DEF-1, a novel Src SH3 binding protein that promotes adipogenesis in fibroblastic cell lines.
(25/178859)
The Src homology 3 (SH3) motif is found in numerous signal transduction proteins involved in cellular growth and differentiation. We have purified and cloned a novel protein, DEF-1 (differentiation-enhancing factor), from bovine brain by using a Src SH3 affinity column. Ectopic expression of DEF-1 in fibroblasts resulted in the differentiation of a significant fraction of the culture into adipocytes. This phenotype appears to be related to the induction of the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma), since DEF-1 NIH 3T3 cells demonstrated augmented levels of PPARgamma mRNA and, when treated with activating PPARgamma ligands, efficient induction of differentiation. Further evidence for a role for DEF-1 in adipogenesis was provided by heightened expression of DEF-1 mRNA in adipose tissue isolated from obese and diabetes mice compared to that in tissue isolated from wild-type mice. However, DEF-1 mRNA was detected in multiple tissues, suggesting that the signal transduction pathway(s) in which DEF-1 is involved is not limited to adipogenesis. These results suggest that DEF-1 is an important component of a signal transduction process that is involved in the differentiation of fibroblasts and possibly of other types of cells. (+info)
Isolation of zebrafish gdf7 and comparative genetic mapping of genes belonging to the growth/differentiation factor 5, 6, 7 subgroup of the TGF-beta superfamily.
(26/178859)
The Growth/differentiation factor (Gdf) 5, 6, 7 genes form a closely related subgroup belonging to the TGF-beta superfamily. In zebrafish, there are three genes that belong to the Gdf5, 6, 7 subgroup that have been named radar, dynamo, and contact. The genes radar and dynamo both encode proteins most similar to mouse GDF6. The orthologous identity of these genes on the basis of amino acid similarities has not been clear. We have identified gdf7, a fourth zebrafish gene belonging to the Gdf5, 6, 7 subgroup. To assign correct orthologies and to investigate the evolutionary relationships of the human, mouse, and zebrafish Gdf5, 6, 7 subgroup, we have compared genetic map positions of the zebrafish and mammalian genes. We have mapped zebrafish gdf7 to linkage group (LG) 17, contact to LG9, GDF6 to human chromosome (Hsa) 8 and GDF7 to Hsa2p. The radar and dynamo genes have been localized previously to LG16 and LG19, respectively. A comparison of syntenies shared among human, mouse, and zebrafish genomes indicates that gdf7 is the ortholog of mammalian GDF7/Gdf7. LG16 shares syntenic relationships with mouse chromosome (Mmu) 4, including Gdf6. Portions of LG16 and LG19 appear to be duplicate chromosomes, thus suggesting that radar and dynamo are both orthologs of Gdf6. Finally, the mapping data is consistent with contact being the zebrafish ortholog of mammalian GDF5/Gdf5. (+info)
Analysis of two cosmid clones from chromosome 4 of Drosophila melanogaster reveals two new genes amid an unusual arrangement of repeated sequences.
(27/178859)
Chromosome 4 from Drosophila melanogaster has several unusual features that distinguish it from the other chromosomes. These include a diffuse appearance in salivary gland polytene chromosomes, an absence of recombination, and the variegated expression of P-element transgenes. As part of a larger project to understand these properties, we are assembling a physical map of this chromosome. Here we report the sequence of two cosmids representing approximately 5% of the polytenized region. Both cosmid clones contain numerous repeated DNA sequences, as identified by cross hybridization with labeled genomic DNA, BLAST searches, and dot matrix analysis, which are positioned between and within the transcribed sequences. The repetitive sequences include three copies of the mobile element Hoppel, one copy of the mobile element HB, and 18 DINE repeats. DINE is a novel, short repeated sequence dispersed throughout both cosmid sequences. One cosmid includes the previously described cubitus interruptus (ci) gene and two new genes: that a gene with a predicted amino acid sequence similar to ribosomal protein S3a which is consistent with the Minute(4)101 locus thought to be in the region, and a novel member of the protein family that includes plexin and met-hepatocyte growth factor receptor. The other cosmid contains only the two short 5'-most exons from the zinc-finger-homolog-2 (zfh-2) gene. This is the first extensive sequence analysis of noncoding DNA from chromosome 4. The distribution of the various repeats suggests its organization is similar to the beta-heterochromatic regions near the base of the major chromosome arms. Such a pattern may account for the diffuse banding of the polytene chromosome 4 and the variegation of many P-element transgenes on the chromosome. (+info)
An effective approach for analyzing "prefinished" genomic sequence data.
(28/178859)
Ongoing efforts to sequence the human genome are already generating large amounts of data, with substantial increases anticipated over the next few years. In most cases, a shotgun sequencing strategy is being used, which rapidly yields most of the primary sequence in incompletely assembled sequence contigs ("prefinished" sequence) and more slowly produces the final, completely assembled sequence ("finished" sequence). Thus, in general, prefinished sequence is produced in excess of finished sequence, and this trend is certain to continue and even accelerate over the next few years. Even at a prefinished stage, genomic sequence represents a rich source of important biological information that is of great interest to many investigators. However, analyzing such data is a challenging and daunting task, both because of its sheer volume and because it can change on a day-by-day basis. To facilitate the discovery and characterization of genes and other important elements within prefinished sequence, we have developed an analytical strategy and system that uses readily available software tools in new combinations. Implementation of this strategy for the analysis of prefinished sequence data from human chromosome 7 has demonstrated that this is a convenient, inexpensive, and extensible solution to the problem of analyzing the large amounts of preliminary data being produced by large-scale sequencing efforts. Our approach is accessible to any investigator who wishes to assimilate additional information about particular sequence data en route to developing richer annotations of a finished sequence. (+info)
Molecular cloning of a cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from liver of Sparus aurata: nutritional regulation of enzyme expression.
(29/178859)
A cDNA clone encoding full-length 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2, 6-P2ase) was isolated and sequenced from a Sparus aurata liver cDNA library. The 2527 bp nucleotide sequence of the cDNA contains a 73 bp 5'-untranslated region (5'-UTR), an open reading frame that encodes a 469 amino acid protein and 1041 bp at the 3'-UTR. The deduced amino acid sequence is the first inferred 6PF-2-K/Fru-2, 6-P2ase in fish. The kinase and bisphosphatase domains, where the residues described as crucial for the mechanism of reaction of the bifunctional enzyme are located, present a high degree of homology with other liver isoenzymes. However, within the first 30 amino acids at the N-terminal regulatory domain of the fish enzyme a low homology is found. Nutritional regulation of the 6-phosphofructo-2-kinase activity, together with immunodetectable protein and mRNA levels of 6PF-2-K/Fru-2,6-P2ase, was observed after starvation and refeeding. In contrast to results previously described for rat liver, the decrease in immunodetectable protein and kinase activity caused by starvation was associated in the teleostean fish to a decrease in mRNA levels. (+info)
Identification of DNA polymorphisms associated with the V type alpha1-antitrypsin gene.
(30/178859)
alpha1-Antitrypsin (alpha1-AT) is a highly polymorphic protein. The V allele of alpha1-AT has been shown to be associated with focal glomerulosclerosis (FGS) in Negroid and mixed race South African patients. To identify mutations and polymorphisms in the gene for the V allele of alpha1-AT in five South African patients with FGS nephrotic syndrome DNA sequence analysis and restriction fragment length polymorphisms of the coding exons were carried out. Four of the patients were heterozygous for the BstEII RFLP in exon III [M1(Val213)(Ala213)] and one patient was a M1(Ala213) homozygote. The mutation for the V allele was identified in exon II as Gly-148 (GGG)-->Arg (AGG) and in all patients was associated with a silent mutation at position 158 (AAC-->AAT). The patient who was homozygous for (Ala213) also had a silent mutation at position 256 in exon III (GAT-->GAC) which was not present in any of the other four patients. Although the V allele of alpha1-AT is not associated with severe plasma deficiency, it may be in linkage disequilibrium with other genes on chromosome 14 that predispose to FGS. Furthermore, the associated silent mutation at position 158 and the Ala213 polymorphism are of interest, as these could represent an evolutionary intermediate between the M1(Ala213) and M1(Val213) subtypes. (+info)
ATF-2-binding regulatory element is responsible for the Ly49A expression in murine T lymphoid line, EL-4.
(31/178859)
To understand the mechanism of Ly49A-expression and its significance in T-cell differentiation, we analyzed the 5'-flanking region of the Ly49A gene in a search for the Ly49A-regulatory element. Since very few known regulatory elements have been found in this region, presumably a novel regulatory sequence(s) could exist. Accordingly, we defined the 13-bp regulatory element, 5'-ATGACGAGGAGGA-3', restricted to Ly49A-expression in EL-4 cells in comparison with two other representative cell lines tested. This element, designated as EL13, proved to be previously undiscovered by homology search and is highly homologous with several virus DNAs. Using EL13 as a probe we have cloned a cDNA encoding a binding protein to EL13. Its deduced nucleotide sequence revealed that EL13-binding protein is almost identical with rat ATF-2. Although ATF-2 is known to bind to cyclic AMP responsive element (CRE), EL13 shares five out of eight nucleotides with this consensus sequence. Our results suggested that ATF-2 may play an important role via binding to EL13 for the expression of Ly49A. These data will provide useful information for understanding T-cell and NK-cell differentiation in murine immune system. (+info)
Induction of AT-specific DNA-interstrand crosslinks by bizelesin in genomic and simian virus 40 DNA.
(32/178859)
Bizelesin is a bifunctional AT-specific DNA alkylating drug. Our study characterized the ability of bizelesin to induce interstrand crosslinks, a potential lethal lesion. In genomic DNA of BSC-1 cells, bizelesin formed from approx. 0.3 to 6.03+/-0.85 interstrand crosslinks per 106 base pairs, at 5-100 nM drug concentration, respectively, comparable to the number of total adducts previously determined in the same system (J.M. Woynarowski, M.M. McHugh, L.S. Gawron, T.A. Beerman, Biochemistry 34 (1995) 13042-13050). Bizelesin did not induce DNA-protein crosslinks or strand breaks. A model defined target, intracellular simian virus 40 (SV40) DNA, was employed to map at the nucleotide level sites of bizelesin adducts, including potential interstrand crosslinks. Preferential adduct formation was observed at AT tracts which are abundant in the SV40 matrix associated region and the origin of replication. Many sites, including each occurrence of 5'-T(A/T)4A-3', co-mapped on both DNA strands suggesting interstrand crosslinks, although monoadducts were also formed. Bizelesin adducts in naked SV40 DNA were found at similar sites. The localization of bizelesin-induced crosslinks in AT-rich tracts of replication-related regions is consistent with the potent anti-replicative properties of bizelesin. Given the apparent lack of other types of lesions in genomic DNA, interstrand crosslinks localized in AT-rich tracts, and to some extent perhaps also monoadducts, are likely to be lethal effects of bizelesin. (+info)