Cloning,sequencing and expression of porcine CD40 ligand in Escherichia coli and human and porcine cells

The CD40L ligand (CD40L) plays an important role in the interaction between antigen-specific T lymphocytes and antigen-presenting cells. The porcine CD40L encoding gene was isolated from porcine peripheral blood mononuclear cells (PBMC) using RT-PCR. Sequence analysis of the cloned CD40L gene showed an open reading frame of 786 base pairs encoding a 262 amino acid protein with a predicted molecular mass of 29 kD. The deduced amino acid sequence of the porcine CD40L shared 82%, 88% and 93% similarity with the CD40L protein of mouse, human and cattle. The isolated CD40L sequence was expressed as a hexahistidine fusion protein in Escherichia coli and purified by affinity chromatography. The analysis of the CD40L-expression in human 293 and porcine MAX cells by immunofluorescence showed its location on the cell surface.     

Molecular cloning, characterization, and chromosomal assignment of porcine cationic amino acid transporter-1

We have cloned and characterized the gene encoding the porcine cationic amino acid transporter, member 1 (CAT-1) (HGMW-approved gene symbol SLC7A1) from porcine pulmonary artery endothelial cells. The porcine SLC7A1 encodes 629 deduced amino acid residues showing a higher degree of sequence similarity with the human counterpart (91.1%) than with the rat (87.3%) and mouse (87.6%) counterparts. Confocal microscopic examination of porcine CAT-1-GFP-expressing HEK293 cells revealed that porcine CAT-1 localizes on the plasma membrane. Amino acid uptake studies in Xenopus oocytes injected with cRNA encoding this protein demonstrated transport properties consistent with system y(+). Radiation hybrid mapping data indicate that the porcine SLC7A1 maps to the distal end of the short arm of pig chromosome 11 (SSC11). This map location is consistent with the known conservation of genome organization between human and pig and provides further confirmation that we have characterized the porcine orthologue of the human SLC7A1.     

Live cell imaging of X chromosome reactivation during somatic cell reprogramming

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.     

Tumorigenesis in mice with a fusion of the leukaemia oncogene Mll and the bacterial lacZ gene

Many different chromosomal translocations occur in man at chromosome 11q23 in acute leukaemias. Molecular analyses revealed that the MLL gene (also called ALL-1, HRX or HTRX) is broken by the translocations, causing fusion with genes from other chromosomes. The diversity of MLL fusion partners poses a dilemma about the function of the fusion proteins in tumour development. The consequence of MLL truncation and fusion has been analysed by joining exon 8 of Mll with the bacterial lacZ gene using homologous recombination in mouse embryonic stem cells. We show that this fusion is sufficient to cause embryonic stem cell-derived acute leukaemias in chimeric mice, and these tumours occur with long latency compared with those found in MLL-Af9 chimeric mice. These findings indicate that an MLL fusion protein can contribute to tumorigenesis, even if the fusion partner has no known pathogenic role. Thus, truncation and fusion of MLL can be sufficient for tumorigenesis, regardless of the fusion partner.     

Regulation of murine telomere length by Rtel: an essential gene encoding a helicase-like protein

Little is known about the genes that regulate telomere length diversity between mammalian species. A candidate gene locus was previously mapped to a region on distal mouse Chr 2q. Within this region, we identified a gene similar to the dog-1 DNA helicase-like gene in C. elegans. We cloned this Regulator of telomere length (Rtel) gene and inactivated its expression in mice. Rtel(-/-) mice died between days 10 and 11.5 of gestation with defects in the nervous system, heart, vasculature, and extraembryonic tissues. Rtel(-/-) embryonic stem cells showed telomere loss and displayed many chromosome breaks and fusions upon differentiation in vitro. Crosses of Rtel(+/-) mice with Mus spretus showed that Rtel from the Mus musculus parent is required for telomere elongation of M. spretus chromosomes in F1 cells. We conclude that Rtel is an essential gene that regulates telomere length and prevents genetic instability.     

Targeted chromosome elimination from ES-somatic hybrid cells

To engineer a stem cell genome, we developed a technology for targeted elimination of chromosomes from mouse embryonic stem (ES)-somatic hybrid cells. Here we demonstrate the use of a universal chromosome elimination cassette (CEC) for elimination of a single embryonic stem cell (ESC)-derived chromosome 11 or 12, and also both copies of chromosome 6, which harbor pluripotency-associated genes including Nanog. We attribute hybrid-cell pluripotency to the expression of Nanog from the reprogrammed somatic-cell nuclei.     

Sequence and structural organization of the human gene encoding ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a potent polypeptide hormone whose actions appear to be restricted to the nervous system where it promotes survival, neurotransmitter synthesis and neurite outgrowth in certain neuronal populations. We have cloned the gene encoding human CNTF (hCNTF) and have characterized its structure and organization. The hCNTF gene appears to be a unique-copy gene with a simple genetic organization, since only a single intron interrupts the coding domain. The hCNTF gene is located on chromosome 11, as determined using human-hamster somatic cell hybrids. The CNTF protein is highly conserved in evolution. The amino acid (aa) sequences of rat and rabbit CNTF translated from cDNAs display approx. 85% homology with the deduced aa sequence encoding hCNTF.     

Characterization of the cDNA coding for mouse plasminogen and localization of the gene to mouse chromosome 17

A full-length cDNA coding for mouse plasminogen has been isolated and characterized. The cDNA is 2720 bp in length (excluding the poly(A) tail) and contains a 24-bp 5' noncoding region, an open reading frame of 2436 bp, and a 3' noncoding region of 257 bp. The open reading frame codes for 812 amino acids and includes a signal peptide that is likely 19 amino acids in length and the mature protein of 793 amino acids. The calculated Mr of mouse plasminogen is 88,706 excluding carbohydrate. There are two potential N-linked carbohydrate addition sites; one of which is glycosylated in human, bovine, and porcine plasminogens. Mouse plasminogen was found to contain two additional amino acids compared to the human protein. In addition, mouse and human plasminogens were found to be 79 and 76% identical at the protein and DNA levels, respectively. Analysis of the segregation of two allelic forms, Plgb and Plgd, of plasminogen DNA in three sets of recombinant inbred strains has allowed the localization of the mouse plasminogen gene to the proximal end of mouse chromosome 17 within the t complex and close to the locus D17Rp17. The Plg gene is deleted in the semidominant deletion mutant, hair-pintail (Thp).     

Embryonic stem cells can be used to construct hybrid cell lines containing a single, selectable murine chromosome

Microcell-mediated chromosome transfer is a useful technique for the study of gene function, gene regulation, gene mapping, and functional cloning in mammalian cells. Complete panels of donor cell lines, each containing a different human chromosome, have been developed. These donor cell lines contain a single human chromosome marked with a dominant selectable gene in a rodent cell background. However, a similar panel does not exist for murine chromosomes. To produce mouse monochromosomal donor hybrids, we have utilized embryonic stem (ES) cells with targeted gene disruptions of known chromosomal location as starting material. ES cells with mutations in aprt, fyn, and myc were utilized to generate monochromosomal hybrids with neomycin phosphotransferase-marked murine Chr 8, 10, or 15 respectively in a hamster or rat background. This same methodology can be used to generate a complete panel of marked mouse chromosomes for somatic cell genetic experimentaion. Received: 28 July 1998 / Accepted: 15 December 1998     

Cloning and characterization of the rabbit POU5F1 gene.

The product of the POUSF1 gene, Oct4, plays an important role both in embryonic development and in the self-renewal and differentiation of totipotent cells. To understand the function of Oct4 in rabbit ES cells, we cloned and sequenced the rabbit POU5F1 gene, as well as the cDNA encoded by the gene. The Oct4 cDNA contains a 1083 bp ORF encoding a 360 aa protein and a 241 bp 3' UTR sequence. Oct4 mRNA was expressed at a high level in rabbit ES cells and was barely detectable in the adult spleen, kidney, brain and muscle tissues. The POU5F1 gene is approximately 6 kb in length and includes five exons and four introns. Gene organization is similar to that of the mouse, human and bovine orthologs. Sequencing of the gene revealed an 82% (mouse), 90% (human) and 89% (bovine) overall identity at the protein level. The rabbit POUSF1 gene was mapped to chromosome 12q1.1 by PCR amplification of DNA from two putative POU5F1-containing BAC clones, which were previously mapped to chromosome 12q1.1. The cloning of the rabbit POU5F1 gene will facilitate studies on its roles in rabbit embryogenesis and ES cells.     

Yeast importin-β is required for nuclear import of the Mig2 repressor

Background

In interphase nuclei of a wide range of species chromosomes are organised into their own specific locations termed territories. These chromosome territories are non-randomly positioned in nuclei which is believed to be related to a spatial aspect of regulatory control over gene expression. In this study we have adopted the pig as a model in which to study interphase chromosome positioning and follows on from other studies from our group of using pig cells and tissues to study interphase genome re-positioning during differentiation. The pig is an important model organism both economically and as a closely related species to study human disease models. This is why great efforts have been made to accomplish the full genome sequence in the last decade.

Results

This study has positioned most of the porcine chromosomes in in vitro cultured adult and embryonic fibroblasts, early passage stromal derived mesenchymal stem cells and lymphocytes. The study is further expanded to position four chromosomes in ex vivo tissue derived from pig kidney, lung and brain.

Conclusions

It was concluded that porcine chromosomes are also non-randomly positioned within interphase nuclei with few major differences in chromosome position in interphase nuclei between different cell and tissue types. There were also no differences between preferred nuclear location of chromosomes in in vitro cultured cells as compared to cells in tissue sections. Using a number of analyses to ascertain by what criteria porcine chromosomes were positioned in interphase nuclei; we found a correlation with DNA content.     

Isolation, characterization, and chromosomal mapping of mouse P450 17 alpha-hydroxylase/C17-20 lyase.

Cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P45017 alpha) catalyzes the conversion of C-21 steroids to C-19 steroids in gonads. A full-length mouse cDNA encoding P450 17 alpha was isolated from a mouse Leydig cell library and characterized by restriction mapping and sequencing. The predicted amino acid sequence has 83% homology to rat, 66% homology to human, and 62% homology to bovine P45017 alpha amino acid sequences. The protein is 507 amino acids in length, which is 1 amino acid shorter than the human protein and 2 amino acids shorter than the bovine protein. The structural gene encoding P450 17 alpha (Cyp17) was localized utilizing an interspecific testcross to mouse chromosome 19, distal to Got-1. Another cytochrome P450, P4502c (Cyp2c), also is located at the distal end of chromosome 19. CYP17, CYP2c, and GOT1 have been mapped to human chromosome 10, with CYP2C and GOT1 mapped to the distal region, q24.3 and q25.3, respectively. The data in the present study indicate conserved syntenic loci on mouse chromosome 19 and human chromosome 10 and predict that the structural gene encoding P45017 alpha will be found distal to GOT1 on human chromosome 10.