Development and Characterization of cDNA Resources for the Common Marmoset: One of the Experimental Primate Models

The common marmoset is a new world monkey, which has become a valuable experimental animal for biomedical research. This study developed cDNA libraries for the common marmoset from five different tissues. A total of 290 426 high-quality EST sequences were obtained, where 251 587 sequences (86.5%) had homology (1E⁻¹⁰⁰) with the Refseqs of six different primate species, including human and marmoset. In parallel, 270 673 sequences (93.2%) were aligned to the human genome. When 247 090 sequences were assembled into 17 232 contigs, most of the sequences (218 857 or 15 089 contigs) were located in exonic regions, indicating that these genes are expressed in human and marmoset. The other 5578 sequences (or 808 contigs) mapping to the human genome were not located in exonic regions, suggesting that they are not expressed in human. Furthermore, a different set of 118 potential coding sequences were not similar to any Refseqs in any species, and, thus, may represent unknown genes. The cDNA libraries developed in this study are available through RIKEN Bio Resource Center. A Web server for the marmoset cDNAs is available at http://marmoset.nig.ac.jp/index.html, where each marmoset EST sequence has been annotated by reference to the human genome. These new libraries will be a useful genetic resource to facilitate research in the common marmoset.     

ISOLATION OF PHASE-SPECIFIC COMPLE-MENTARY DNAS FROM CARPOSPOROPHYTES OF GRACILARIOPSIS LEMA-NEIFORMIS USING MAGNETIC BEADS AND PCR

Kamiya, M.¹, Moon, D. A.², Kawai, H.¹ & Goff, L. J.^{2 1}Kobe University Research Center for Inland Seas, 2746 Iwaya, Awaji-cho 656-2401 Japan; ²Department of Biology, University of California, Santa Cruz, CA 95064 USA Although the morphology and developmental patterns of carposporophyte stage have been investigated well, there are few molecular, genetic, or biochemical data about this stage. The greatest obstacle to this research has been that the conventional methods to isolate tissue-specific genes require a lot of tissues, but the carposporophyte is very tiny and mostly embedded in female gametophyte tissues. Recent advanced techniques have allowed the subtractive cloning of differentially expressed genes from small amounts of tissue or cells. We applied the subtractive hybridization method using magnetic beads and PCR to the analysis of phase-specific cDNAs from carpo-sporophytes of Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta). A hundred cystocarps were dissected to isolate gonimoblast tissues, and total RNAs were extracted from the gonimoblast tissues and the female gametophyte branches, respectively. Messenger RNAs were captured on paramagnetic oligo-dT beads, followed by first-strand cDNA synthesis on the beads. Three rounds of subtractive hybridization between the amplified second-strand carposporophyte cDNA in solution and the first-strand gametophyte cDNA attached to magnetic beads were sufficient to remove common genes present in both gametophyte and carposporophyte stages. A specific PCR product from the nuclear GAPDH gene was readily amplified from gametophyte and carposporophyte cDNA, but no amplification was observed using the subtracted carposporophyte cDNA as template. This control PCR product demonstrates that the hybridization steps successfully removed the common GAPDH cDNA, which is found in both stages, giving confidence that the remaining genes cloned from the subtracted carposporophyte cDNA library are stage-specific.     

NHE10, an osteoclast-specific member of the Na+/H+ exchanger family, regulates osteoclast differentiation and survival [corrected

Bone homeostasis is tightly regulated by the balanced actions of osteoblasts (OBs) and osteoclasts (OCs). We previously analyzed the gene expression profile of OC differentiation using a cDNA microarray, and identified a novel osteoclastogenic gene candidate, clone OCL-1-E7 [J. Rho, C.R. Altmann, N.D. Socci, L. Merkov, N. Kim, H. So, O. Lee, M. Takami, A.H. Brivanlou, Y. Choi, Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis, DNA Cell Biol. 21 (2002) 541-549]. In this study, we have isolated full-length cDNAs corresponding to this clone from mice and humans to determine the functional roles of this gene in osteoclastogenesis. The full-length cDNA of OCL-1-E7 encodes 12 membrane-spanning domains that are typical of isoforms of the Na⁺/H⁺ exchangers (NHEs), indicating that this clone is a novel member of the NHE family (hereafter referred to as NHE10). Here, we show that NHE10 is highly expressed in OCs in response to receptor activator of nuclear factor-κB ligand signaling and is required for OC differentiation and survival.     

A nonradioactive method for isolating complementary DNAs endocing calmodulin-binding proteins

Calmodulin labeled with¹²⁵I or³⁴S has been used to screen expression libraries to isolate cDNAs encoding calmodulin-binding proteins (CBPs) from several eukaryotic systems. The use of radiolabeled calmodulin has, however, several disadvantages. We have developed a nonradiactive method to isolate cDNAs for CBPs using biotinylated calmodulin. Screening of a cDNA library in an expression vector with biotinylated calmodulin resulted in the isolation of cDNAs encoding CBPs. Avidin and biotin blocking steps, prior to incubation of the filters with biotinylated calmodulin, are found to be essential to eliminate the cDNAs that code for biotin-containing polypeptides. The cDNA clones isolated using this nonradioactive method bound calmodulin in a calcium-dependent manner. The binding of biotinylated calmodulin to these clones was completely abolished by ethylene glycolbis(\-aminoethylether)-N,N′-tetraacetic acid (EGTA), a calcium chelator. Furthermore, the isolated cDNAs were confirmed by probing the clones with³⁵S-labeled calmodulin. All the isolated clones bound to radiolabeled calmodulin in the presence of calcium but not in the presence of EGTA. The method described here is simple, fast, and does not involve preparation and handing of radiolabeled calmodulin. All the materials used in this method are commercially available; hence, this procedure should be widely applicable to isolate cDNAs encoding CBPs from any eukaryotic organism.     

Biologically relevant effects of mRNA amplification on gene expression profiles

Background  

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results.     

Combination of PCR subtraction and cDNA microarray for differential gene expression profiling.

PCR subtraction hybridization has been used effectively to enrich and single out differentially expressed genes. However identification of these genes by means of cloning and sequencing individual cDNAs is a tedious and lengthy process. In this report, an attempt has been made to combine the use of PCR select cDNA subtraction hybridization and cDNA microarrays to identify differentially expressed genes using a nonradioactive chemiluminescent detection method. mRNA from human prolactin (hPRL) or human prolactin antagonist (hPRL-G129R) treated and non-treated breast cancer cells was isolated, and cDNAs were synthesized and used for the PCR subtraction to enrich the differentially expressed genes in the treated cells. The PCR-amplified and subtracted cDNA pools were purified and labeled using the digoxigenin method. Labeled cDNAs were hybridized to a human apoptosis cDNA microarray membrane and identified by chemiluminescence. The results suggest that the strategy of combining all three methods will allow for a more efficient, nonradioactive way of identifying differentially expressed genes in target cells.     

A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins II: public release of inaugural version of InGaP database containing gene/protein expression profiles for 127 mouse KIAA genes/proteins.

The inaugural version of the InGaP database (Integrative Gene and Protein expression database; http://www.kazusa.or.jp/ingap/index.html) is a comprehensive database of gene/protein expression profiles of 127 mKIAA genes/proteins related to hypothetical ones obtained in our ongoing cDNA project. Information about each gene/protein consists of cDNA microarray analysis, subcellular localization of the ectopically expressed gene, and experimental data using anti-mKIAA antibody such as Western blotting and immunohistochemical analyses. KIAA cDNAs and their mouse counterparts, mKIAA cDNAs, were mainly isolated from cDNA libraries derived from brain tissues, thus we expect our database to contribute to the field of neuroscience. In fact, cDNA microarray analysis revealed that nearly half of our gene collection is predominantly expressed in brain tissues. Immunohistochemical analysis of the mouse brain provides functional insight into the specific area and/or cell type of the brain. This database will be a resource for the neuroscience community by seamlessly integrating the genomic and proteomic information about the mouse KIAA genes/proteins.     

Gene expression profiles analyzed by DNA sequencing of cDNA clones constructed from porcine preadipocytes and adipocytes

As a step towards understanding the molecular mechanism of adipogenesis in pigs, preadipocytes purified from the back fat of 1 day-old female piglets were used for in vitro culture. Normalized cDNA libraries were constructed with 1.6×10⁷ and 1.1×10⁷ independent clones from preadipocyte and mature adipocyte mRNAs, respectively. Polymerase chain reaction (PCR) result using primers T3 and T7 (universal primer) confirmed the presence of the insert in the vector. Sequencing of 2,112 randomly selected clones from each cDNA library identified 217 clusters, 1,169 singletons, and 216 contigs in preadipocytes and 231 clusters, 1,100 singletons, and 233 contigs in mature adipocytes. Expressed sequence tag (EST) identified 24 genes with known annotation highly expressed in adipocytes and 21 in preadipocytes by at least four EST number. Among those 45 genes, when analyzed by real time RT-PCR, 76% of the gene showed significant difference between preadipocytes and mature adipocytes. Highly expressed genes in mature adipocytes were related to adipogenesis, extracellular matrix control and oncogenes, whereas cytoskeleton-related genes were down-regulated. An interesting similarity found during gene profile studies indicated a correlation between cancer and adipogenesis.