha glucosidase.

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II. To study the disease at the molecular level, we have previously isolated and sequenced the cDNA (3.6 kb) for human GAA. We have now isolated the structural gene, mapped and determined the position and size of the exons containing the entire cDNA, and determined the sequence of the intron-exon junctions. The structural gene is approximately 28 kb and contains 20 exons. The first exon has only 5' untranslated sequence and is separated by an approximately 2.7-kb intron from the second exon that contains the initiation ATG. The second as well as the last exon are quite large (578 and 607 bp) with the remainder of the exons ranging from 85-187 bp. Additionally, two new restriction fragment length (RFLPs) for Xba I and Stu I are described at the GAA locus, one of which is most 5' of the eight RFLPs we have previously described.     

Structure and evolution of the human prosaposin chromosomal gene.

The gene for prosaposin was characterized by sequence analysis of chromosomal DNA to gain insight into the evolution of this locus that encodes four highly conserved sphingolipid activator proteins or saposins. The 13 exons ranged in size from 57 to 1200 bp, while the introns were from 91 to 3812 bp in length. The regions encoding saposins A, B, and D each had three exons, while that for saposin C had only two. This sequence included the regions that encode the carboxy terminus of the signal peptide, the four mature prosaposin proteins, and the 3' untranslated region. Primer extension studies indicated that over 99% of the coding sequence was contained in these 19,985 bp. Use of PCR and reverse PCR techniques indicated that the most 5' coding approximately 140 bp contained large introns and at least two small exons. Analyses of the intronic positions in the saposin regions indicated that this gene evolved from an ancestral gene by two duplication events and at least one gene rearrangement involving a double crossover after introns had been inserted into the gene.     

Genomic structure of murine mitochondrial DNA polymerase-gamma

We have sequenced a genomic clone of the gene encoding the mouse mitochondrial DNA polymerase. The gene consists of 23 exons, which span approximately 13.2 kb, with exons ranging in size from 53 to 768 bp. All intron-exon boundaries conform to the GT-AG rule. By comparison with the human genomic sequence, we found remarkable conservation of the gene structure; the intron-exon borders are in almost identical locations for the 22 introns. The 5' upstream region contains approximately 300 bp of homology between the mouse and human sequences that presumably contain the promoter element. This region lacks any obvious TATA domain and is relatively GC rich, consistent with the housekeeping function of the mitochondrial DNA polymerase. Finally, within the 5' flanking region, both mouse and human genes have a region of 73 bp with high homology to the tRNA-Arg gene.     

Characterization of the gene for prostate-specific antigen, a human glandular kallikrein

The gene for the human glandular kallikrein, prostate-specific antigen, has been cloned. The sequence of 7130 nucleotides encompassing the gene and 633 bp of 5' and 639 bp of 3' flanking DNA has been determined. The translation initiation site was slightly heterogeneous, yielding 5' non-translated leader sequences of 41 and 35 bp. The gene is divided into five exons, with introns located at positions identical with those found in other glandular kallikrein genes. The nucleotide sequence is very similar to that of the human kallikrein gene hGK-1, with 76 to 93% of the nucleotides being identical in the exons and 76 to 87% in the introns. The similarity also extends approximately 200 bp into the sequence flanking the 5' end of hGK-1 and several other, both human and rodent, glandular kallikrein genes.     

Molecular characterization of the human protein kinase C θ * gene locus (PRKCQ)

Members of the protein kinase C (PKC) family of serine/threonine kinases, in particular PKCθ, play critical roles in the regulation of differentiation and proliferation of T lymphocytes. In this study the genomic structure of the human PRKCQ gene that encodes PKCθ was determined. Two genomic P1 clones were isolated from human P1 libraries using the PKCθ cDNA as a probe and have been used to confirm the assignment of the single PRKCQ locus to chromosome 10p15 by FISH analysis. The PRKCQ locus, the first mammalian PKC gene locus characterized so far, spans approximately 62 kb and is composed of 15 coding exons and 14 introns, varying in size between 98 and 16?000 bp. All exon-intron boundaries have been determined by long-range PCR and subsequent DNA sequence analysis. Comparison with other known genomic PKC genes reveals a high degree of homology to the genomic organization of the Drosophila melanogaster dPRKC gene. Alignment of the intron positions in the PRKCQ gene with the intron locations in the dPRKC gene indicates that the sites of seven of the 14 PRKCQ introns are exactly conserved. Exons 5 (32 bp), 11 (174 bp) and 12 (92 bp) share highest similarity in size, organization and primary structure with their counterparts in the Drosophila gene. On the basis of this knowledge of the genomic PRKCQ locus, a directed search for potential genetic polymorphisms and/or genetic abnormalities involved in human genetic disease(s) can now be initiated.     

Genomic organization of human histo-blood group ABO genes

We have isolated human genomic DNA clones encompassing 30 kbpof the histo-blood group ABO locus. The locations of the exonshave been mapped and the nucleotide sequences of the exon-intronboundaries have been determined. The human ABO genes consistof at least seven exons, and the coding sequence in the sevencoding exons spans over 18 kb of the genomic DNA. The exonsrange in size from 28 to 688 bp, with most of the coding sequencelying in exon 7. ABO genomic glycosyltransferase histo-blood group transfusion     

Molecular characterization of the human protein kinase C θ * gene locus (PRKCQ)

Members of the protein kinase C (PKC) family of serine/threonine kinases, in particular PKCθ, play critical roles in the regulation of differentiation and proliferation of T lymphocytes. In this study the genomic structure of the human PRKCQ gene that encodes PKCθ was determined. Two genomic P1 clones were isolated from human P1 libraries using the PKCθ cDNA as a probe and have been used to confirm the assignment of the single PRKCQ locus to chromosome 10p15 by FISH analysis. The PRKCQ locus, the first mammalian PKC gene locus characterized so far, spans approximately 62 kb and is composed of 15 coding exons and 14 introns, varying in size between 98 and 16 000 bp. All exon-intron boundaries have been determined by long-range PCR and subsequent DNA sequence analysis. Comparison with other known genomic PKC genes reveals a high degree of homology to the genomic organization of the Drosophila melanogaster dPRKC gene. Alignment of the intron positions in the PRKCQ gene with the intron locations in the dPRKC gene indicates that the sites of seven of the 14 PRKCQ introns are exactly conserved. Exons 5 (32 bp), 11 (174 bp) and 12 (92 bp) share highest similarity in size, organization and primary structure with their counterparts in the Drosophila gene. On the basis of this knowledge of the genomic PRKCQ locus, a directed search for potential genetic polymorphisms and/or genetic abnormalities involved in human genetic disease(s) can now be initiated. Received: 6 February 1998 / Accepted: 25 May 1998     

Cloning and characterization of the mouse short-chain acyl-CoA dehydrogenase gene

Short-chain acyl-CoA dehydrogenase (SCAD) is one of four straight-chain length specific enzymes involved in the first step of fatty acid β-oxidation. To further understand the similarities between the members of this gene family, to characterize how the gene is regulated, and to determine if there is coordinate regulation between these similar genes, we have isolated genomic clones containing the mouse Acads gene. We show that Acads is a compact, single-copy gene approximately 5000 bp in size. We sequenced the entire coding portion of the gene, all of the intron/exon junctions, and an 850-bp segment upstream of the translation start site. We have determined that the gene consists of 10 exons ranging in size from 57 bp to 703 bp, and 9 introns ranging in size from 80 bp to approximately 700 bp. The 5′ region of the mouse Acads gene lacks a TATA box or a CAAT box, is GC rich, and also lacks any similarity to the related gene, medium-chain acyl-CoA dehydrogenase. This is the initial report of the gene structure and 5′ regulatory sequence of the short-chain acyl-CoA dehydrogenase gene in any species. Received: 20 September 1995 / Accepted: 4 December 1995     

Nucleotide sequence analysis of human hypoxanthine phosphoribosyltransferase (HPRT) gene deletions.

We have determined the nucleotide sequences of 10 intragenic human HPRT gene deletion junctions isolated from thioguanine-resistant PSV811 Werner syndrome fibroblasts or from HL60 myeloid leukemia cells. Deletion junctions were located by fine structure blot hybridization mapping and then amplified with flanking oligonucleotide primer pairs for DNA sequence analysis. The junction region sequences from these 10 HPRT mutants contained 13 deletions ranging in size from 57 bp to 19.3 kb. Three DNA inversions of 711, 368, and 20 bp were associated with tandem deletions in two mutants. Each mutant contained the deletion of one or more HPRT exon, thus explaining the thioguanine-resistant cellular phenotype. Deletion junction and donor nucleotide sequence alignments suggest that all of these HPRT gene rearrangements were generated by the nonhomologous recombination of donor DNA duplexes that share little nucleotide sequence identity. This result is surprising, given the potential for homologous recombination between copies of repeated DNA sequences that constitute approximately a third of the human HPRT locus. No difference in deletion structure or complexity was observed between deletions isolated from Werner syndrome or from HL60 mutants. This suggests that the Werner syndrome deletion mutator uses deletion mutagenesis pathway(s) that are similar or identical to those used in other human somatic cells.     

Genomic organization,alternative splicing,and expression of human and mouse N-RAP,a nebulin-related LIM protein of striated muscle

Linkage analysis identifies 10q24-26 as a disease locus for dilated cardiomyopathy (DCM), a region including the N-RAP gene. N-RAP is a nebulin-like LIM protein that may mediate force transmission and myofibril assembly in cardiomyocytes. We describe the sequence, genomic structure, and expression of human N-RAP, as well as an initial screen to determine whether N-RAP mutations cause cardiomyopathy. Human expressed sequence tag databases were searched with the published 3,528-bp mouse N-RAP open reading frame (ORF). Putative cDNA sequences were interrogated by direct sequencing from cardiac and skeletal muscle RNA. We identified two human N-RAP isoforms with ORFs of 5,085 bp (isoform C) and 5,190 bp (isoform S), encoding products of 193-197 kDa. Genomic database searches localize N-RAP to human chromosome 10q25.3 and match isoforms C and S to 41 and 42 exons. Only isoform C is detected in human cardiac RNA; in skeletal muscle, approximately 10% is isoform C and approximately 90% is isoform S. We investigated apparent differences between human N-RAP cDNA and mouse sequences. Two mouse N-RAP isoforms with ORFs of 5,079 and 5,184 bp were identified with approximately 85% similarity to human isoforms; published mouse sequences include cloning artifacts truncating the ORF. Murine and human isoforms have similar gene structure, tissue specificity, and size. N-RAP is especially conserved within its nebulin-like and LIM domains. We expressed both N-RAP isoforms and the previously described truncated N-RAP in embryonic chick cardiomyocytes. All constructs targeted to myofibril precursors and the cell periphery, and inhibited myofibril assembly. Several human N-RAP polymorphisms were detected, but none were unique to cardiomyopathy patients. N-RAP is highly conserved and exclusively expressed in cardiac and skeletal muscle. Genetic abnormalities remain excellent candidate causes for cardiac and skeletal myopathies.     

Complete intron/exon organization of DNA encoding the alpha' chain of human C3

The third component of human complement (C3), a central molecule in both the classical and alternative pathways of complement, is comprised of two polypeptides, termed the alpha and beta chains. Activation of C3 cleaves the alpha chain into two fragments, C3a, an inflammatory peptide, and the alpha' chain which remains covalently linked to the beta chain. Proteolytic fragments derived from the alpha' chain during activation and regulation of complement play a significant role in host defense and regulation of the immune response. Two cosmid clones covering the alpha' chain region were used to characterize the structure of this portion of the C3 gene. The alpha' chain is encoded by 24 exons, which range in size from 52 to 213 base pairs (bp) with an average size of 115 bp. The splice donor sequence at the beginning of intron 12 has a rare sequence variant of GC instead of the usual GT sequence. Ten introns have been completely sequenced and were surprisingly short, ranging in size from 85 to 242 bp with an average of 140 bp. Other introns range in size from 250 bp to over 4 kilobases in length. The gene size for this portion of C3 is estimated to be 23-24 kilobases. Comparison of exon structure with protein domains and with peptide mapping studies demonstrates that several binding sites on C3 are encoded by single exons. These data support the hypothesis that individual exons can code for functional protein domains.     

Sequence analysis of two exons from the murine dystrophin locus

We have isolated two genomic clones from the murine dystrophin locus, containing single exons encoding protein sequence from the putative actin-binding domain of the amino-terminus and the terminal portion of the triple helical domain. Using interspecific backcross progeny mice, both clones were shown to be X-linked. Sequence analysis indicated that the amino-terminal clone contains a 173 bp exon exhibiting 90% nucleotide sequence identity to human dystrophin exon 6, whilst the C-terminal clone contains a 61 bp exon with 93% nucleotide sequence identity to the human cDNA sequence.