Showing papers by "Nancy A. Jenkins published in 1994"

Limb alterations in brachypodism mice due to mutations in a new member of the TGFβ-superfamily

Abstract: THE mutation brachypodism (bp) alters the length and number of bones in the limbs of mice but spares the axial skeleton1,2. It illustrates the importance of specific genes in controlling the morphogenesis of individual skeletal elements in the tetrapod limb3,4. We now report the isolation of three new members of the transforming growth factor-β (TGF-β) superfamily5 (growth/differentiation factors (GDF) 5, 6 and 7) and show by mapping, expression patterns and sequencing that mutations in Gdf5 are responsible for skeletal alterations in bp mice. GDF5 and the closely related GDF6 and GDF7 define a new subgroup of factors related to known bone- and cartilage-inducing molecules, the bone morpho-genetic proteins (BMPs)6. Studies of Bmp5 mutations in short ear mice have shown that at least one other BMP gene is also required for normal skeletal development7. The highly specific skeletal alterations in bp and short ear mice suggest that different members of the BMP family control the formation of different morphological features in the mammalian skeleton.

Lymphotactin: a cytokine that represents a new class of chemokine

Abstract: In this study, the cytokine-producing profile of progenitor T cells (pro-T cells) was determined. During screening of a complementary DNA library generated from activated mouse pro-T cells, a cytokine designated lymphotactin was discovered. Lymphotactin is similar to members of both the Cys-Cys and Cys-X-Cys chemokine families but lacks two of the four cysteine residues that are characteristic of the chemokines. Lymphotactin is also expressed in activated CD8+ T cells and CD4-CD8- T cell receptor alpha beta + thymocytes. It has chemotactic activity for lymphocytes but not for monocytes or neutrophils. The gene encoding lymphotactin maps to chromosome one. Taken together, these observations suggest that lymphotactin represents a novel addition to the chemokine superfamily.

Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1 beta-converting enzyme.

Abstract: By subtraction cloning we previously identified a set of mouse genes (named Nedd1 through Nedd10) with developmentally down-regulated expression in brain. We now show that one such gene, Nedd2, encodes a protein similar to the mammalian interleukin-1 beta-converting enzyme (ICE) and the product of the Caenorhabditis elegans cell death gene ced-3 (CED-3). Both ICE and CED-3 are known to encode putative cysteine proteases and induce apoptosis when overexpressed in cultured cells. Overexpression of Nedd2 in cultured fibroblast and neuroblastoma cells also resulted in cell death by apoptosis, which was suppressed by the expression of the human bcl-2 gene, indicating that Nedd2 is functionally similar to the ced-3 gene in C. elegans. We also show that during embryonic development, Nedd2 is highly expressed in several types of mouse tissue undergoing high rates of programmed cell death such as central nervous system and kidney. Our data suggest that Nedd2 is an important component of the mammalian programmed cell death machinery.

microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family.

Abstract: The microphthalmia (mi) gene appears essential for pigment cell development and/or survival, based on its mutation in mi mice. It has also been linked to the human disorder Waardenburg Syndrome. The mi gene was recently cloned and predicts a basic/helix-loop-helix/leucine zipper (b-HLH-ZIP) factor with tissue-restricted expression. Here, we show that Mi protein binds DNA as a homo- or heterodimer with TFEB, TFE3, or TFEC, together constituting a new MiT family. Mi can also activate transcription through recognition of the M box, a highly conserved pigmentation gene promoter element, and may thereby determine tissue-specific expression of pigmentation enzymes. Six mi mutations shown recently to cluster in the b-HLH-ZIP region produce surprising and instructive effects on DNA recognition and oligomerization. An alternatively spliced exon located outside of the b-HLH-ZIP region is shown to significantly modulate DNA recognition by the basic domain. These findings suggest that Mi's critical roles in melanocyte survival and pigmentation are mediated by MiT family interactions and transcriptional activities.

Targeted disruption of the neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues.

Abstract: The neurofibromatosis (NF1) gene shows significant homology to mammalian GAP and is an important regulator of the ras signal transduction pathway. To study the function of NF1 in normal development and to try and develop a mouse model of NF1 disease, we have used gene targeting in ES cells to generate mice carrying a null mutation at the mouse Nf1 locus. Although heterozygous mutant mice, aged up to 10 months, have not exhibited any obvious abnormalities, homozygous mutant embryos die in utero. Embryonic death is likely attributable to a severe malformation of the heart. Interestingly, mutant embryos also display hyperplasia of neural crest-derived sympathetic ganglia. These results identify new roles for NF1 in development and indicate that some of the abnormal growth phenomena observed in NF1 patients can be recapitulated in neurofibromin-deficient mice

Molecular basis of mouse microphthalmia ( mi ) mutations helps explain their developmental and phenotypic consequences

Abstract: Mutations in the mouse microphthalmia (mi) gene affect the development of a number of cell types including melanocytes, osteoclasts and mast cells. Recently, mutations in the human mi gene (MITF) were found in patients with Waardenburg Syndrome type 2 (WS2), a dominantly inherited syndrome associated with hearing loss and pigmentary disturbances. We have characterized the molecular defects associated with eight murine mi mutations, which vary in both their mode of inheritance and in the cell types they affect. These molecular data, combined with the extensive body of genetic data accumulated for murine mi, shed light on the phenotypic and developmental consequences of mi mutations and offer a mouse model for WS2.

The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.

Abstract: Mice harboring the waved-1 {wa-1) and waved-2 {wa-2) mutations exhibit skin and eye abnormalities that are strikingly similar to those of TGF-a-deficient mice, and wa-1 and TGF-a were recently shown to be allelic. Because the wa-2 mutation was mapped previously to the vicinity of the EGF/TGF-a receptor (EGFR) gene on mouse chromosome 11, we hypothesized that the wa-2 phenotype might result from a defect in either the expression or activity of EGFR, or both. In the present report, we show that EGFR mRNA and protein of normal size are expressed in wa-2 liver and skin at levels that are comparable to those in the corresponding normal tissues, and that the ability of wa-2 EGFR to bind ligand is unaltered. However, ligand-dependent autophosphorylation of wa-2 EGFR is diminished 5- to 10-fold in vitro, and the ability of wa-2 EGFR to phosphorylate an exogenous substrate is reduced by >90% compared with that of the control receptor. EGF-induced tyrosine phosphorylation, including that of EGFR itself, is also diminished in skin, particularly at lower doses of exogenous EGF. To establish the nature of the wa-2 mutation, we determined the nucleotide sequence of the coding region of normal and wa-2 murine EGFR cDNAs. A comparison of these sequences revealed a single-nucleotide transversion resulting in the substitution of a glycine for a conserved valine residue near the amino terminus of the tyrosine kinase domain. The importance of this mutation was confirmed by showing that its introduction into an otherwise normal EGFR markedly reduced the receptor's tyrosine kinase activity in transfected Chinese hamster ovary cells. Finally, in situ hybridization analysis demonstrated expression of EGFR predominantly in the outer root sheath of active hair follicles in neonatal mice. As we previously localized TGF-a mRNA to the inner root sheath, this pattern of EGFR expression is consistent with the effect of the wa-2 mutation on hair structure, and together with our previous characterization of TGF-a-deficient mice, reveals a critical role for signaling by this ligand/receptor system in skin.

A Mef2 gene that generates a muscle-specific isoform via alternative mRNA splicing

Abstract: Members of the myocyte-specific enhancer-binding factor 2 (MEF2) family of transcription factors bind a conserved A/T-rich sequence in the control regions of numerous muscle-specific genes. Mammalian MEF2 proteins have been shown previously to be encoded by three genes, Mef2, xMef2, and Mef2c, each of which gives rise to multiple alternatively spliced transcripts. We describe the cloning of a new member of the MEF2 family from mice, termed MEF2D, which shares extensive homology with other MEF2 proteins but is the product of a separate gene. MEF2D binds to and activates transcription through the MEF2 site and forms heterodimers with other members of the MEF2 family. Deletion mutations show that the carboxyl terminus of MEF2D is required for efficient transactivation. MEF2D transcripts are widely expressed, but alternative splicing of MEF2D transcripts gives rise to a muscle-specific isoform which is induced during myoblast differentiation. The mouse Mef2, Mef2c, and Mef2d genes map to chromosomes 7, 13, and 3, respectively. The complexity of the MEF2 family of regulatory proteins provides the potential for fine-tuning of transcriptional responses as a consequence of combinatorial interactions among multiple MEF2 isoforms encoded by the four Mef2 genes.

The mouse CD69 gene. Structure, expression, and mapping to the NK gene complex.

Abstract: CD69 is a rapidly induced T cell activation Ag that is also expressed in an inducible fashion on cells of most, if not all, hematopoietic lineages. Molecular cloning has shown that CD69 is a type II membrane glycoprotein that is a member of the C-type lectin family. In this report we have shown that induction of CD69 mRNA in activated murine thymocytes and T cells is very rapid, peaking between 30 and 60 min poststimulation, and transient, dropping to nearly resting levels by 8 h. An analysis of the mouse CD69 gene structure showed the gene to consist of 5 exons and have a phorbol ester-inducible promoter element within the first 700 bp upstream of the start of transcription. Chromosomal mapping placed the mouse CD69 gene on the long arm of chromosome 6 near the NK gene complex that contains the related NKR-P1 and Ly-49 gene families. The human CD69 gene mapped to chromosome 12p13 near the related NKG2 gene cluster and in a region associated with rearrangements in approximately 10% of cases of childhood acute lymphocytic leukemia.

Expression of the mouse corticotropin-releasing hormone gene in vivo and targeted inactivation in embryonic stem cells.

Abstract: Corticotropin-releasing hormone (CRH), one of the primary regulators of the hypothalamic-pituitary-adrenal (HPA) axis, exhibits abnormal regulation in pathologic states such as depression and anorexia nervosa. Analysis of the role of CRH in regulation of the HPA axis would be facilitated by the creation of animal models in which CRH gene structure and function could be manipulated. We have determined the DNA sequence of the mouse CRH gene. Using a highly sensitive reverse transcription-polymerase chain reaction method, we have found expression of CRH mRNA in adrenal, ovary, testis, gut, heart, anterior pituitary, lung, and spleen, in addition to cerebral cortex and hypothalamus. Within the spleen, CRH mRNA is localized specifically to T-lymphocytes. We mapped the chromosomal location of mouse CRH via interspecific mouse backcrosses to chromosome 3, which is not the site of any naturally occurring mutations consistent with CRH deficiency. Because of this, we inactivated a CRH allele in mouse embryonic stem (ES) cells by homologous recombination with a mutant mouse CRH gene lacking the entire coding region of preproCRH. Mice chimeric for each of two ES clones with an inactivated CRH allele are being used to generate animals with complete CRH deficiency.

Mapping of two genes encoding members of a distinct subfamily of MAX interacting proteins: MAD to human chromosome 2 and mouse chromosome 6, and MXI1 to human chromosome 10 and mouse chromosome 19.

Abstract: Both the MAD and the MXI1 genes encode basic-helix-loop-helix-leucine zipper (bHLH-Zip) transcription factors which bind Max in vitro, forming a sequence-specific DNA-binding complex similar to the Myc-Max heterodimer. Mad and Myc compete for binding to Max. In addition, Mad has been shown to act as a transcriptional repressor while Myc appears to function as an activator. Mxi1 also appears to lack a transcriptional activation domain. Therefore, Mxi1 and Mad might antagonize Myc function and are candidate tumor suppressor genes. We report here the mapping of the MAD and MXI1 genes in human and mouse by fluorescence in situ hybridization (FISH) and by recombination mapping. The MAD gene was mapped to human chromosome 2 at band p13 by FISH and to mouse chromosome 6 by meiotic mapping. The MXI1 gene was mapped to human chromosome 10 at band q25 and on mouse chromosome 19 at region D by FISH. There was a second site of hybridization on mouse chromosome 2 at region C, which may represent a pseudogene or a related sequence. The mapping results confirm regions of conservation between human chromosome 2p13 and mouse chromosome 6 and between chromosome 10q25 and mouse chromosome 19D. Human chromosomes 2p13 and 10q25 have been involved in specific tumors where the role of Mad and Mxi1 can now be investigated.

In situ localization and chromosomal mapping of the AG1 (Dmp1) gene.

Abstract: Dentinogenesis is being used as a model for understanding the biomineralization process. The odontoblasts synthesize a structural matrix comprised of Type I collagen fibrils which define the basic architecture of the tissue. The odontoblasts also synthesize and deliver a number of dentin-specific acidic macromolecules into the extracellular compartment. These acidic macromolecules may be involved in regulating the ordered deposition of hydroxyapatite crystals within the matrix. AG1 is the first tooth-specific acidic macromolecule to have been cloned and sequenced. To identify which cells of the rat incisor pulp/odontoblast complex were responsible for synthesis of AG1, in situ hybridization was used. Digoxigenin labeled sense and anti-sense AG1 riboprobes were prepared. The AG1 mRNA was found to be expressed in the mature secretory odontoblasts. Neither pulp cells nor pre-odontoblasts showed any staining with the anti-sense probes. Chromosomal localization studies placed the AG1 gene on mouse chromosome 5q21, in tight linkage with Fgf5. AG1 has been renamed Dmp1 (dentin matrix protein 1) in accordance with present chromosomal nomenclature. Mouse 5q21 corresponds to the 4q21 locus in humans. This is the locus for the human tooth mineralization disorder dentinogenesis imperfecta Type II (DI-II). These data suggest that the Dmp1 gene is involved in mineralization and is a candidate gene for DI-II.

Maintenance of Wnt-3 expression in Purkinje cells of the mouse cerebellum depends on interactions with granule cells

Abstract: Wnt genes encode secreted proteins implicated in cell fate changes during development. To define specific cell populations in which Wnt genes act, we have examined Wnt expression in the cerebellum. This part of the brain has a relatively simple structure and contains well-characterized cell populations. We found that Wnt-3 is expressed during development of the cerebellum and that expression is restricted to the Purkinje cell layer in the adult. Wnt-3 expression in Purkinje cells increases postnatally as granule cells start to make contacts with Purkinje cells. To investigate whether interactions with granule cells influence Wnt-3 expression in Purkinje cells, we examined gene expression in several mouse mutants, using the expression of En-2 to follow the fate of granule cells. In the weaver mutant, in which granule cells fail to migrate and subsequently die in the external granular layer, Wnt-3 expression was normal at postnatal day 15 (P15). At that time, some granule cells are still present in the external granular layer. At P28, however, when granule cells could no longer be detected, Wnt-3 expression was almost absent. In the meander tail mutant, in which the anterior cerebellar lobes lack granule cells, Wnt-3 expression was only detected in the normal posterior lobes. Since En genes are implicated in cell-cell interactions mediated by Wnt genes, we examined En-2/En-2 mutant mice, finding normal Wnt-3 expression, indicating that the effect of granule cells on the maintenance of Wnt-3 is not mediated by En-2. Our results show that Wnt-3 expression in Purkinje cells is modulated by their presynaptic granule cells at the time of neuronal maturation.

Isolation of an allele of reeler by insertional mutagenesis.

Abstract: Reeler (rl) is an autosomal recessive mutation that affects migration of postmitotic neurons in the mouse central nervous system. The reeler (rl/rl) mouse displays a disruption of laminar structures in both the cerebellum and the forebrain and it exhibits tremors, dystonia, and ataxia. The molecular basis of the reeler phenotype is unknown because the gene involved has not yet been identified. We report here the isolation and characterization of an allele of rl, reelertransgene (rltg). This allele was generated by the fortuitous insertion of a transgene, supfos (sf), into the mouse rl locus. Crosses between rl/+ and rltg/+ mice yielded offspring that exhibited the reeler phenotype, indicating that rl and rltg are allelic. We cloned the genomic sequences flanking the transgene insertion site from the rltg/rltg mouse genome. Chromosomal mapping studies revealed that the 5' flanking cellular sequence maps to a locus, D5Gmr1, that lies in a region of mouse chromosome 5 that also contains the rl locus. Southern blot analysis using a probe derived from the D5Gmr1 locus revealed no gross structural rearrangement in the rl locus. Thus, unlike the two rl alleles described previously, rltg provides a molecular probe that can now be used to identify and isolate the rl gene.

Identification and chromosomal mapping of a receptor tyrosine kinase with a putative phospholipid binding sequence in its ectodomain.

Abstract: We have cloned a novel receptor tyrosine kinase that has an unusual ectodomain. The extracellular sequence consists of 416 amino acids and has none of the structural motifs that have been found in other receptor tyrosine kinases. The 150 amino acids in the amino terminus of the receptor is homologous to a putative phospholipid-binding sequence that is found also in other cell adhesion molecules such as the neuronal A5 antigen and coagulation factors V and VIII. The kinase domain has a short cytoplasmic tail and contains a short insert between subdomains I and II. The structure of this receptor kinase suggests that it belongs to a new family of receptors involved in cell-cell interactions. The cell adhesion kinase (Cak) is expressed at low levels in most adult tissues and expression is highest in the brain and lung. Using fluorescence in situ hybridization and interspecific backcross mapping, the Cak gene was localized to human chromosome 6 and mouse chromosome 17.

The molecular basis for dominant yellow agouti coat color mutations

Abstract: Agouti expression during the middle portion of the mouse hair growth cycle induces melanocytes to synthesize yellow instead of black pigment, generating black hairs with a yellow band. Dominant agouti alleles increase the amount of yellow pigment in the coat and are associated with pleiotropic effects including obesity, diabetes and increased tumor susceptibility. Four dominant agouti alleles (Aiapy, Aiy, and Avy) were recently shown to result from insertions that cause ubiquitous expression of chimeric transcripts encoding a wild-type agouti protein(1,2). Three insertions were identified as intracisternal A-particles, which helps explain the variable coat colors and parental imprinting effects associated with some dominant agouti alleles.

Identification of Evi-3, a novel common site of retroviral integration in mouse AKXD B-cell lymphomas.

Abstract: We have identified a novel common site of ecotropic viral integration called ecotropic viral integration site 3 (Evi-3) in B-cell lineage lymphomas of the AKXD recombinant inbred strains of mice A number of virally induced pre-B-, B-, myeloid, and T-cell lymphomas were screened for viral rearrangements at Evi-3; rearrangements were found in pre-B- and B-cell lymphomas but not in other hematopoietic tumors Genetic mapping studies localized Evi-3 to mouse chromosome 18, distinct from proto-oncogene and common viral integration loci identified previously in the mouse Each proviral integration at Evi-3 is contained within a 200-bp region that lies inside a CpG island All but one of the proviruses have integrated in the same 5'-to-3' transcriptional orientation Transcripts from Evi-3 are expressed in a developmentally regulated manner in B cells Taken together, these data suggest that Evi-3 represents a novel proto-oncogene involved in mouse B-cell lymphomas

The genes encoding the glutamate receptor subunits KA1 and KA2 (GRIK4 and GRIK5) are located on separate chromosomes in human, mouse, and rat

Abstract: The chromosomal localization of the human and rat genes encoding the kainate-preferring glutamate receptor subunits KA1 and KA2 (GRIK4 and GRIK5, respectively) was determined by Southern analysis of rat x mouse and human x mouse somatic cell hybrid panels and by fluorescence in situ hybridization The localization of the mouse genes (Grik4 and Grik5) was established by interspecific backcross mapping GRIK4 and GRIK5 are located on separate chromosomes (Chrs) in all species GRIK4 mapped to human Chr 11q223, mouse Chr 9, and rat Chr 8 GRIK5 mapped to human Chr 19q132, mouse Chr 7, and rat Chr 1 The genes encoding the (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-preferring subunit GluR4, or GluRD (GRIA4), the neural cell adhesion molecule (NCAM), the D2 dopamine receptor (DRD2), and the Thy-1 cell surface antigen (THY1) have all been previously mapped to the human Chr 11q22 region The mapping of the human GRIK4 and GRIK5 genes confirms and extends the relationship between human Chr 11 and mouse Chr 9 and also human Chr 19 and mouse Chr 7 GRIK4 is the fifth gene shared by human Chr 11 and rat Chr 8, whereas GRIK5 is 1 out of the 12 genes that are located on both human Chr 19 and rat Chr 1 Our data extend the conserved synteny established between certain human, mouse, and rat Chrs

Effects of provirus integration in the Tpl-1/Ets-1 locus in Moloney murine leukemia virus-induced rat T-cell lymphomas: levels of expression, polyadenylation, transcriptional initiation, and differential splicing of the Ets-1 mRNA.

Abstract: The Tpl-1 locus was defined as a genomic DNA region which is targeted by provirus insertion during progression of Moloney murine leukemia virus-induced rat T-cell lymphomas. Using a panel of 156 (Mus musculus x Mus spretus) x Mus musculus interspecific backcross mice, we mapped Tpl-1 to mouse chromosome 9 at a distance of 1.2 +/- 0.9 centimorgans from the Ets-1 proto-oncogene (S.E. Bear, A. Bellacosa, P.A. Lazo, N.A. Jenkins, N.G. Copeland, C. Hanson, G. Levan, and P.N. Tsichlis, Proc. Natl. Acad. Sci. USA 86:7495-7499, 1989). In this report, we present evidence that all the known Tpl-1 provirus insertions occurred immediately 5' of the first exon of Ets-1 (exon A) and that the earlier detected distance between Tpl-1 and Ets-1 was due to the high frequency of meiotic recombination in the region between the site of provirus integration and exon III. Northern (RNA) blot analysis of polyadenylated RNA from normal adult rat tissues and Moloney murine leukemia virus-induced T-cell lymphomas and hybridization to a Tpl-1/Ets-1 probe derived from the 5' end of the gene revealed two lymphoid cell-specific RNA transcripts, of 5.5 and 2.2 kb. Sequence analysis of a near-full-length (4,991-bp) cDNA clone of the 5.5-kb RNA revealed a 441-amino-acid open reading frame encoding a protein identical to the human and mouse Ets-1 proteins with the exception of five and nine species-specific conservative amino acid differences, respectively. The steady-state level of the Tpl-1/Ets-1 RNA and of the Ets-1 protein was modestly elevated in tumors carrying a provirus in the Tpl-1 locus. The relative ratio of the two Ets-1 transcripts, which were shown to arise by differential polyadenylation, was not affected by provirus insertion. Moreover, the major site of transcriptional initiation, which was localized by primer extension 250 bp upstream of the 5' end of the Ets-1 cDNA clone, was shown to be identical in normal cells and tumors carrying a provirus in the Tpl-1 locus. Finally, the differential splicing of Ets-1 exon VII was shown by RNase protection to occur at a rate of 15 to 26% and to remain unaffected by provirus insertion. The subtlety of these effects, in contrast to the strong growth selection of cells with a provirus in the Tpl-1/Ets-1 locus, suggests that provirus insertion may affect the fine regulation of the gene, perhaps during cell cycle progression.