Mouse Clones Research Articles

Rab Coupling Protein (RCP), a Novel Rab4 and Rab11 Effector Protein

Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11. We name this protein Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family.

Untimely ends and new beginnings in mouse cloning

A new study shows that the majority of mice cloned from somatic cells die prematurely. Of the six dead clones analyzed, four exhibited extensive necrosis of the liver, two had tumors and all had pneumonia, suggestive of immune impairment. These latest cloning-associated phenotypes will facilitate a better understanding of the mechanisms underlying nuclear-transfer cloning in mammals.

Human CREB-binding Protein/p300-interacting Transactivator with ED-rich Tail (CITED) 4, a New Member of the CITED Family, Functions as a Co-activator for Transcription Factor AP-2

Members of the CREB-binding protein/p300-interacting transactivator with ED-rich tail (CITED) family bind CREB-binding protein and p300 with high affinity and regulate gene transcription. Gene knockout studies indicate that CITED2 is required for neural crest and neural tube development and that it functions as a co-activator for transcription factor AP-2 (TFAP2). Here we describe human CITED4, a new member of this family, which is encoded by a single exon mapping to chromosome 1p34--1p35. CITED4 and p300/CREB-binding protein are present in endogenous naturally occurring complexes, indicating that they interact physiologically. The interaction occurs between the cysteine-histidine-rich domain 1 of p300 and the carboxyl terminus of CITED4. In keeping with this, CITED4 functions as a transactivator when artificially targeted to a promoter element. CITED4 physically interacts with all TFAP2 isoforms in vitro and strongly co-activates all TFAP2 isoforms in Hep3B cells. Co-activation of TFAP2 requires amino-terminal and carboxyl-terminal residues of CITED4. In HepG2 cells, CITED4 is significantly weaker than CITED2 for TFAP2C co-activation. These results suggest that CITED4 may function as a co-activator for TFAP2. They also suggest the existence of cell type- and TFAP2 isoform-specific co-activation by CITED2 and CITED4, which may result in differential modulation of TFAP2 function.

Homologues of amino acid permeases: cloning and tissue expression of XAT1 and XAT2

The l-type (LAT) family of amino acid transporters is composed of exchangers for neutral, cationic, and anionic amino acids. They form functional heterodimers with membrane glycoproteins, rBAT or 4F2hc/CD98, to which they are linked by a disulphide bond. We report the molecular cloning and tissue expression of new mouse and human homologues of the LAT family, termed mXAT1, mXAT2 and hXAT2. The latter two proteins may correspond to ortholog genes in mouse and human. The hXAT2 gene is located on chromosome 8q21.3. The cloned X amino acid transporter (XAT) cDNAs are predicted to encode proteins of about 50 kDa. From a phylogenetic point of view, the three XAT proteins cluster together, but sequence comparison and secondary structure prediction show that they are also related to the members of the LAT family. Like these transporters, the XAT proteins show 12 transmembrane domains and a conserved cysteine residue, located in the second extracellular loop. This conserved cysteine is involved in the disulphide bond formed between the known members of the LAT family and 4F2hc or rBAT. The mXAT1 and hXAT2 mRNAs are expressed in the kidney but they are not detectable in a variety of other tissues. The corresponding proteins were efficiently translated following transfection of their cDNAs in Chinese hamster ovary (CHO) cells. However, cDNA transfection in CHO cells did not induce amino acid uptake, even when cotransfected with vectors expressing 4F2hc or rBAT. This could be related to the fact that mXAT1 and hXAT2 did not form detectable disulphide-linked heterodimers with 4F2hc or rBAT when they were co-expressed in CHO cells. Identification of other putative partner(s) of these LAT family-related transporters may be necessary to understand their role in renal physiology.

Cloning and characterization of guanine deaminase from mouse and rat brain

A search for genes differentially expressed in the rat striatum revealed a gene fragment with a ventral to dorsal striatal expression pattern. The sequence of the fragment was used to isolate mouse and rat clones that upon sequencing were identified as homologous to human guanine deaminase. Here we report the distribution of guanine deaminase in the rodent brain. In situ hybridization localization of the encoding mRNA showed a distribution primarily in forebrain areas including cortical pyramidal neurons, ventral striatal medium spiny neurons, hippocampal pyramidal neurons in CA3–CA1 and granule cells in the dentate gyrus, and neurons of the amygdala. Immunohistochemistry using antibodies raised against peptide fragments derived from the guanine deaminase protein sequence showed localization of guanine deaminase in areas predicted by the mRNA distribution. In addition to immunolabeling of neurons in the cerebral cortex, hippocampus, striatum and amygdala there was also labeling in the terminal fields of these neurons including the thalamus, globus pallidum and substantia nigra. A functional histochemical assay that demonstrates the site of guanine deamination shows guanine deaminase activity in a pattern that matched the immunohistochemical localization. The cellular distribution of guanine deaminase to distal areas of the cell including terminals and dendrites was additionally demonstrated by the expression of recombinant guanine deaminase in transformed cortical neurons in culture. In summary we have described the isolation and characterization of mouse and rat guanine deaminase. The expression of guanine deaminase is primarily restricted to forebrain neurons. A histochemical assay was used to localize guanine deaminase activity to the dendrites and axons of neurons expressing guanine deaminase.

FANTOM DB: database of Functional Annotation of RIKEN Mouse cDNA Clones.

FANTOM DB, the database of Functional Annotation of RIKEN Mouse cDNA Clones, is designed to store sequence information of RIKEN full-length enriched mouse cDNA clones, graphical views of sequence analysis results, curated functional annotation information and additional descriptions, including Gene Ontology terms. RIKEN's Mouse Gene Encyclopedia Project aims to collect full-length enriched cDNA clones from various mouse tissues, determine the full-length nucleotide sequences, infer their chromosomal locations by computer and characterize gene expression patterns. FANTOM DB has been developed to facilitate this work and to facilitate functional genomic studies such as positional candidate cloning, cDNA microarrays and protein interaction analyses. FANTOM DB contains 21 076 full-length cDNA sequences with rich functional annotations and is publicly available. FANTOM DB thus provides curated functional annotation to RIKEN full-length enriched mouse clones, and has links to other public resources. FANTOM DB can be accessed at http://fantom.gsc.riken.go.jp/db/.

Molecular Cloning of Novel Mouse and Human Putative Citrate Lyase β-Subunit

Using a fluorescent differential display (FDD) technique, a novel cDNA was identified by screening for gene expressed differentially between the Dunn osteosarcoma cell line and the LM8 cell line, an isolated variant of the Dunn cell line that has high metastatic potential to the lung. Molecular cloning of the cDNA revealed the clone has similarity to a bacterial fermentation enzyme, the citrate lyase β-subunit (CL-β). Northern blot and competitive reverse transcription-PCR (RT-PCR) analysis revealed up-regulation of the gene in the LM8 cell line. An RNA Master blot indicated that the mRNA encoding CL-β is expressed abundantly in murine heart, liver, and kidney. A human expressed sequence tag (EST) database search suggested that a similar cDNA is expressed in humans. A gene with identical sequence is located on chromosome 13 in the genome database (Sanger centre, UK). These data suggest that a citrate fermentation pathway may exist in eukaryotes including mammals.

Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer.

When neural cells were collected from the entire cerebral cortex of developing mouse fetuses (15.5-17.5 days postcoitum) and their nuclei were transferred into enucleated oocytes, 5.5% of the reconstructed oocytes developed into normal offspring. This success rate was the highest among all previous mouse cloning experiments that used somatic cells. Forty-four percent of live embryos at 10.5 days postcoitum were morphologically normal when premature and early-postmitotic neural cells from the ventricular side of the cortex were used. In contrast, the majority (95%) of embryos were morphologically abnormal (including structural abnormalities in the neural tube) when postmitotic-differentiated neurons from the pial side of the cortex were used for cloning. Whereas 4.3% of embryos cloned with ventricular-side cells developed into healthy offspring, only 0.5% of those cloned with differentiated neurons in the pial side did so. These facts seem to suggest that the nuclei of neural cells in advanced stages of differentiation had lost their developmental totipotency. The underlying mechanism for this developmental limitation could be somatic DNA rearrangements in differentiating neural cells.

Restricted expression of LUZP in neural lineage cells: a study in embryonic stem cells.

A novel protein LUZP with 3 leucine zipper motifs at its amino terminus is predominantly expressed in the adult brain. A modified gene targeting approach was employed in an attempt to establish in vitro and in vivo models in which Luzp is knock-out (KO) for phenotype assessment and a reporter gene lacZ is knock-in (KI) for tracing its expression. We report in this study the molecular cloning of the Luzp gene, its targeting vector construction and Luzp-KO/lacZ-KI embryonic stem (ES) clone selection. Since LUZP is also expressed in ES cells, the possibility of embryonic lethality cannot be excluded when attempting to establish Luzp-null mutant mice. We have therefore examined the development of homozygous Luzp-KO/lacZ-KI clones in nude mice. Tissue types derived from all three embryonic germ layers were observed in teratomas developed in nude mice. In situ X-gal staining further revealed restricted expression of LUZP in neural lineage cells.

Cloning, characterization, and tissue expression pattern of mouse Nma/BAMBI during odontogenesis.

Degenerate oligonucleotides to consensus serine kinase functional domains previously identified a novel, partial rabbit tooth cDNA (Zeichner-David et al., 1992) that was used in this study to identify a full-length mouse clone. A 1390-base-pair cDNA clone was isolated encoding a putative 260-amino-acid open reading frame containing a hydrophobic 25-amino-acid potential transmembrane domain. This clone shares some homology with the TGF-beta type I receptor family, but lacks the intracellular kinase domain. DNA database analysis revealed that this clone has 86% identity to a newly isolated human gene termed non-metastatic gene A and 80% identity to a Xenopus cDNA clone termed BMP and activin membrane bound inhibitor. Here we report the mouse Nma/BAMBI cDNA sequence, the tissue expression pattern, and confirmed expression in dental cell lines. This study demonstrates that Nma/BAMBI is a highly conserved protein across species and is expressed at high levels during odontogenesis.

Cell cycle effects of IL-10 on malignant B-1 cells.

IL-10 is overexpressed in human chronic lymphocytic leukemia (CLL), and is an autocrine growth factor involved in the development of malignant B1 clones in NZB mice, a murine model for CLL. Antisense IL-10 oligonucleotide treatment induces apoptosis and cell cycle disruption in these cells both in vitro and in vivo. In addition, NZB IL-10 knock-out mice fail to develop the B-1 clones. Dampening of IL-10 protein production via antisense IL-10 oligonucleotide treatment is correlated with decreased p27/Kip1 protein expression which results in increased cyclin D2, cyclin E and cyclin A associated kinase activity. The action of the antisense oligonucleotides is through alterations in cell cycle regulation, resulting in accelerated cell cycle progression, a G2/M block which culminates in apoptosis induction in the malignant cells. This implies that the role of IL-10 as an autocrine growth factor in malignant B-1 cells lies in its ability to inhibit apoptosis induction through the maintenance of sustainable cell cycle progression in malignant cells.

Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei.

Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide (DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis (cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% (v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.

Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei

Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide (DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis (cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% (v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.

Cloning, Mapping, Genomic Organization, and Expression of Mouse M-LP, a New Member of the Peroxisomal Membrane Protein Mpv17 Domain Family

We have identified a mouse full-length cDNA and gene encoding a novel protein (M-LP), based on an expressed sequence tag (EST) sequence (GenBank Accession No. AI482564) obtained by differential display screening of age-dependently expressed genes in mouse kidney. The ML-P gene is composed of three exons, ranges over 5 kb on mouse chromosome 16B1-B2 and is expressed as two transcripts (1455 and 3058 bp), both of which include the same open-reading frame encoding 194 amino acids. M-LP is expressed mainly in kidney and spleen and shows age-dependent expression. M-LP has sequence homologies and membrane topologies very similar to the Mpv17 protein, a peroxisomal protein involved in the development of early-onset glomerulosclerosis. Search of the protein domain family database (ProDom) revealed that M-LP is a new member of the Mpv17 domain family (PD008400).

Cloning, expression and localization of human BM88 shows that it maps to chromosome 11p15.5, a region implicated in Beckwith-Wiedemann syndrome and tumorigenesis.

Porcine BM88 is a neuron-specific protein that enhances neuroblastoma cell differentiation in vitro and may be involved in neuronal differentiation in vivo. Here we report the identification, by Western blotting, of homologous proteins in human and mouse brain and the isolation of their respective cDNAs. Several human and mouse clones were identified in the EST database using porcine BM88 cDNA as a query. A human and a mouse EST clone were chosen for sequencing and were found both to predict a protein of 149 amino acids, with 79.9% reciprocal identity, and 76.4% and 70.7% identities to the porcine protein, respectively. This indicated that the clones corresponded to the human and mouse BM88 homologues. In vitro expression in a cell-free system as well as transient expression in COS7 cells yielded polypeptide products that were recognized by anti-BM88 antibodies and were identical in size to the native BM88 protein. Northern-blot analysis showed a wide distribution of the gene in human brain whereas immunohistochemistry on human brain sections demonstrated that the expression of BM88 is confined to neurons. The initial mapping assignment of human BM88 to chromosome 11p15.5, a region implicated in Beckwith-Wiedemann syndrome and tumorigenesis, was retrieved from the UniGene database maintained at the National Centre for Biotechnology Information (NCBI, Bethesda, MD, U.S.A.). We confirmed this localization by performing fluorescence in situ hybridization on BM88-positive cosmid clones isolated from a human genomic library. These results suggest that BM88 may be a candidate gene for genetic disorders associated with alterations at 11p15.5.

Mouse cDNA library

A collaboration between the Riken Genome Exploration Research Group Phase II team and the FANTOM Consortium has produced a collection of full-length complementary DNAs, their sequences and their physical mapping to the mouse genome (Nature, 8 February). The authors report on a cDNA set of 21 076 mouse clones (some of which are derived from the same transcripts). The work forms the basis for the production of microarrays and ways to identify new genes in this very useful animal model. D.M.

DSCAM, a Highly Conserved Gene in Mammals, Expressed in Differentiating Mouse Brain

Down Syndrome Cell Adhesion molecule (DSCAM) is a member of the immunoglobulin superfamily, and represents a novel class of neuronal cell adhesion molecules. In order to understand the cellular functions of DSCAM, we isolated full-length mouse and human cDNA clones, and analysed its expression during mouse development and differentiation. Sequence analysis of the human DSCAM cDNA predicted at least 33 exons that are distributed over 840 kb. When compared to human DSCAM, the mouse homologue showed 90 and 98% identity at the nucleotide and amino acid levels, respectively. In mouse, DSCAM is located on 16C, the syntenic region for human chromosome band 21q22 and also the region duplicated in mouse DS models. DSCAM gene is predicted to encode an ∼220-kDa protein, and its expression shows dynamic changes that correlate with neuronal differentiation during mouse development. Our results suggest that DSCAM may play critical roles in the formation and maintenance of specific neuronal networks in brain.

The Growth of the Very Large CD8+ T Cell Clones in Older Mice Is Controlled by Cytokines

Older humans and mice frequently contain very large clones of CD8(+) T cells. In mice these cells are phenotypically very similar to memory CD8(+) T cells. Like memory CD8(+) T cells, most members of the clones are in continuous slow division, apparently independently of Ag stimulation. Proliferation of the CD8(+) clonal T cells is inhibited in mice treated with Ab to the IL-2R beta-chain that blocks signaling by either IL-2 or IL-15. However, inhibition of IL-2 increases the numbers of dividing clonal cells. Therefore, like normal memory CD8(+) T cells, expansion of the clones is driven by IL-15 and inhibited by IL-2 and is probably limited by the amounts of IL-15 and IL-2 present in the host. Control by these two cytokines may account for the fact that, although the clones can be very large, they do not overwhelm or kill their hosts. Nevertheless the clonal cells compete successfully with normal memory CD8(+) T cells for growth. Perhaps the clonal cells use IL-15 more effectively or are more resistant to the inhibitory effects of IL-2. Thus they might affect the immune response of their hosts by competing for factors that stimulate and inhibit normal CD8(+) memory T cells.

Restricted Expression of LUZP in Neural Lineage Cells: A Study in Embryonic Stem Cells

A novel protein LUZP with 3 leucine zipper motifs at its amino terminus is predominantly expressed in the adult brain. A modified gene targeting approach was employed in an attempt to establish in vitro and in vivo models in which Luzp is knock-out (KO) for phenotype assessment and a reporter gene lacZ is knock-in (KI) for tracing its expression. We report in this study the molecular cloning of the Luzp gene, its targeting vector construction and Luzp-KO/lacZ-KI embryonic stem (ES) clone selection. Since LUZP is also expressed in ES cells, the possibility of embryonic lethality cannot be excluded when attempting to establish Luzp-null mutant mice. We have therefore examined the development of homozygous Luzp-KO/lacZ-KI clones in nude mice. Tissue types derived from all three embryonic germ layers were observed in teratomas developed in nude mice. In situ X-gal staining further revealed restricted expression of LUZP in neural lineage cells.

Comparative architectural aspects of regions of conserved synteny on human chromosome 11p15.3 and mouse chromosome 7 (including genes WEE1 and LMO1)

Human chromosome 11p15.3 is associated with chromosome aberrations in the Beckwith Wiedemann Syndrome and implicated in the pathogenesis of different tumor types including lung cancer and leukemias. To date, only single tumor-relevant genes with linkage to this region (e.g. LMO1) have been found suggesting that this region may harbor additional potential disease associated genes. Although this genomic area has been studied for years, the exact order of genes/chromosome markers between D11S572 and the WEE1 gene locus remained unclear. Using the FISH technique and PAC clones of the flanking markers we determined the order of the genomic markers. Based on these clones we established a PAC contig of the respective region. To analyse the chromosome area in detail the synteny of the orthologous region on distal mouse chromosome 7 was determined and a corresponding mouse clone contig established, proving the conserved order of the genes and markers in both species: “cen–WEE1–D11S2043–ZNF143–RANBP7–CEGF1– ST5–D11S932–LMO1–D11S572–TUB–tel”, with inverted order of the murine genes with respect to the telomere/centromere orientation. The region covered by these contigs comprises roughly 1.6 MB in human as well as in mouse. The genomic sequence of the two subregions (around WEE1 and LMO1) in both species was determined using a shotgun sequencing strategy. Comparative sequence analysis techniques demonstrate that the content of repetitive elements seems to decline from centromere to telomere (52.6% to 34.5%) in human and in the corresponding murine region from telomere to centromere (41.87% to 27.82%). Genomic organisation of the regions around WEE1 and LMO1 was conserved, although the length of gene regions varied between the species in an unpredictable ratio. CpG islands were found conserved in putative promoter regions of the known genes but also in regions which so far have not been described as harboring expressed sequences.