Genomic imprinting of Mash2, a mouse gene required for trophoblast development.

The mouse gene Mash2 encodes a transcription factor required for development of trophoblast progenitors. Mash2-homozygous mutant embryos die at 10 days postcoitum from placental failure. Here we show that Mash2 is genomically imprinted. First, Mash2+/- embryos inheriting a wild-type allele from their father die at the same stage as -/- embryos, with a similar placental phenotype. Second, the Mash2 paternal allele is initially expressed by groups of trophoblast cells at 6.5 and 7.5 days post-coitum, but appears almost completely repressed by 8.5 days post-coitum. Finally, we have genetically and physically mapped Mash2 to the distal region of chromosome 7, within a cluster of imprinted genes, including insulin-2, insulin-like growth factor-2 and H19.

Evolution of the mammalian G protein alpha subunit multigene family.

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. The multigene family of G protein alpha subunits, which interact with receptors and effectors, exhibit a high level of sequence diversity. In mammals, 15 G alpha subunit genes can be grouped by sequence and functional similarities into four classes. We have determined the murine chromosomal locations of all 15 G alpha subunit genes using an interspecific backcross derived from crosses of C57BL/6J and Mus spretus mice. These data, in combination with mapping studies in humans, have provided insight into the events responsible for generating the genetic diversity found in the mammalian alpha subunit genes and a framework for elucidating the role of the G alpha subunits in disease.

The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

A genetic linkage map of the mouse: current applications and future prospects.

Technological advances have made possible the development of high-resolution genetic linkage maps for the mouse. These maps in turn offer exciting prospects for understanding mammalian genome evolution through comparative mapping, for developing mouse models of human disease, and for identifying the function of all genes in the organism.

Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes.

The otd/Otx gene family encodes paired-like homeodomain proteins that are involved in the regulation of anterior head structure and sensory organ development. Using the yeast one-hybrid screen with a bait containing the Ret 4 site from the bovine rhodopsin promoter, we have cloned a new member of the family, Crx (Cone rod homeobox). Crx encodes a 299 amino acid residue protein with a paired-like homeodomain near its N terminus. In the adult, it is expressed predominantly in photoreceptors and pinealocytes. In the developing mouse retina, it is expressed by embryonic day 12.5 (E12.5). Recombinant Crx binds in vitro not only to the Ret 4 site but also to the Ret 1 and BAT-1 sites. In transient transfection studies, Crx transactivates rhodopsin promoter-reporter constructs. Its activity is synergistic with that of Nrl. Crx also binds to and transactivates the genes for several other photoreceptor cell-specific proteins (interphotoreceptor retinoid-binding protein, beta-phosphodiesterase, and arrestin). Human Crx maps to chromosome 19q13.3, the site of a cone rod dystrophy (CORDII). These studies implicate Crx as a potentially important regulator of photoreceptor cell development and gene expression and also identify it as a candidate gene for CORDII and other retinal diseases.

Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites.

Neuronal activity is an essential stimulus for induction of plasticity and normal development of the CNS. We have used differential cloning techniques to identify a novel immediate-early gene (IEG) cDNA that is rapidly induced in neurons by activity in models of adult and developmental plasticity. Both the mRNA and the encoded protein are enriched in neuronal dendrites. Analysis of the deduced amino acid sequence indicates a region of homology with alpha-spectrin, and the full-length protein, prepared by in vitro transcription/translation, coprecipitates with F-actin. Confocal microscopy of the native protein in hippocampal neurons demonstrates that the IEG-encoded protein is enriched in the subplasmalemmal cortex of the cell body and dendrites and thus colocalizes with the actin cytoskeletal matrix. Accordingly, we have termed the gene and encoded protein Arc (activity-regulated cytoskeleton-associated protein). Our observations suggest that Arc may play a role in activity-dependent plasticity of dendrites.

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

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.

Spontaneous germ line virus infection and retroviral insertional mutagenesis in eighteen transgenic Srev lines of mice.

SWR/J-RF/J hybrid mice spontaneously acquire new germ line ecotropic proviruses at high frequency. In the studies described here, we used these hybrids to produce 18 transgenic mouse lines, each carrying a single newly acquired Srev locus (SWR/J-RF/J ecotropic proviral locus). All of the newly acquired proviruses identified in mosaic founder SWR/J-RF/J mice that could be transmitted through the germ line were also present in somatic tissues, demonstrating that viral integration occurred before the germ line was set aside from the somatic lineages. Quantitative analysis of proviral DNA copy numbers in somatic and germinal tissues of mosaic founder parents combined with structural analysis of Srev loci indicated that these proviruses are acquired after multiple rounds of somatic viral reinfection and that most of these viral integration events occurred after DNA replication in the zygote and before DNA replication in the four-cell embryo. The frequency of provirus acquisition in Srev lines that expressed the infectious ecotropic virus was similar to that in SWR.RF mice carrying Emv-16 and Emv-17, suggesting that the chromosomal integration site of the parental locus is not an important determinant for high-frequency provirus acquisition. The frequency of recessive lethal mutations induced by spontaneous viral integration was 5%, which was similar to that induced by preimplantation embryo infection. This approach represents a simple and viable strategy for inducing and studying mutations that affect mammalian development.

Structure, expression, and chromosome location of the gene for the beta subunit of brain-specific Ca2+/calmodulin-dependent protein kinase II identified by transgene integration in an embryonic lethal mouse mutant.

The transgenic mouse strain CAT40 carries in its germ line one copy of a DNA construct consisting of the chloramphenicol acetyltransferase gene and the immunoglobulin heavy-chain enhancer. We show that transgene integration has resulted in a recessive lethal mutation that leads to death of homozygous CAT40 embryos shortly after implantation. The transgene has integrated adjacent to the 3' end of the gene coding for the beta subunit of the brain-specific Ca2+/calmodulin-dependent protein kinase II (Camk-2). The complete cDNA sequence of the Camk-2 gene and most of its exon/intron structure was determined. The deduced amino acid sequence is highly homologous to the previously described rat protein. The chromosomal location of the Camk-2 locus was mapped by interspecific backcross analysis to the proximal region of mouse chromosome 11. This region lacks previously identified recessive embryonic lethal mutations. During embryonic development, Camk-2-specific transcripts are first seen in the head section of 12.5-day-old embryos, and in adult mice the gene is expressed almost exclusively in the brain. Although transcription of the Camk-2 gene in heterozygous CAT40 mice is affected by transgene integration, it is unlikely that this gene is responsible for the mutant phenotype, since it is not expressed in blastocysts and the first transcripts during normal development are detected after the death of homozygous CAT40 embryos. Transgene integration is accompanied by a large deletion of cellular DNA; death is therefore most likely caused by the loss of a gene or genes that are important for early postimplantation development.

Zac1 (Lot1), a potential tumor suppressor gene, and the gene for epsilon-sarcoglycan are maternally imprinted genes: identification by a subtractive screen of novel uniparental fibroblast lines.

Imprinted genes are expressed from one allele according to their parent of origin, and many are essential to mammalian embryogenesis. Here we show that the epsilon-sarcoglycan gene (Sgce) and Zac1 (Lot1) are both paternally expressed imprinted genes. They were identified in a subtractive screen for imprinted genes using a cDNA library made from novel parthenogenetic and wild-type fibroblast lines. Sgce is a component of the dystrophin-sarcoglycan complex, Zac1 is a nuclear protein inducing growth arrest and/or apoptosis, and Zac1 is a potential tumor suppressor gene. Sgce and Zac1 are expressed predominantly from their paternal alleles in all adult mouse tissues, except that Zac1 is biallelic in the liver and Sgce is weakly expressed from the maternal allele in the brain. Sgce and Zac1 are broadly expressed in embryos, with Zac1 being highly expressed in the liver primordium, the umbilical region, and the neural tube. Sgce, however, is strongly expressed in the allantoic region on day 9.5 but becomes more widely expressed throughout the embryo by day 11.5. Sgce is located at the proximal end of mouse chromosome 6 and is a candidate gene for embryonic lethality associated with uniparental maternal inheritance of this region. Zac1 maps to the proximal region of chromosome 10, identifying a new imprinted locus in the mouse, homologous with human chromosome 6q24-q25. In humans, unipaternal disomy for this region is associated with fetal growth retardation and transient neonatal diabetes mellitus. In addition, loss of expression of ZAC has been described for a number of breast and ovarian carcinomas, suggesting that ZAC is a potential tumor suppressor gene.

Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation.

Interferon regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STAT1) and/or 113 kDa (STAT2). Recent studies have suggested that these proteins are substrates of Janus kinases and that proteins related in STAT1 are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-3 (IL-3). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STAT1. Unlike STAT1, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jak1 and Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma interferon (IFN-gamma)-activated sequence of the interferon regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat1 gene, suggesting that the genes arose by gene duplication. Unlike Stat1, neither IFN-alpha nor IFN-gamma activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-3, granulocyte colony-stimulating factor, stem cell factor, colon-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.

PEBP2 alpha B/mouse AML1 consists of multiple isoforms that possess differential transactivation potentials.

A murine transcription factor, PEBP2, is composed of two subunits, alpha and beta. There are two genes in the mouse genome, PEBP2 alpha A and PEBP2 alpha B, which encode the alpha subunit. Two types of the alpha B cDNA clones, alpha B1 and alpha B2, were isolated from mouse fibroblasts and characterized. They were found to represent 3.8- and 7.9-kb transcripts, respectively. The 3.8-kb RNA encodes the previously described alpha B protein referred to as alpha B1, while the 7.9-kb RNA encodes a 387-amino-acid protein, termed alpha B2, which is identical to alpha B1 except that it has an internal deletion of 64 amino acid residues. Both alpha B1 and alpha B2 associate with PEBP2 beta and form a heterodimer. The alpha B2/beta complex binds to the PEBP2 binding site two- to threefold more strongly than the alpha B1/beta complex does. alpha B1 stimulates transcription through the PEBP2 site about 40-fold, while alpha B2 is only about 25 to 45% as active as alpha B1. Transactivation domain is located downstream of the 128-amino-acid runt homology region, referred to as the Runt domain. Mouse chromosome mapping studies revealed that alpha A, alpha B, and beta genes are mapped to chromosomes 17, 16, and 8, respectively. The last two genes are syntenic with the human AML1 on chromosome 21q22 and PEBP2 beta/CBF beta on 16q22 detected at the breakpoints of characteristic chromosome translocations of the two different subtypes of acute myeloid leukemia. These results suggest that previously described chimeric gene products, AML1/MTG8(ETO) and AML1-EAP generated by t(8;21) and t(3;21), respectively, lack the transactivation domain of AML1.

Molecular cloning reveals that the p160 Myb-binding protein is a novel, predominantly nucleolar protein which may play a role in transactivation by Myb.

We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

Death-associated protein kinase 2 is a new calcium/calmodulin-dependent protein kinase that signals apoptosis through its catalytic activity.

We have identified and characterized a new calcium/calmodulin (Ca2+/CaM) dependent protein kinase termed death-associated protein kinase 2 (DAPK2) that contains an N-terminal protein kinase domain followed by a conserved CaM-binding domain with significant homologies to those of DAP kinase, a protein kinase involved in apoptosis. DAPK2 mRNA is expressed abundantly in heart, lung and skeletal muscle. The mapping results indicated that DAPK2 is located in the central region of mouse chromosome 9. In vitro kinase assay revealed that DAPK2 is autophosphorylated and phosphorylates myosin light chain (MLC) as an exogenous substrate. DAPK2 binds directly to CaM and is activated in a Ca2+/CaM-dependent manner. A constitutively active DAPK2 mutant is generated by removal of the CaM-binding domain (deltaCaM). Treatment of agonists that elevate intracellular Ca2+-concentration led to the activation of DAPK2 and transfection studies revealed that DAPK2 is localized in the cytoplasm. Overexpression of DAPK2, but not the kinase negative mutant, significantly induced the morphological changes characteristic of apoptosis. These results indicate that DAPK2 is an additional member of DAP kinase family involved in apoptotic signaling.

Unp, a mouse gene related to the tre oncogene.

We have cloned cDNAs from a novel gene designated Unp. Unp cDNAs contain a large open reading frame that would encode a protein of 89 kDa. The predicted protein contains a putative nuclear localization signal, as well as consensus sequences for binding to the retinoblastoma gene product. The latter elements are contained within a region having strong similarity to the human tre oncogene. We have localized the Unp gene to mouse chromosome 9 in a region of homology with human chromosome 3p. This region has been implicated in a number of human malignancies.

Cloning and developmental expression of the murine homolog of the acute leukemia proto-oncogene AF4.

AF4 is the 4q21 gene involved in the acute lymphoblastic leukemia associated t(4;11)(q21;q23) where it forms a fusion gene with MLL. In order to gain insight into AF4's role in leukemogenesis we have studied its functional domains and expression pattern during murine development. We have cloned the murine homolog, Af4. We have demonstrated that 5' half of Af4 encodes a region with transcriptional transactivation activity which is disrupted by the t(4;11) in human leukemias. We have also localized the murine AF4 protein to the nucleus supporting a role for AF4 in transcription. The developmental expression pattern of Af4 was determined in situ hybridization and suggests Af4 plays an important role in the development of the hematopoietic, cardiovascular, skeletal and central nervous systems. A repeating pattern of Af4 expression in development is down-regulation with differentiation of a tissue. Among the cell types where this pattern of down-regulation is noted are B-lymphocytes. These findings raise the possibility that the disruption of normal AF4 function by the translocation may contribute to leukemogenesis.

Expression of a novel form of Tec kinase in hematopoietic cells and mapping of the gene to chromosome 5 near Kit.

The Tec kinase was initially identified as a novel cytoplasmic protein tyrosine kinase that is preferentially expressed in the liver and is highly homologous to the Drosophila Dsrc28C src-related tyrosine kinase. In screening of interleukin 3 (IL-3)-dependent myeloid leukemia cells for protein tyrosine kinases, we observed that all cell lines examined expressed high levels of Tec transcripts. However, characterization of Tec cDNAs indicated that they differed significantly from the published sequence. Most strikingly, an insertion of 41 bp in the 5' region affects the initiation codon and results in replacing the published 13 amino acid amino-terminal sequences with 94 amino acids. Using polymerase chain reaction (PCR) analysis, only the form containing the insertion was detected in hematopoietic cells. In addition, we found an in-frame insertion of 66 bp that introduces an additional 22 amino acids into the SH3 domain. This insertion restores conserved SH3 sequences that are found in the src gene family and in the Dsrc28C gene. By PCR analysis, approximately equal levels of Tec transcripts containing the intact SH3 domain and containing the 22 amino acid deletion were found in hematopoietic cells. Lastly, by interspecies backcross analysis, we show that the Tec gene is tightly linked to the c-Kit gene on mouse chromosome 5.

Molecular characterization of mouse and rat CPP32 beta gene encoding a cysteine protease resembling interleukin-1 beta converting enzyme and CED-3.

Interleukin-1 beta converting enzyme (ICE) defines a new class of mammalian cysteine protease that shares strong homology with the Caenorhabditis elegans death gene ced-3. Both ICE and CED-3, when introduced into cultured cells, induce apoptosis, indicating that this type of cysteine protease may play an important role in the process of programmed cell death. Here, we report the cloning of a mouse and rat gene encoding a novel cysteine protease. The putative proteins encoded by these cDNAs contain the conserved sequence (QACRG) necessary for covalent linkage to the substrate as well as the three amino acids responsible for substrate binding and catalysis in ICE. Amino acid sequence analysis indicates that this rodent cysteine protease is the homolog of human CPP32 beta. Mouse CPP32 beta mRNA is highly expressed in spleen, and to a lesser degree in brain, lung, liver, and kidney. The mouse CPP32 beta genomic locus spans a region of approximately 20 kb, including seven exons and six introns. Mouse interspecific backcross mapping allowed localization of CPP32 beta to the central region of mouse chromosome 8, linked to Scvr, Lpl, Jund1 and Mlr.

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

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.

Structure, chromosome mapping, and expression of the mouse Lyl-1 gene.

The mouse Lyl-1 gene was cloned and shown to consist of four exons with extensive nucleotide and structural homology to the human LYL1 gene. The Lyl-1 gene was localized to the central region of mouse chromosome 8 which defines a new region of synteny with human chromosome 19p. The predicted mouse Lyl-1 protein is 78% identical to human LYL1. The region of highest similarity occurs in the basic DNA binding and helix-loop-helix dimerization motifs which are nearly identical in mouse and man differing by only one conservative amino acid substitution. Expression of the Lyl-1 gene was found to be low in murine spleen and undetectable in other tissues by Northern blot analysis. In lymphoid cell lines, Lyl-1 was expressed in most B lineage cells but downregulated during terminal differentiation and was not expressed in most T lineage cells. In a human T ALL cell line carrying a translocation that juxtaposed LYL1 with the beta TCR gene, the translocated LYL1 gene was transcriptionally active whereas the nontranslocated gene was transcriptionally silent. We conclude that LYL1 has the properties of a lineage- and differentiation-specific HLH protein that contributes to T-cell neoplasia through its deregulated expression following chromosomal translocation.