Genomic organization,chromosomal localization,and alternative splicing of the human phosphodiesterase 8B gene

We have characterized the gene for human phosphodiesterase 8B, PDE8B, and cloned the full-length cDNA for human PDE8B (PDE8B1) and two splice variants (PDE8B2 and PDE8B3). The PDE8B gene is mapped to the long arm of chromosome 5 (5q13) and is composed of 22 exons spanning over approximately 200kb. The donor and acceptor splice site sequences match the consensus sequences for the exon-intron boundaries of most eukaryotic genes. PDE8B1 encodes an 885 amino acid enzyme, containing an N-terminal REC domain, a PAS domain, and a C-terminal catalytic domain. PDE8B2 and PDE8B3 both have deletion in the PAS domain and encode 838 and 788 amino acid proteins, respectively. RT-PCR analysis revealed that while PDE8B1 is the most abundant variant in thyroid gland, PDE8B3, but not PDE8B1, is the most abundant form in brain. These findings suggest that selective usage of exons produces three different PDE8B variants that exhibit a tissue-specific expression pattern.     

Characterization of the bovine PRKAG3 gene: structure, polymorphism, and alternative transcripts

The bovine PRKAG3 gene encodes the AMPK gamma3 subunit, one isoform of the regulatory gamma subunit of the AMP-activated protein kinase (AMPK). The AMPK plays a major role in the regulation of energy metabolism and mutations affecting the genes encoding the gamma subunits have been shown to influence AMPK activity. The gamma3 subunit is involved in the regulation of AMPK activity in skeletal muscle and strongly influences glycogen metabolism. Glycogen content in muscle is correlated to meat quality in livestock because it influences postmortem maturation process and ultimate pH. Naturally occurring mutations in the porcine PRKAG3 gene highly affect meat quality by influencing glycogen content before slaughter. We present the characterization of the bovine PRKAG3 gene and a polymorphism analysis in three cattle breeds. Thirty-two SNPs were identified among which 13 are in the coding region, one is in the 3' UTR, and 18 are in the introns. Five of them change an amino acid in the PRKAG3 protein sequence. Allelic frequencies were determined in the three breeds considered, and mutant alleles affecting the coding sequence are found at a very low frequency. Alternative splicing sites were identified at two positions of the gene, introducing heterogeneity in the population of proteins translated from the gene.     

Molecular structure of the human argininosuccinate synthetase gene: occurrence of alternative mRNA splicing.

The human genome contains one expressed argininosuccinate synthetase gene and ca. 14 pseudogenes that are dispersed to at least 11 human chromosomes. Eleven clones isolated from a human genomic DNA library were characterized extensively by restriction mapping, Southern blotting, and nucleotide sequencing. These 11 clones represent the entire expressed argininosuccinate synthetase gene that spans 63 kilobases and contains at least 13 exons. The expressed gene codes for two mRNAs that differ in their 5' untranslated sequences and arise by alternative splicing involving the inclusion or deletion of an entire exon. In normal human liver and cultured fibroblasts, the predominant mature argininosuccinate synthetase mRNA lacks sequences encoded by exon 2 in the expressed gene. In contrast, the predominant argininosuccinate synthetase mRNA in baboon liver contains exon 2 sequences. A transformed canavanine-resistant human cell line in which argininosuccinate synthetase activity is 180-fold higher than that in wild-type cells contains abundant amounts of both forms of the argininosuccinate synthetase mRNA. The mRNA lacking exon 2 sequences is the more abundant mRNA species in the canavanine-resistant cells. These observations show that splicing of the argininosuccinate synthetase mRNA is species specific in primates and varies among different human cell types.     

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP-Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.     

Characterization on the alternative splicing, expression and gene phylogenesis of PTPR4 family in Japanese flounder, Paralichthys olivaceus

One mechanism of eukaryotic signaling is protein phosphorylation by protein tyrosine phosphatases (PTPs). Here we have identified the PTP Receptor-Type IV (PTPR4) family, including one form of PTPalpha and two forms of PTPepsilon (PTPepsilon M and PTPepsilon C) in flounder. The existence of PTPepsilon C has not been reported in non-mammalian animals. Semi-quantitative RT-PCR revealed independent expression patterns and levels of PTPalpha and the two forms of PTPepsilon in various tissues. The sequence of PTPepsilon C was identical to that of PTPepsilon M except for its 5'-terminal regions. Southern blot analysis proved that there existed only one PTPepsilon gene in flounder genome, indicating that the two isoforms of PTPepsilon might have been derived from alternative splicing of the single gene. Phylogenetic analysis of PTP domain D2 and part of D1 of PTPR4 showed that flounder was first joint with other teleost fish and then tetrapods, and also provided evidence that the gene duplication from the ancestor gene to PTPalpha and PTPepsilon occurred before the divergence of Gnathastomata and Agnatha. These results showed that the functional evolution of protein phosphorylation is promoted by not only genome duplication, but also elaborate regulation of gene expression.