New gene family defined by MORC, a nuclear protein required for mouse spermatogenesis.

Mammalian spermatogenesis is a complex developmental process. The analysis of mouse mutations has provided insight into biochemical pathways required for completion of this process. We previously described the autosomal recessive mouse morc TgN(Tyr)1Az(microrchidia) mutation, a serendipitous transgenic insertional mutation which causes arrest of spermatogenesis prior to the pachytene stage of meiosis prophase I. We now report the molecular characterization of the morc locus and positional cloning of a gene disrupted by the morc TgN(Tyr)1Az mutation. This gene, which we term Morc, encodes a 108 kDa protein expressed specifically in male germ cells. The transgene integrated within the first intron of Morc and was accompanied by an intragenic deletion of approximately 13 kb of genomic sequences, removing exons 2-4 and abrogating expression of the wild-type transcript. Analysis of the MORC protein sequence revealed putative nuclear localization signals, two predicted coiled-coil structural motifs and limited homology to GHL (GyraseB, Hsp90, MutL) ATPase. Epitope-tagged MORC protein expressed in COS7 cells localized to the nucleus. We also cloned the human MORC homolog and show that it too is testis-specific, but closely related human genes are transcribed in multiple somatic tissues. Homologous proteins are also present in zebrafish, nematodes, slime mold and plants. Thus, cloning of Morc defines a novel gene family whose members are likely to serve important biological functions in both meiotic and mitotic cells of multicellular organisms.

Identification of morc (microrchidia), a mutation that results in arrest of spermatogenesis at an early meiotic stage in the mouse.

The microrchidia, or morc, autosomal recessive mutation results in the arrest of spermatogenesis early in prophase I of meiosis. The morc mutation arose spontaneously during the development of a mouse strain transgenic for a tyrosinase cDNA construct. Morc -/- males are infertile and have grossly reduced testicular mass, whereas -/- females are normal, indicating that the Morc gene acts specifically during male gametogenesis. Immunofluorescence to synaptonemal complex antigens demonstrated that -/- male germ cells enter meiosis but fail to progress beyond zygotene or leptotene stage. An apoptosis assay revealed massive numbers of cells undergoing apoptosis in testes of -/- mice. No other abnormal phenotype was observed in mutant animals, with the exception of eye pigmentation caused by transgene expression in the retina. Spermatogenesis is normal in +/- males, despite significant transgene expression in germ cells. Genomic analysis of -/- animals indicates the presence of a deletion adjacent to the transgene. Identification of the gene inactivated by the transgene insertion may define a novel biochemical pathway involved in mammalian germ cell development and meiosis.

Gene targeting in embryonic stem cells: the new physiology and metabolism.

The development of transgenic technology, whereby genes (or mutations) can be stably introduced into the germline of experimental mammals, now allows investigators to create mice of virtually any genotype and to assess the consequences of these mutations in the context of a developing and intact mammal. In contrast to traditional "gain-of-function" mutations, typically created by microinjection of the gene of interest into the one-celled zygote, gene targeting via homologous recombination in pluripotential embryonic stem cells allows one to modify precisely the gene of interest. The purpose of this review is to introduce the reader to the history of development of embryonic stem cell technology, the current methods employed to create "knock-out" mice, and the application of these methods to solve problems in biology. While the technology promises to provide enormous insight into mammalian development genetics, our desire is that this review will stimulate the application of gene targeting in embryonic stem cells to begin to unravel problems in complex regulatory pathways, specifically intermediary metabolism and physiology.

Pluripotential rabbit embryonic stem (ES) cells are capable of forming overt coat color chimeras following injection into blastocysts.

The isolation of pluripotent embryonic stem (ES) cell lines from preimplantation rabbit embryos and their in vitro properties have been previously described. In the present investigation, these ES cell lines were further characterized and their capacity to contribute to formation of adult, fertile animals upon injection into recipient New Zealand White blastocysts demonstrated. The efficiency of chimera formation was low (5% of live born), but the degree of chimerism, as assessed by coat color contribution from the Dutch belted strain, was high (10-50%). Thus a significant step is taken toward the development of gene-targeting technology in the rabbit, an animal whose physiology and size lend itself to unique applications in biomedical research.

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.

Cloning and characterization of the murine homolog of the sno proto-oncogene reveals a novel splice variant.

The cellular function(s) of the SNO protein remain undefined. To gain a better understanding of possible developmental roles of this cellular proto-oncogene, we have cloned two murine sno cDNAs and have investigated their expression patterns in embryonic and postnatal tissues. A single major transcript of 7.5 kb is detected in multiple tissues by Northern blot. However, reverse transcriptase polymerase chain reaction (RT-PCR) and RNAse protection assays revealed a novel splice variant in every tissue examined. Two isoforms, termed sno N and sno-dE3 (dE3, deletion within exon 3), were identified. The sno-dE3 isoform employs a novel 5' splice site located within the coding region of the third exon and deletes potential kinase recognition motifs. Transcripts of both sno isoforms accumulate ubiquitously but are most abundant in the developing central nervous system. The in situ hybridization patterns of sno expression during murine development suggest potential roles in tissues with a high degree of cellular proliferation. Expression in terminally differentiated tissues such as muscle and neurons indicates that SNO may have multiple functional activities.

Structural characterization and regulatory element analysis of the heart isoform of cytochrome c oxidase VIa.

In order to investigate the mechanism(s) governing the striated muscle-specific expression of cytochrome c oxidase VIaH we have characterized the murine gene and analyzed its transcriptional regulatory elements in skeletal myogenic cell lines. The gene is single copy, spans 689 base pairs (bp), and is comprised of three exons. The 5'-ends of transcripts from the gene are heterogeneous, but the most abundant transcript includes a 5'-untranslated region of 30 nucleotides. When fused to the luciferase reporter gene, the 3.5-kilobase 5'-flanking region of the gene directed the expression of the heterologous protein selectively in differentiated Sol8 cells and transgenic mice, recapitulating the pattern of expression of the endogenous gene. Deletion analysis identified a 300-bp fragment sufficient to direct the myotube-specific expression of luciferase in Sol8 cells. The region lacks an apparent TATA element, and sequence motifs predicted to bind NRF-1, NRF-2, ox-box, or PPAR factors known to regulate other nuclear genes encoding mitochondrial proteins are not evident. Mutational analysis, however, identified two cis-elements necessary for the high level expression of the reporter protein: a MEF2 consensus element at -90 to -81 bp and an E-box element at -147 to -142 bp. Additional E-box motifs at closely located positions were mutated without loss of transcriptional activity. The dependence of transcriptional activation of cytochrome c oxidase VIaH on cis-elements similar to those found in contractile protein genes suggests that the striated muscle-specific expression is coregulated by mechanisms that control the lineage-specific expression of several contractile and cytosolic proteins.

Nuclear transfer of putative rabbit embryonic stem cells leads to normal blastocyst development.

Rabbit embryonic stem-like cells, characterized by embryoid body formation and differentiation into cell types representative of all three germ layers, were studied for their ability to promote early embryonic development after nuclear transfer. After culture of the reconstructed embryos, 23% (n = 35) developed successfully into morulae or blastocysts, compared with 34% (n = 62) for cloned embryos derived from nuclear transfer with embryonic blastomeres. The cloned embryos from the embryonic stem-like cells appeared normal, with an average of 26% inner cell mass cells, similar to that of control non-manipulated embryos (25%) or cloned embryos from blastomeres (25%). Thus, nuclear transfer of rabbit embryonic stem-like cells leads to early embryonic development that is indistinguishable from blastomere fusion. These results have implications for the development of gene targeting in a species (rabbit) that may be a more suitable model for studying certain human diseases. In addition, this technique may be applicable to other species from which putative embryonic stem cells have been derived, particularly agriculturally important animals.

Mice expressing both B7-1 and viral glycoprotein on pancreatic beta cells along with glycoprotein-specific transgenic T cells develop diabetes due to a breakdown of T-lymphocyte unresponsiveness.

T lymphocytes have been implicated in the onset of many autoimmune diseases; however, the mechanisms underlying T-cell activation toward self antigens are poorly understood. To study whether T-lymphocyte costimulation can overcome the immunologic unresponsiveness observed in an in vivo model, we have created transgenic mice expressing the costimulatory mouse molecule B7-1, a ligand for the CD28 receptor, on pancreatic beta cells. We now report that triple-transgenic mice expressing both B7-1 and a viral glycoprotein on their beta cells, along with T cells expressing the viral-glycoprotein-specific transgenic T-cell receptor, all develop insulitis (lymphocytic infiltration of the pancreatic islets) and diabetes. In striking contrast, the T cells in double-transgenic mice expressing the same viral glycoprotein (but no B7) on their pancreatic beta cells and the transgenic T-cell receptor on their T cells, reported earlier, remain indifferent to the glycoprotein-expressing beta cells. In fact, all three transgenes are required to initiate immune-mediated destruction of the beta cells. Mice expressing any of the transgenes alone, or any two in combination, maintain normal islet architecture and never spontaneously develop insulitis or diabetes. Our results show that aberrant B7 expression on peripheral tissues may play an important role in the activation of self-reactive T cells and further suggest that abnormal expression of costimulatory receptors may be involved in various T-cell-mediated autoimmune diseases.

Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos.

We have derived putative embryonic stem (ES) cell lines from preimplantation rabbit embryos and report here their initial characterization. Two principal cell types emerged following serial passage of explanted embryos, and each has subsequently given rise to immortalized cell lines. One cell type has morphology identical to primary outgrowths of trophectoderm, is strictly feeder-cell dependent, and spontaneously forms trophectodermal vesicles at high cell density. The second type appears to represent pluripotent ES cells derived from the inner cell mass as evidenced by 1) ability to grow in an undifferentiated state on feeder layers, 2) maintenance of a predominantly normal karyotype through serial passage (over 1 year), and 3) ability to form embryoid bodies, which form terminally differentiated cell types representative of ectoderm, mesoderm, and endoderm. These ES cells may ultimately be suitable for introduction of germline mutations (via homologous recombination). The rabbit's size, reproductive capability, and well-characterized physiology make it suitable for a wide range of investigations, particularly for development of large animal models of human disease.

Gradients of transgene expression directed by the human myoglobin promoter in the developing mouse heart.

Prior studies using transient transfection assays in cultured avian and murine skeletal myotubes indicate that the proximal 2-kb segment of the 5' flanking region of the human myoglobin gene contains transcriptional control elements sufficient to direct muscle-specific and developmentally regulated expression of reporter genes. To examine the function of the human myoglobin gene promoter during development of skeletal and cardiac myocytes in the intact animal, a 2.0-kb myoglobin gene upstream fragment was fused to an Escherichia coli lacZ reporter gene and injected into fertilized mouse oocytes. beta-Galactosidase (beta-gal) activity was detected selectively in cardiac and skeletal myocytes of fetal and adult transgenic mice. A distinctive spatial pattern of myoglobin promoter activity was observed in fetal hearts: beta-gal staining was more pronounced within the left ventricular subendocardium than within the subepicardium and was essentially undetectable in the ventricular trabeculae or atria. Expression of endogenous myoglobin mRNA and protein, assessed by in situ hybridization and immunohistochemistry, demonstrated a similar spatial pattern. In contrast, hearts from adult transgenic mice demonstrated essentially homogeneous expression of beta-gal and of endogenous myoglobin mRNA and protein throughout the myocardium, including the trabeculae and atria. These data indicate that the 2.0-kb upstream region of the human myoglobin gene includes cis-acting regulatory elements sufficient to direct transgene expression during murine cardiac development that is myocyte-specific and responsive to positional cues in a similar manner to the endogenous myoglobin gene.

Cutaneous lymphoproliferation and lymphomas in interleukin 7 transgenic mice.

To investigate the role of interleukin 7 (IL-7) in the development of the lymphoid system, we have generated two lines of transgenic mice carrying an IL-7 cDNA fused to an immunoglobulin heavy chain promoter and enhancer. This transgene is expressed in the bone marrow, lymph nodes, spleen, thymus, and skin provoking a perturbation of T cell development characterized by a marked reduction of CD4+ CD8+ (double-positive) thymocytes. Quite unexpectedly, however, both lines also develop a progressive cutaneous disorder involving a dermal lymphoid infiltrate that results in progressive alopecia, hyperkeratosis, and exfoliation. Although the infiltrate is primarily composed of T lineage cells, its development is not impeded in the athymic nu/nu background. Furthermore, the phenotype can be transmitted horizontally by transplanting lymphoid tissues or skin to syngeneic wild-type mice. Thus, the phenotype is conveyed by skin-homing, mobile cells (presumably the infiltrating lymphocytes) in a cell-autonomous fashion. In addition to the skin phenotype, this transgene also provokes the development of a lymphoproliferative disorder that induces B and T cell lymphomas within the first 4 mo of life. These findings suggest potential physiologic actions of IL-7 in T cell development and in cutaneous immunity. They also demonstrate that IL-7 can act as an oncogene in the living organism.

Binary system for regulating transgene expression in mice: targeting int-2 gene expression with yeast GAL4/UAS control elements.

We have developed a binary transgenic system that activates an otherwise silent transgene in the progeny of a simple genetic cross. The system consists of two types of transgenic mouse strains, targets and transactivators. A target strain bears a transgene controlled by yeast regulatory sequences (UAS) that respond only to the yeast transcriptional activator GAL4. A transactivator strain expresses an active GAL4 gene that can be driven by any selected promoter. The current paradigm uses the murine growth factor int-2 cDNA as the target gene and the GAL4 gene driven by the mouse mammary tumor virus long terminal repeat as the transactivator. Both target and transactivator strains are phenotypically normal. By contrast, the bigenic offspring of these two strains express high levels of the target int-2 gene in each organ expressing the GAL4 transactivator. They also display a characteristic dominant int-2 phenotype that consists of epithelial hyperplasia in mammary and salivary glands, as well as prostatic and epididymal hypertrophy, which results in male sterility.

Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex.

Deficiency of the iron-sulfur clusters of mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase (complex I) in an infant with congenital lactic acidosis.

We report the case of an infant with hypoglycemia, progressive lactic acidosis, an increased serum lactate/pyruvate ratio, and elevated plasma alanine, who had a moderate to profound decrease in the ability of mitochondria from four organs to oxidize pyruvate, malate plus glutamate, citrate, and other NAD+-linked respiratory substrates. The capacity to oxidize the flavin adenine dinucleotide-linked substrate, succinate, was normal. The most pronounced deficiency was in skeletal muscle, the least in kidney mitochondria. Enzymatic assays on isolated mitochondria ruled out defects in complexes II, III, and IV of the respiratory chain. Further studies showed that the defect was localized in the inner membrane mitochondrial NADH-ubiquinone oxidoreductase (complex I). When ferricyanide was used as an artificial electron acceptor, complex I activity was normal, indicating that electrons from NADH could reduce the flavin mononucleotide cofactor. However, electron paramagnetic resonance spectroscopy performed on liver submitochondrial particles showed an almost total loss of the iron-sulfur clusters characteristic of complex I, whereas normal signals were noted for other mitochondrial iron-sulfur clusters. This infant is presented as the first reported case of congenital lactic acidosis caused by a deficiency of the iron-sulfur clusters of complex I of the mitochondrial electron transport chain.

Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria.

The purification and kinetic characterization of an NAD(P)+-malic enzyme from 22aH mouse hepatoma mitochondria are described. The enzyme was purified 328-fold with a final yield of 51% and specific activity of 38.1 units/mg of protein by employing DEAE-cellulose chromatography and an ATP affinity column. Sephadex G-200 chromatography yielded a native Mr = 240,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major subunit with Mr = 61,000, suggesting a tetrameric structure, and also showed that the preparation contained less than 10% polypeptide impurities. Use of the ATP affinity column required the presence of MnCl2 and fumarate (an allosteric activator) in the elution buffers. In the absence of fumarate, the Michaelis constants for malate, NAD+, and NADP+ were 3.6 mM, 55 microM, and 72 microM, respectively; in the presence of fumarate (2 mM), the constants were 0.34 mM, 9 microM, and 13 microM, respectively. ATP was shown to be an allosteric inhibitor, competitive with malate. However, the inhibition by ATP displayed hyperbolic competitive kinetics with a KI (ATP) of 80 microM (minus fumarate) and 0.5 mM (plus 2 mM fumarate). The allosteric properties of the enzyme are integrated into a rationale for its specific role in the pathways of malate and glutamate oxidation in tumor mitochondria.

The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)+-dependent malic enzyme.

Little evidence has been available on the oxidative pathways of glutamine and glutamate, the major respiratory substrates of cancer cells. Glutamate formed from glutamine by phosphate-dependent glutaminase undergoes quantitative transamination by aerobic tumor mitochondria to yield aspartate. However, when malate is also added there is a pronounced decrease in aspartate production and a large formation of citrate and alanine, in both state 3 and 4 conditions. In contrast, addition of malate to normal rat heart, liver, or kidney mitochondria oxidizing glutamate causes a marked increase in aspartate production. Further analysis showed that extramitochondrial malate is oxidized almost quantitatively to pyruvate + CO2 by NAD(P)+-linked malic enzyme, present in the mitochondria of all tumors tested, but absent in heart, liver, and kidney mitochondria. On the other hand intramitochondrial malate generated from glutamate is oxidized quantitatively to oxalacetate by mitochondrial malate dehydrogenase of tumors. Acetyl-CoA derived from extramitochondrial malate via pyruvate and oxalacetate derived from glutamate via intramitochondrial malate are quantitatively converted into citrate, which is extruded. No evidence was found that malic enzyme of tumor mitochondria converts glutamate-derived malate into pyruvate as postulated in other reports. Possible mechanisms for the integration of mitochondrial malic enzyme and malate dehydrogenase activities in tumors are discussed.