Bernhard G. Baumgartner

Novel polysome messages and changes in translational activity appear after induction of adipogenesis in 3T3-L1 cells

BackgroundControl of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control. We presume that translational control is an essential process in the control of adipogenesis, especially in the first hours after hormonal stimulation. 3T3-L1 preadipocytes were cultured to confluency and adipogenesis was induced by standard protocols using a hormonal cocktail. Cells were harvested before and 6 hours after hormonal induction. mRNAs attached to ribosomes (polysomal mRNAs) were separated from unbound mRNAs by velocity sedimentation. Pools of polysomal and unbound mRNA fractions were analyzed by microarray analysis. Changes in relative abundance in unbound and polysomal mRNA pools were calculated to detect putative changes in translational activity. Changes of expression levels of selected genes were verified by qPCR and Western blotting.ResultsWe identified 43 genes that shifted towards the polysomal fraction (up-regulated) and 2 genes that shifted towards free mRNA fraction (down-regulated). Interestingly, we found Ghrelin to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (eIF4B, HSF1, IRF6, MYC, POLR2a, RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa, TSC22d3), form part of ribosomes (RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa), act on the regulation of translation (eIF4B) or transcription (HSF1, IRF6, MYC, TSC22d3). Others act as chaperones (BAG3, HSPA8, HSP90ab1) or in other metabolic or signals transducing processes.ConclusionsWe conclude that a moderate reorganisation of the functionality of the ribosomal machinery and translational activity are very important steps for growth and gene expression control in the initial phase of adipogenesis.

Molecular cloning and chromosomal assignment of the porcine 54 and 56 kDa vacuolar H(+)-ATPase subunit gene (V-ATPase)

Abstract. Vacuolar proton-translocating ATPases (V-ATPase) are multisubunit enzyme complexes located in the membranes of eukaryotic cells regulating cytoplasmic pH. So far, nothing is known about the genomic organization and chromosomal location of the various subunit genes in higher eukaryotes. Here we describe the isolation and analysis of a cDNA coding for the 54- and 56-kDa porcine V-ATPase subunit alpha and beta isoforms. We have determined the genomic structure of the V-ATPase subunit gene spanning at least 62 kb on Chromosome (Chr) 4q14-q16. It consists of 14 exons with sizes ranging from 54 bp to 346 bp, with a non-coding first exon and an alternatively spliced seventh exon leading to two isoforms. The 5′ end of the V-ATPase cDNA was isolated by RACE-PCR. The V-ATPase alpha isoform mRNA, lacking the seventh exon, has an open reading frame of 1395 nucleotides encoding a hydrophilic protein of 465 amino acids with a calculated molecular mass of 54.2 kDa and a pI of 7.8, whereas the beta isoform has a length of 1449 nucleotides encoding a protein of 483 amino acids with a calculated molecular mass of 55.8 kDa. Amino acid and DNA sequence comparison revealed that the porcine V-ATPase subunit exhibits a significant homology to the VMA13 subunit of Saccharomyces cerevisiae V-ATPase complex and V-ATPase subunit of Caenorhabditis elegans.

Molecular cloning, mapping, and functional analysis of the bovine sulfate transporter SLC26a2 gene

Sulfate is one of the most important macronutrients in cells and the major sulfur source in many organisms as well as one of the most abundant anions in the serum. As sulfate is a hydrophilic anion, movement across the lipid bilayer is mediated by transporters that regulate efflux and influx. Here, we report the molecular cloning, mapping, and functional analysis of the bovine solute carrier/sulfate transporter SLC26a2 gene, the first member of this family to be cloned in cattle. A recombinant phage library was screened, and single phages harbouring the SLC26a2 gene was isolated and sequenced. A fragment of 6295 base pairs (bp) of the bovine SLC26a2 gene harbouring exon 2 and exon 3 was used for further analysis. Similar to the human, ovine, mouse, and rat SLC26a2 gene, the bovine ortholog consists of two coding exons. The open reading frame harbours 2202 nucleotides (nt), coding for a protein of 734 amino acids with a calculated molecular weight of 81.5 kilodaltons (kDa) and a statistical isoelectric point (pI) of 8.77. The bovine SLC26a2 gene was mapped to chromosome 7q23-q24 (BTA 7q23-q24) by fluorescence in situ hybridisation (FISH) analysis. Two point mutations were identified comparing the DNAs of 300 Holstein Frisian cattle, one of them resulting in an isoleucine to serine amino acid exchange at position 520. The Ile520Ser exchange influences the sulfate uptake as measured in primary fibroblasts isolated from testis and in immortalized fibroblastoid bovine cell lines.

Molecular analysis of the porcine proteolipid protein (PLP) gene

Abstract. The proteolipid protein (PLP) gene codes for the most abundant protein in the central nervous system (CNS) myelin of higher vertebrates. Its function in the myelin sheath is not clear however, a series of point mutations have been shown to have devastating effects on the myelin. The structure of the PLP genes is highly conserved, comprising seven exons that code for an open reading frame of 277 amino acids. We determined a total of 20,957 bp of the porcine PLP gene and compared this sequence with the human PLP sequence. A very high similarity was detected between the non-coding regions of the PLP genes of human and pig, interrupted primarily by several transposable elements. The porcine PLP gene was assigned to the long arm of Chromosome (Chr) X (SSXq2.2-2.4). The analysis of the PLP transcripts revealed three transcription start sites within 160 bp upstream of the translation start codon. Functional studies of the 3′ region showed the use of several polyadenylation signals. Three main transcripts were detected in adult pigs in the range of 3200, 2400, and 1600 nucleotides with Northern blot analysis. The usage of an alternative splice site within exon 3 was shown.

Structural and functional analysis of the porcine secretory carrier membrane protein 1 gene (SCAMP1).

Abstract. The secretory carrier membrane proteins (SCAMPs) are highly conserved integral vesicle membrane components of the post-Golgi secretory and endocytic pathways. We have isolated and characterized the porcine SCAMP1 cDNA and gene coding for a variant of the SCAMP family. The SCAMP1 cDNA has a length of 3827 bp including a 133-bp 5′ and 2701-bp 3′ untranslated region. The mRNA has an open reading frame of 1014 nt coding for a protein of 338 amino acids with a calculated molecular mass of 37.9 kDa and a pI of 7.9. The porcine SCAMP1 is 97.04% identical with the human and rat paralogs, respectively. The SCAMP1 gene consists of nine exons with sizes ranging from 78 to 2842 bp and spans at least 70 kb of genomic DNA on porcine Chromosome (Chr) 2q21–q22. The promoter of the SCAMP1 gene is TATA-box-less, and transcription starts at a G-nucleotide 133 nt upstream the start codon.

Dynamics of mRNA and polysomal abundance in early 3T3-L1 adipogenesis.

BackgroundAdipogenesis is a complex process, in which immature pre-adipocytes change morphology, micro-anatomy and physiology to become mature adipocytes. These store and accumulate fat and release diverse hormones. Massive changes in protein content and protein composition of the transforming cell take place within a short time-frame.In a previous study we analyzed changes in the abundance of free and polysomal, i.e. ribosome bound, RNAs in the first hours of adipogenesis in the murine cell line 3T3-L1. Here we analyze changes of mRNA levels and their potential contribution to the changing protein pool by determination of mRNA levels and ribosome binding to mRNAs in 3T3-L1 cells stimulated for adipogenesis. We grouped mRNA species into categories with respect to up- or down-regulated transcription and translation and analyzed the groups regarding specific functionalities based on Gene Ontology (GO).ResultsA shift towards up-regulation of gene expression in early adipogenesis was detected. Genes up-regulated at the transcriptional (TC:up) and translational (TL:up) level (TC:up/TL:up) are very likely involved in control and logistics of translation. Many of them are known to contain a TOP motif. In the TC:up/TL:unchanged group we detected most of the metal binding proteins and metal transporters. In the TC:unchanged/TL:up group several factors of the olfactory receptor family were identified, while in TC:unchanged/TL:down methylation and repair genes are represented. In the TC:down/TL:up group we detected many signaling factors. The TC:down/TL:unchanged group mainly consists of regulatory factors.ConclusionsWithin the first hours of adipogenesis, changes in transcriptional and translational regulation take place. Notably, genes with a specific biological or molecular function tend to cluster in groups according to their transcriptional and translational regulation.

Phenotype selection reveals coevolution of muscle glycogen and protein and PTEN as a gate keeper for the accretion of muscle mass in adult female mice.

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.

Extrinsic and intrinsic regulation of DOR/TP53INP2 expression in mice: effects of dietary fat content, tissue type and sex in adipose and muscle tissues

BackgroundDOR/TP53INP2 acts both at the chromosomal level as a nuclear co-factor e.g. for the thyroid hormone receptor and at the extrachromosomal level as an organizing factor of the autophagosome. In a previous study, DOR was shown to be down-regulated in skeletal muscle of obese diabetic Zucker fa/fa rats.MethodsTo identify sites of differential DOR expression in metabolically active tissues, we measured differences in DOR expression in white adipose tissue (WAT), brown adipose tissue (BAT), skeletal muscle (SM) and heart muscle (HM) by qPCR. To assess whether DOR expression is influenced in the short term by nutritional factors, NMRI mice were fed different fat rich diets (fat diet, FD: 18% or high fat diet, HFD: 80% fat) for one week and DOR expression was compared to NMRI mice fed a control diet (normal diet, ND: 3.3% fat). Additionally, DOR expression was measured in young (45 days old) and adult (100 days old) genetically obese (DU6/DU6i) mice and compared to control (DUKs/DUKsi) animals.ResultsANOVA results demonstrate a significant influence of diet, tissue type and sex on DOR expression in adipose and muscle tissues of FD and HFD mice. In SM, DOR expression was higher in HFD than in FD male mice. In WAT, DOR expression was increased compared to BAT in male FD and HFD mice. In contrast, expression levels in female mice were higher in BAT for both dietary conditions.DOR expression levels in all tissues of 100 days old genetically obese animals were mainly influenced by sex. In HM, DOR expression was higher in male than female animals.ConclusionsDOR expression varies under the influence of dietary fat content, tissue type and sex. We identified target tissues for further studies to analyze the specific function of DOR in obesity. DOR might be part of a defense mechanism against fat storage in high fat diets or obesity.

Mouse genes encoding DNA topoisomerase I

We have initiated a genetic analysis of the physiologically important enzyme type I DNA topoisomerase in mouse. The exon-intron structures of the 5′ part and the 3′ part of the active gene, Top-1, were determined and shown to be quite similar to those of the previously determined human gene TOP1. The active mouse gene was mapped to the distal Chromosome (Chr) 2. In addition, the mouse genome contains one truncated processed topoisomerase-I-related pseudogene (retroposon), Top-1ps, on Chr 16. The Top-1ps locus, together with the immunoglobulin-lambda-light-chain locus, defines and additional conserved linkage group common to murine Chr 16 and human Chr 22, the site of the human pseudogene TOP1P2. The mapping data suggest that the pseudogene was established before mammalian radiation. Structural features, shared by the mouse and the human pseudogene, support this possibility.

Structural analysis and transcript processing of the bovine proteolipid protein (PLP) gene.

In this study we present the complete genomic structure of the bovine PLP gene and its assignment to the long arm of the X-chromosome (BTXq2.1). We determined a total of 18, 767 bp of the bovine PLP gene and compared it to the human heterolog. A very high similarity was detected between the non-coding regions, interrupted primarily by several transposable elements. A deletion of 13 bp in the vicinity to the translation start signal in the promoter of the bovine PLP gene was found. Functional studies of the 3′ region showed the use of several polyadenylation signals. Three main transcripts were detected in adult cattle in the range of 3200, 2400, and 1600 nucleotides using Northern blot analysis. An additional shorter transcript was detected in the cerebrum of calves.