Isolation and identification of novel protein kinase genes from the round-spotted pufferfish(Tetraodon fluviatilis) genomic DNA

The round-spotted pufferfishTetraodon fluviatilis has a genome size of 380 Mb which is slightly smaller than that of another pufferfish,Fugu rubripes rubripes (Fugu). Due to their compact genome and small introns, both pufferfishes have been proposed as model organisms for genome studies. In this study, we have used genomic DNA as template to perform PCR to screen for protein kinase (pk) genes. Forty-oneT. fluviatilis pk genes encoding 7 receptor tyrosine kinases, 14 nonreceptor tyrosine kinases, 16 serine/threonine kinases, 1 dual kinase and 3 novel kinases have been identified. The success of this approach depends on the size and location of the introns. Most of the identifiedpk gene fragments contain introns, ranging from 71 to 300 bp, with an average of 120 bp. It is noteworthy that the intron/exon boundaries of certain genes which belong to the same family are identical. We also analyzed by specific RT-PCR primers the expression profile of those 3 novel genes as well as some selectedpk genes in a variety of tissues. We found thaterbB3,pku , mrk, CaMK I,CaMKII, and two novel kinase genes (133 and 3–26) are expressed in all tissues examined. However, the novel clone 146 is strongly expressed in the brain and weakly in the intestine, kidney and heart.     

Cytogenetic and molecular analysis of the pufferfish Tetraodon fluviatilis (Osteichthyes)

In view of their compact genome, pufferfish (Tetraodontiformes) have been proposed as model animal for the study of the vertebrate genome. Despite such interest, cytogenetic information about puffers is still scanty. To fill this gap, a cytogenetic analysis of T. fluviatilis has been performed using both classical and molecular techniques. C-banding, followed by DAPI staining, evidenced that in T. fluviatilis, like all other puffer species so far examined, heterochromatin is essentially AT-rich and it is located at centromeres, whereas staining with CMA₃, silver staining and FISH with a 28S ribosomal RNA gene DNA probe showed 2–4 nucleolar organizing regions (NORs) located in heterochromatic regions in the considered puffer species. FISH with the 5S probe put in evidence both in T. fluviatilis and in T. nigroviridis only a 5S cluster per haploid genome that is physically unlinked with the major ribosomal RNA genes including the 28S rRNA genes. Hybridization with the (TTAGGG)_n probe showed in all the puffers brightly fluorescent signals uniform both in size and intensity at the end of all the chromosomes. Finally, mariner-like elements (MLEs) have been identified in T. fluviatilis and they have located into the NOR-associated heterochromatin.     

Analysis of the spermine synthase gene region in Fugu rubripes, Tetraodon fluviatilis, and Danio rerio

A prerequisite to understanding the evolution of the human X chromosome is the analysis of synteny of X-linked genes in different species. We have focused on the spermine synthase gene in human Xp22. 1. We show that whereas the human gene spans a genomic region of 54 kb, the Fugu rubripes gene is encompassed in a 4.7-kb region. However, we could not find conserved synteny between this region of human Xp22 and the equivalent F. rubripes region. A cosmid clone containing the F. rubripes gene does not contain other X-linked genes. Instead we identified homologs of human genes that are autosomally localized: the ryanodine receptor type I (RYRI), which is implicated in malignant hyperthermia and central core disease, and the HE6 gene. Comparison of the F. rubripes, Tetraodon fluviatilis, mouse, human, and Danio rerio 5'UTRs of spermine synthase highlights conserved sequences potentially involved in regulation. Interestingly, pseudogenes of this gene that are present in the human and mouse genomes seem to be absent in the compact F. rubripes genome. Analysis of a D. rerio PAC clone containing spermine synthase shows an intermediate genomic size in this fish. Sequence analysis of this PAC clone did not reveal other known genes: neither the RYRI gene, nor the HE6 gene, nor other human Xp22 genes were identified.     

Genomic organization and characterization of the promoter of the human ATP-binding cassette transporter-G1 (ABCG1) gene

The ATP-binding cassette transporter G1 (ABCG1) was recently identified as a regulator of macrophage cholesterol and phospholipid transport. This transporter together with ABCA1 belongs to a group of sterol-sensitive ABC proteins which are induced by lipid loading or specific oxysterols. We report here the genomic structure of ABCG1 along with the 5' flanking sequence using library screening and BLAST search analysis. The ABCG1 gene spans more than 70 kb and contains 15 exons. The exon size is between 30 and 1081 bp and the introns range in size from 137 bp to more than 45 kb. All exon-intron boundaries display the canonical GT/AG sequences. Using promoter-luciferase reporter assays in the myeloid cell lines THP-1 and RAW246.7 and the hepatoma cell line HepG2 we could demonstrate the functionality of the ABCG1 promoter and the minimal sequence requirements for gene expression. The TATA-less proximal promoter contains multiple Sp1 binding sites and a consensus sequence for sterol regulatory element binding protein.