Fine structure of the human ceruloplasmin gene

We characterized the genomic region corresponding to the human ceruloplasmin cDNA previously reported. Using PCR-direct sequencing methods, we determined precise intron/exon boundaries and intron-exon composition of the gene in the region. The gene region spanned about 50 kb and was composed of 19 exons and 18 introns. The lengths of exons and introns range from 107 to over 267 bp and from 0.44 to 10.0 kb, respectively. The translation initiation codon and the termination codon were located in exons 1 and 19, respectively. The nucleotide sequences of the introns were also determined in the region around the intron/exon boundaries for 24-220 bp. All the sequences around the intron/exon boundaries were consistent with the 5' and 3' consensus sequences for splice junctions of transcribed genes. Putative lariat sequences were identified between -17 and -42 nucleotides from the 3' splice junction for all 18 introns.     

Partial structure of the gene encoding the 78 kDa gastrin binding protein excludes a close relationship with the peroxisomal trifunctional enzyme

A 10.9 kb porcine genomic clone encoding nucleotides 124-732 of the cDNA for the porcine 78 kDa gastrin-binding protein has been isolated and characterized. The coding sequence is interrupted by 7 introns, which vary in length from 93 to 3000bp. The positions of the intron/exon junctions are different from the junctions in the gene encoding the rat peroxisomal trifunctional enzyme. Despite 33% amino acid sequence identity between the two proteins it is concluded that the porcine gastrin binding protein is not closely related to the rat trifunctional enzyme.     

In vitro mutagenicity of the plasmacytomagenic agent pristane (2,6,10,14-tetramethylpentadecane)

Cystatin A is a cysteine proteinase inhibitor with a molecular mass of 11 kDa, and is located mainly in the keratohyaline granules of the stratum granulosum and the cornified envelope of the stratum corneum in the epidermis. In this study, we demonstrated the genomic structure of this proteinase inhibitor in which there were three exons of 111 bp, 102 bp and 226 bp in length, while the lengths of the 1st and 2nd intron were approximately 14 Kbp and 4 Kbp, respectively. The conserved sequence of QVVAG was encoded in the 2nd exon and was not inserted by any introns. There were binding sites for SP-1 and AP-2 in the promoter region and an AP-1 binding site in the 1st intron. The successful amplification of each exon of cystatin A may possibly contribute to the detection of the genomic abnormality of some skin disorders e.g. keratinization disorder, chronic bacterial infection or photophobia.     

The heat-shock gene hsp83 of Drosophila auraria: genomic organization, nucleotide sequence, and long antiparallel coupled ORFs (LAC ORFs)

The genomic organization of the hsp83 gene of Drosophila auraria, a far-eastern endemic species belonging to the montium subgroup of the melanogaster species group, is presented here. Based on in situ hybridization on polytene chromosomes, cDNA and genomic clone mapping, nucleotide sequencing, and genomic Southern analysis, hsp83 is shown to be present as a single-copy gene at locus 64B on the 3L chromosome arm in D. auraria. This gene is organized into two exons separated by a 929-bp intron. The first exon represents the mRNA leader sequence and is not translated, while the coding region, having a length of 2,151 bp, is solely included in the second exon. Nucleotide sequence comparisons of D. auraria hsp83 with homologous sequences from other organisms show high conservation of the coding region (88-92% identity) in the genus Drosophila, in addition to the conserved genomic organization of two-exons-one-intron, of comparable size and arrangement. A phylogenetic tree based on the protein sequences of homologous genes from representative organisms is in accord with the accredited phylogenetic position of D. auraria. In the hsp83 gene region, a second case of long antiparallel coupled open reading frames (LAC ORFs) for this species was found. The antiparallel to the hsp83 gene ORF is 1,554 bases long, while the two ORFs overlap has a size of 1,548 bp. The anti-hsp83 ORF does not show significant homology to any known gene sequences. In addition, no similar LAC ORF structures were found in homologous gene regions of other organisms.     

A mechanism for unsplicing and exon skipping in human alpha- and beta-globin mutant pre-mRNA splicing

In both of normal human alpha- and beta-globin pre-mRNAs, two introns are spliced correctly, giving mRNA products of (exon 1)-(exon 2)-(exon 3). If 5'-splice site of the second intron is mutated, mRNA of the beta-globin is composed of (exon 1)-(exon 3), skipping the whole exon 2 sequence. However, if 5'-splice site of the second intron is mutated in the alpha-globin, mRNA is (exon 1)-(exon 2)-(intron 2)-(exon 3), unsplicing intron 2. In both of the alpha- and beta-globin pre-mRNAs, strengths of 3'-splice signals of the first and second introns were calculated by the quantification method reported previously, which explains a reason for exon skipping and unsplicing in terms of relative strengths of those signals.     

The gene for the major protein (SVSP109) of bovine semen: structure and promoter analysis

We have characterized the gene of SVSP109, specifying the bovine secretory protein SVSP109, which is synthesized in a tissue- as well as species-specific manner. Approximately 1.3 kb upstream of the SVSP109 gene, the 3'-end of another gene designated HG5 was identified. The HG5 gene fragment comprises exon (n-1) and exon (n), separated by an intron. Exon (n) contains the complete 3'-untranslated region, whereas exon (n-1) encodes the C-terminal part of a hitherto unknown protein with high homology to SVSP109. The sequenced region of 6956 bp of a bovine genomic clone comprised the complete SVSP109 gene, which is made up of five exons and four introns. Primer extension analysis and RTPCR of poly(A+)RNA from seminal vesicle revealed that the first exon 1 extends to a position 34 bp downstream of the TATA sequence. Employing a CAT assay, a definitive but weak promoter activity was detected in pCATeSVSP15 (base pairs -639 to +574) and pCATeSVSP10 (base pairs -639 to +198); pCATeSVSP6 (base pairs -262 to +65) displayed promoter activity similar to the positive control. We conclude from these results that the TATA sequence located at position -34 is part of the functional promoter of the SVSP109 gene.     

Nucleotide sequence diversity at the alcohol dehydrogenase 1 locus in wild barley (Hordeum vulgare ssp. spontaneum): an evaluation of the background selection hypothesis

The background selection hypothesis predicts a reduction in nucleotide site diversity and an excess of rare variants, owing to linkage associations with deleterious alleles. This effect is expected to be amplified in species that are predominantly self-fertilizing. To examine the predictions of the background selection hypothesis in self-fertilizing species, we sequenced 1,362 bp of adh1, a gene for alcohol dehydrogenase (Adh; alcohol:NAD+ oxidoreductase, EC 1.1.1.1), in a sample of 45 accessions of wild barley, Hordeum vulgare ssp. spontaneum, drawn from throughout the species range. The region sequenced included 786 bp of exon sequence (part of exon 4, all of exons 5-9, and part of exon 10) and 576 bp of intron sequence (all of introns 4-9). There were 19 sites polymorphic for nucleotide substitutions, 8 in introns, and 11 in exons. Of the 11 nucleotide substitutions in codons, 4 were synonymous and 7 were nonsynonymous, occurring uniquely in the sample. There was no evidence of recombination in the region studied, and the estimated effective population size (Ne) based on synonymous sites was approximately 1.8-4.2 x 10(5). Several tests reveal that the pattern of nonsynonymous substitutions departs significantly from neutral expectations. However, the data do not appear to be consistent with recovery from a population bottleneck, recent population expansion, selective sweep, or strong positive selection. Though several features of the data are consistent with background selection, the distributions of polymorphic synonymous and intron sites are not perturbed toward a significant excess of rare alleles as would be predicted by background selection.     

Genomic characterization of the Neurofibromatosis Type 1 gene of Fugu rubripes

The genomic structure of the Neurofibromatosis Type1 (NF1) gene of Fugu rubripes was investigated by sequence analysis of two overlapping cosmids. The Fugu NF1 gene spans 27 kb and is 13 times smaller than the human counterpart owing primarily to reduced intron size. The predicted amino acid sequence is highly related to that of human neurofibromin, exhibiting an overall similarity of 91.5%. Nearly all exons described for the human NF1 gene could be identified, except exon 12b and the alternatively spliced exons 9br and 48a. With the exception of the splice acceptor site in front of exon 16, all splice sites are in identical positions to those found in the human gene. Intron 1, which is 100-140 kb long in humans, spans 2575 bp in the Fugu NF1 gene. Another large intron of the human NF1 gene, intron 27b (45-50 kb), is 3942 bp of size in Fugu. Sequences related to the OMgp gene (Oligodendrocyte-Myelin-glycoprotein) or the EVI2A gene (ecotropic viral integration site), which are inserted into human NF1 intron 27b, were not detected in the corresponding Fugu intron. However, a single exon gene with similarity to the human EVI2B gene has been found on the reverse strand of Fugu intron 27b. This suggests that the human EVI2B gene and the Fugu gene in intron 27b have a common ancestor. We found the expression of this inserted gene in liver and kidney, but not in brain tissue of Fugu rubripes.     

Sequence, genomic organization, and chromosomal location of the mouse Müllerian-inhibiting substance type II receptor gene

We have determined the sequence and structure of the mouse Müllerian-inhibiting substance (MIS) type II receptor gene. Sequence comparisons demonstrate that the mouse, rat, rabbit, and human MIS type II receptors are highly conserved. The mouse MIS type II receptor gene is encoded by 11 exons and spans approximately 9-kb. Only half of the intron/exon boundaries of its kinase domain are conserved in comparison to the kinase domain of the related activin type II receptor. Whereas the activin type II receptor gene contains large introns (> 40-kb), the largest intron of the MIS type II receptor gene is only 4.3-kb. The MIS type II receptor gene (Amhr) is closely linked to Hoxc on mouse chromosome 15. Knowledge of the sequence and genomic structure of Amhr provides important information for the genetic manipulation of the Amhr locus.     

Four novel mutations of sterol 27-hydroxylase gene in Italian patients with cerebrotendinous xanthomatosis

We report the characterization of eight mutations of sterol 27-hydroxylase gene (CYP27) in five Italian patients with cerebrotendinous xanthomatosis, who were found to be compound heterozygotes. Four mutations (C --> T at nt 45 of exon 4, G(+1) --> A in intron 6, G(+5) --> T in intron 7, and G(-1) --> A in intron 7) are novel. The C --> T at nt 45 of exon 4 converts the arginine codon into a stop codon thus generating a truncated protein of 198 amino acids. The three splice site mutations reduced the content of CYP27 mRNA in skin fibroblasts to very low or undetectable levels and generated minute amounts of abnormal mRNAs. The G(+1) --> A transition in intron 6 produced three abnormal mRNAs. In the first, the 5' half of exon 6 joins to exon 7, skipping 89 bp of exon 6, and in the second, exon 5 joins directly to exon 7. The predicted translation products of these mRNAs are truncated proteins. In the third abnormal mRNA, exon 5 joins to exon 8 with an in-frame deletion of 246 bp. The G(+5) --> T transversion in intron 7 generates a single abnormal mRNA in which exon 6 joins directly to exon 8, with a frameshift and a premature stop codon. In the G(-1) --> A transition in intron 7, two mRNAs are generated. In the first, the retention of the whole intron 7 causes a frameshift and a premature stop codon; in the second, the joining of exon 7 to exon 8 is associated with an in-frame deletion of the first 6 nucleotides. All these novel mutations are predicted to produce structurally abnormal enzymatic proteins with no measurable biological activity.     

The exon/intron organization and chromosomal mapping of the mouse ADAMTS-1 gene encoding an ADAM family protein with TSP motifs

ADAM is a recently discovered gene family that encodes proteins with a disintegrin and metalloproteinase. ADAMTS-1 is a gene encoding a new member protein of the ADAM family with the thrombospondin (TSP) type I motif, the expression of which is associated with inflammatory processes. In the present study, we have characterized the exon/intron organization of the mouse ADAMTS-1 gene. The ADAMTS-1 gene is composed of nine exons, all of which are present within the 9.2-kb genomic region. Among the nine exons, exons 1, 5, and 6 encode a proprotein domain, a disintegrin-like domain, and a TSP type I motif, respectively, of the ADAMTS-1 protein, suggesting that there is a correlation between exon/intron organization and functional domains. In addition, the exon/ intron organization of the ADAMTS-1 gene is very different from that of the metalloproteinase-like/disintegrin-like/ cysteine-rich protein gene (MDC) (ADAM11), suggesting that the genomic structure of ADAM family genes is not necessarily conserved. Furthermore, fluorescence in situ hybridization revealed that the ADAMTS-1 gene is located in region C3-C5 of chromosome 16, to which none of the previously identified ADAM genes have been mapped.     

Genomic organization of IFI16, an interferon-inducible gene whose expression is associated with human myeloid cell differentiation: correlation of predicted protein domains with exon organization

The human IFI16 gene is a member of an interferon-inducible family of mouse and human genes closely linked on syntenic regions of chromosome 1. Expression of these genes is largely restricted to hemopoietic cells, and is associated with the differentiation of cells of the myeloid lineages. As a prelude to defining the mechanisms governing IFI16 expression, we have deduced its genomic organization using a combination of genomic cloning and polymerase chain reaction amplification of genomic DNA. IFI16 consists of ten exons and nine intervening introns spanning at least 28 kilobases (kb) of DNA. The reiterated domain structure of IFI16 protein is closely reflected in its intron/exon boundaries, and may represent the evolutionary fusion of several independent functional domains. Thus, exon 1 consists of 5' untranslated (UT) sequences and contains sequence motifs that may confer interferon-inducibility, and exon 2 encodes the lysine-rich amino-terminal ("K") region, which possesses DNA-binding activity. Exon 3 codes for a domain which is poorly conserved between family members, except for a strongly retained basic motif likely to provide localization. The first of two 200 amino acid repeat domains that are the hallmark of this family (domain A) is represented jointly on exons 4 and 5, which are reiterated as exons 8 and 9, respectively, to encode the second 200 amino acid domain (B). Two intervening serine-threonine-rich domains (C and C'), unique to IFI16, are each encoded by single exons of identical length (exons 5 and 6). These domains are predicted to encode semi-rigid "spacer" domains between the 200 amino acid repeats. The reiterated nature of exons 4 to 6 and the insertion of introns into a single reading frame strongly suggest that IFI16 and related genes arose by a series of exon duplications, some of which antedated speciation into mouse and humans. Several alternative mRNA cap sites downstream of a TATA consensus sequence were defined, using primer extension analysis of mRNA. Sequencing of approximately 1.7 kb of DNA upstream of this region revealed no recognizable consensus elements for induction by interferon-alpha (interferon-alpha/beta-stimulated response elements), but two motifs resembling interferon-gamma activation sites were located. IFNs alpha and gamma both induce IFI16 mRNA expression in myeloid cells. Interferon-alpha inducibility of IFI16 may be regulated by an interferon-alpha/beta-stimulated response consensus element in the 5' UT exon, as a similar motif is conserved in the corresponding position in the related myeloid cell nuclear differentiation antigen gene.(ABSTRACT TRUNCATED AT 400 WORDS)