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The involvement of dual specificity phosphatases (DSPs) in the mitogen-activated protein kinase (MAPK) signaling has been mostly limited to the inactivation of MAPKs by the direct dephosphorylation of the TXY motif within their activation loop. We report the cloning and characterization of a murine DSP, called JNK pathway-associated phosphatase (JKAP), which lacks the regulatory region present in most other MAP kinase phosphatases (MKPs) and is preferentially expressed in murine Lin−Sca-1+stem cells. Overexpression of JKAP in human embryonic kidney 293T cells specifically activated c-Jun N-terminal kinase (JNK) but not p38 and extracellular signal-regulated kinase 2. Overexpression of a mutant JKAP, JKAP-C88S, blocked tumor necrosis factor-α-induced JNK activation. Targeted gene disruption in murine embryonic stem cells abolished JNK activation by tumor necrosis factor-α and transforming growth factor-β, but not by ultraviolet-C irradiation, indicating that JKAP is necessary for optimal JNK activation. JKAP associated with JNK and MKK7, but not SEK1, in vivo. However, JKAP did not interact with JNK in vitro, suggesting that JKAP exerts its effect on JNK in an indirect manner. Taken together, these studies identify a positive regulator for the JNK pathway and suggest a novel role for DSP in mitogen-activated protein kinase regulation.

Drosophila Flamingo (Fmi) is an evolutionally conserved seven-pass transmembrane receptor of the cadherin superfamily. Fmi plays multiple roles in patterning neuronal processes and epithelial planar cell polarity. To explore the in vivo roles of Fmi homologs in mammals, we previously cloned one of the mouse homologs, mouse flamingo1/Celsr2. Here, we report the results of our study of its embryonic and postnatal expression patterns together with those of two other paralogs, Celsr1 and Celsr3. Celsr1–3 expression was initiated broadly in the nervous system at early developmental stages, and each paralog showed characteristic expression patterns in the developing CNS. These genes were also expressed in several other organs, including the cochlea, where hair cells develop planar polarity, the kidney, and the whisker. The Celsr2 protein was distributed at intercellular boundaries in the whisker and on processes of neuronal cells such as hippocampal pyramidal cells, Purkinje cells, and olfactory neurons. Celsr2 is mapped to a distal region of the mouse chromosome 3. We discussed possible functions of seven-pass transmembrane cadherins in mouse development. © 2002 Wiley-Liss, Inc.

Trophinin has been identified as a membrane protein mediating apical cell adhesion between two human cell lines: trophoblastic HT-H cells, and endometrial epithelial SNG-M cells. Expression patterns of trophinin in humans suggested its involvement in embryo implantation and early placental development. The mouse trophinin gene maps to the distal part of the X chromosome and corresponds to human chromosome Xp11.21–22, the locus where the human trophinin gene maps. Western blot analysis indicates that the molecular weight of mouse trophinin is 110 kDa, which is consistent with the calculated value of 107 kDa. Positive signals for trophinin proteins were detected in preimplantation mouse embryos at the morula and blastocyst stages. Implanting blastocysts do not show detectable levels of trophinin protein, demonstrating that trophinin is not involved in blastocyst adhesion to the uterus in the mouse. Mouse embryo strongly expressed trophinin in the epiblast 1 day after implantation. Trophinin protein was not found in the mouse uteri and placenta after 5.5 days postcoitus (dpc). Targeted disruption of the trophinin gene in the mouse showed a partial embryonic lethality in a 129/SvJ background, but the cause of this lethality remains undetermined. The present study indicates significant differences between mouse and human trophinins in their expression patterns, and it suggests that trophinin is not involved in embryo implantation and placental development in the mouse.

Two classes of cysteinyl leukotriene receptor, CysLT1and CysLT2, have been identified and pharmacologically characterized in human tissues. Although the CysLT1receptor mediates the proinflammatory effects of leukotrienes in human asthma, the physiological roles of CysLT2receptor are not defined, and a suitable mouse model would be useful in delineating function. We report here the molecular cloning and characterization of the mouse CysLT2receptor (mCysLT2R) from heart tissue. mCysLT2R cDNA encodes a protein of 309 amino acids, truncated at both ends compared with the human ortholog (hCysLT2R). The gene resides on the central region of mouse chromosome 14 and is composed of 6 exons with the entire coding region located in the last exon. Two 5′-untranslated region splice variants were identified with the short form lacking exon 3 as the predominant transcript. Although the overall expression of mCysLT2R is very low, the highest expression was detected in spleen, thymus, and adrenal gland by ribonuclease protection assay, and discrete sites of expression in heart were observed by in situhybridization. Intracellular calcium mobilization in response to cysteinyl leukotriene administration was detected in human embryonic kidney 293T cells transfected with recombinant mCysLT2R with a rank order of potency leukotriene C4(LTC4) = LTD4≫ LTE4. [3H]LTD4binding to membranes expressing mCysLT2R could be effectively competed by LTC4and LTD4and only partially inhibited by LTE4and BAYu9773. The identification of mCysLT2R will be useful for establishing CysLT2R-deficient mice and determining novel leukotriene functions.

ABSTRACTJuvenile myelomonocytic leukemia (JMML) is a disease that occurs in young children and is associated with a high mortality rate. In most patients, JMML has a progressive course leading to death by virtue of infection, bleeding, or progression to acute myeloid leukemia (AML). As it is known that children with neurofibromatosis type 1 syndrome have a markedly increased risk of developing JMML, we have previously developed a mouse model of JMML through reconstitution of lethally irradiated mice with hematopoietic stem cells homozygous for a loss-of-function mutation in the Nf1gene (D. L. Largaespada, C. I. Brannan, N. A. Jenkins, and N. G. Copeland, Nat. Genet. 12:137–143, 1996). In the course of these experiments, we found that all these genetically identical reconstituted mice developed a JMML-like disorder, but only a subset went on to develop more acute disease. This result strongly suggests that additional genetic lesions are responsible for disease progression to AML. Here, we describe the production of a unique tumor panel, created using the BXH-2 genetic background, for identification of these additional genetic lesions. Using this tumor panel, we have identified a locus, Epi1, which maps 30 to 40 kb downstream of theMybgene and appears to be the most common site of somatic viral integration in BXH-2 mice. Our findings suggest that proviral integrations at Epi1cooperate with loss of Nf1to cause AML.

LAP2β is an integral membrane protein of the nuclear envelope involved in chromatin and nuclear architecture. Using the yeast two-hybrid system, we have cloned a novel LAP2β-binding protein, mGCL, which contains a BTB/POZ domain and is the mouse homologue of the Drosophila germ-cell-less (GCL) protein. In Drosophila embryos, GCL was shown to be essential for germ cell formation and was localized to the nuclear envelope. Here, we show that, in mammalian cells, GCL is co-localized with LAP2β to the nuclear envelope. Nuclear fractionation studies reveal that mGCL acts as a nuclear matrix component and not as an integral protein of the nuclear envelope. Recently, mGCL was found to interact with the DP3α component of the E2F transcription factor. This interaction reduced the transcriptional activity of the E2F-DP heterodimer, probably by anchoring the complex to the nuclear envelope. We demonstrate here that LAP2β is also capable of reducing the transcriptional activity of the E2F-DP complex and that it is more potent than mGCL in doing so. Co-expression of both LAP2β and mGCL with the E2F-DP complex resulted in a reduced transcriptional activity equal to that exerted by the pRb protein.

SALL1 is a mammalian homolog of the Drosophilaregion-specific homeotic gene spalt (sal); heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. We have isolated a mouse homolog of SALL1 (Sall1) and found that mice deficient in Sall1 die in the perinatal period and that kidney agenesis or severe dysgenesis are present. Sall1 is expressed in the metanephric mesenchyme surrounding ureteric bud; homozygous deletion ofSall1 results in an incomplete ureteric bud outgrowth, a failure of tubule formation in the mesenchyme and an apoptosis of the mesenchyme. This phenotype is likely to be primarily caused by the absence of the inductive signal from the ureter, as the Sall1-deficient mesenchyme is competent with respect to epithelial differentiation. Sall1 is therefore essential for ureteric bud invasion, the initial key step for metanephros development.

Paralemmin is a protein implicated in plasma membrane dynamics. Here we describe the identification of two new paralemmin-related proteins. A partial paralemmin homolog, palmdelphin, is predominantly cytosolic, unlike paralemmin which is lipid-anchored to the plasma membrane through a C-terminal CaaX motif. We have mapped the mouse palmdelphin gene to distal chromosome 3 between Amy2 and Abcd3, in a region homologous to human chromosome 1p22-p21 where the human palmdelphin gene is located. We have also identified a second paralemmin isoform, paralemmin-2. It is expressed from a gene on human chromosome 9q31-q33 which ends only 33 kb upstream of the gene encoding the protein kinase A-binding protein,AKAP2/AKAP-KL. The closely adjacent paralemmin-2 and AKAP2 genes are functionally linked in a very unusual manner. Chimeric mRNAs are expressed, apparently by RNA readthrough and differential splicing, that encode natural fusion proteins in which either the N-terminal coiled-coil region or nearly the complete sequence of paralemmin-2 except its C-terminal CaaX motif is fused to AKAP2/AKAP-KL. The N-terminal coiled-coil region is conserved in paralemmin-1, paralemmin-2/AKAP2, palmdelphin and a fourth, uncharacterized gene, suggesting that it is a modular functional domain.

The Shc gene family is an emerging family, containing at least three members designated Shc/ShcA, Sck/Sli/ShcB, N-Shc/Rai/ShcC in mammals. In this study, we determined the genomic organization of the mouse Shc family. Coding regions of ShcA, B, and C each comprised 12 exons, spanned approximately 6, 20, and 65 kb, and located on chromosome 3, 10, and 13, respectively. Based on this genome analysis, we determined the full-length structure of mouse Sck/ShcB as a 68-kD protein. We found that the 68-kD full-length Sck/ShcB was more efficiently phosphorylated upon EGF treatment than the previously-analyzed CH2-deleted form. We also found that Sck specifically interacted with a 135-kD phosphoprotein (pp135) through its SH2 domain following membrane depolarization. The Sck-pp135 interaction was reduced by Src kinase inhibitors. These results suggest that Sck, but not N-Shc nor Shc, transmit signals in conjunction with pp135 following Src activation and/or calcium entry in the cell.

Many mRNAs in male germ cells lack the canonical AAUAAA but are normally polyadenylated (Wallace, A. M., Dass, B., Ravnik, S. E., Tonk, V., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and MacDonald, C. C. (1999)Proc. Natl. Acad Sci. U. S. A.96, 6763–6768). Previously, we demonstrated the presence of two distinct forms of theMr64,000 protein of the cleavage stimulation factor (CstF-64) in mouse male germ cells and in brain, a somaticMr64,000 form and a variantMr70,000 form. The variant form was specific to meiotic and postmeiotic germ cells. We localized the gene for the somatic CstF-64 to the X chromosome, which would be inactivated during male meiosis. This suggested that the variant CstF-64 was an autosomal homolog activated during that time. We have named the variant form “τ CstF-64,” and we describe here the cloning and characterization of the mouse τCstF-64 cDNA, which maps to chromosome 19. The mouse τCstF-64 protein fits the criteria of the variant CstF-64, including antibody reactivity, size, germ cell expression, and a common proteolytic digest pattern with τCstF-64 from testis. Features of mτCstF-64 that might allow it to promote the germ cell pattern of polyadenylation include a Pro → Ser substitution in the RNA-binding domain and significant changes in the region that interacts with CstF-77.

A unique pattern of LacZ expression was found in a transgenic mouse line, likely due to regulatory elements at the site of integration. Two new genes flanking the transgene were identified. At early stages of development, the transgene is transiently expressed in ventro-lateral demomyotomal cells migrating from the somites into the limb buds. At late developmental stages and in the adult, lacZ staining marks vascular smooth muscle cells throughout the vascular bed, with the exception of the major elastic arteries, and in pericytes. No expression was detected in skeletal and smooth muscles. Different patterns of expression in vascular smooth muscles was observed at distinct levels of the vascular tree, in arteries as well as in veins. Vessel injury, resulting in stimulation of smooth muscle cells proliferation and migration, is associated with transgene down-regulation. After the formation of neointima thickening, it is reactivated. This transgenic insertion may therefore be used as a useful marker to identify novel physiological cues or genetic elements involved in the regulation of the vascular smooth muscle phenotype(s). It may also provide an experimental tool for studying vasculature and the involvement of pericytes in regulating microvascular homeostasis. © 2001 Wiley-Liss, Inc.

We report the cloning and chromosomal localization of murine and human Mix genes, members of a subclass of paired-like homeobox genes of which the Xenopus laevis Mix.1 gene is the founding member. The murine Mix gene was mapped to the distal region of chromosome 1 and the human region to the syntenic region 1q41-42. Northern analysis and RT-PCR of murine adult and embryonic tissues demonstrated that Mix expression was restricted to the early embryo. Whole-mount in situ hybridization revealed patchy but symmetrical Mix expression in visceral endoderm of embryonic day (E)5.5 embryos. In slightly older embryos, the expression was skewed to one side of the embryo and by E6.5, at the onset of gastrulation, expression was seen in the epiblast, visceral endoderm, nascent mesoderm, and the primitive streak. This expression pattern was maintained in mid- and late-streak embryos. In early bud-stage embryos, expression was strongest in the proximal two thirds of the streak, extending to the base of the allantois. By the headfold-stage, expression was confined to the remnant of the primitive streak in the caudal region of the embryo and, after E8.0, in the caudal notochord and tail bud mesoderm. Mix transcripts were no longer detectable after embryonic day 9.5. © 2000 Wiley-Liss, Inc.

RLIM is a RING H2 zinc finger protein that acts as a negative coregulator for LIM homeodomain transcription factors. We have isolated genomic λ clones that cover the entire mouse RLIM-encoding Rnf12gene. The Rnf12gene encompasses 20 kb and consists of at least five exons and four introns. Several transcriptional start sites within a 24-bp region were mapped around 300 nt upstream of the translational start site. Rnf12-specific mRNA can be detected in many tissues as revealed by Northern blot analysis. Transient cotransfections reveal that the proximal Rnf12promoter can be activated in vitroby ubiquitously and more restrictively expressed transcription factors, some of which are known mediators of signal transduction pathways, e.g., mammalian Krüppel-like transcription factors, Sox and ets-related proteins, and RBP-J. We isolated a cDNA encoding human RLIM, which is highly conserved with mouse and chick RLIM. By fluorescence in situhybridization and interspecific backcross analysis, we have localized the Rnf12gene to the central regions of mouse and human chromosome X.

We report the identification and characterization of a novel CC chemokine designated CCL28 and its receptor CCR10, known previously as orphan G-protein-coupled receptor GPR2. Human and mouse CCL28 share 83% identity at the amino acid and 76% at the nucleic acid levels. We also identified the mouse homologues of CCL28 and of CCR10, which map to mouse chromosomes 13 and 11, respectively. CCL28 is expressed in a variety of human and mouse tissues, and it appears to be predominantly produced by epithelial cells. Both human and mouse CCL28 induce calcium mobilization in human and mouse CCR10-expressing transfectants. CCL28 desensitized the calcium mobilization induced in CCR10 transfectants by CCL27, indicating that these chemokines share this new chemokine receptor. In vitro,recombinant human CCL28 displays chemotactic activity for resting CD4 or CD8 T cells.

In a signal sequence trap screening of the murine brain, we identified a new member of the tumor necrosis factor receptor superfamily designated TROY. TROY is a type I membrane protein of 416 amino acids with characteristic cysteine-rich motifs in the extracellular domain and a tumor necrosis factor receptor-associated factor (TRAF) 2 binding sequence in the cytoplasmic domain of 223 amino acids. In fact, activation of nuclear factor κB was induced by the overexpression of TROY and inhibited by dominant negative forms of TRAF2, TRAF5, and TRAF6, indicating that TRAFs and nuclear factor κB are involved in the signal transduction of TROY. We also cloned a cDNA for a human counterpart, which showed a 75% homology with mouse TROY at the amino acid level. The extracellular domain of TROY exhibits an extensive homology with that of Edar, a receptor that specifies hair follicle fate. TROY mRNA is strongly expressed in brain and embryo and moderately expressed in the heart, lung, and liver but not the spleen. In the embryo, the expression level is particularly strong in the skin. Interestingly, in situhybridization analysis of the embryo showed that TROY mRNA was exclusively expressed in the epithelium of many tissues. On the other hand, in neonatal mice, TROY is expressed in hair follicles like Edar as well as in the cerebrum, suggesting pleiotropic functions of TROY in development as well as in the adult mice. The Troygene is located near the waved coat(Wc) locus, a mutant related to abnormalities in skin and hair.

PSMC3 and PSMC4, components of the 19S complex of the 26S proteasome, show a significant degree of amino acid similarity, especially in the conserved ATPase domain (CAD). In this study, we characterized the mouse Psmc3and Psmc4genes. The genomic structures of both genes showed a significant degree of similarity. The Psmc3gene was composed of 12 coding exons, whereas the Psmc4gene had 11 exons. Exons encoding the leucine zipper domain and CAD were identical in number between the Psmc3and Psmc4genes. The Psmc3gene mapped to mouse chromosome 2, whereas Psmc4mapped to chromosome 7. We further addressed the biological roles of Psmc3 and Psmc4 through the generation of gene targeted mice. Both Psmc3- and Psmc4-deficient mice died before implantation, displaying defective blastocyst development. These findings indicate that Psmc3 and Psmc4 have similar and essential roles in early embryogenesis and further that both ATPases have noncompensatory functions in vivo.

cDNA library subtraction techniques were used to identify transcripts expressed by activated mouse αβPTCR+CD4−CD8−(double‐negative; DN) T cells, a subset of natural killer T (NKT) cells. The most frequent cDNAs identified included the chemokines TCA3, macrophage inflammatory protein‐1α (MIP‐1α), MIP‐1β, and lymphotactin (LPTN), the cytokines interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ), and a granzyme. We also identified a new member of the immunoglobulin superfamily (Ig‐SF). This molecule was designated class I‐restricted T cell‐associated molecule (CRTAM) as a result of its restricted expression pattern in T cells. Human CRTAM was also identified, and shares the same expression pattern as the mouse molecule. LPTN and CRTAM exhibit the same expression pattern in T cells, suggesting the existence of a gene expression program common to class I‐MHC‐restricted T cells. J. Leukoc. Biol.67: 725–734; 2000.

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 ɛ-sarcoglycan gene (Sgce) andZac1(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 Zac1is a potential tumor suppressor gene. Sgceand Zac1are expressed predominantly from their paternal alleles in all adult mouse tissues, except that Zac1is biallelic in the liver andSgceis weakly expressed from the maternal allele in the brain. Sgceand Zac1are broadly expressed in embryos, with Zac1being 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. Sgceis 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.Zac1maps 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 ZAChas been described for a number of breast and ovarian carcinomas, suggesting that ZACis a potential tumor suppressor gene.

ABSTRACTImprinted genes are expressed from one allele according to their parent of origin, and many are essential to mammalian embryogenesis. Here we show that the ?-sarcoglycan gene (Sgce) andZac1(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 Zac1is a potential tumor suppressor gene. Sgceand Zac1are expressed predominantly from their paternal alleles in all adult mouse tissues, except that Zac1is biallelic in the liver andSgceis weakly expressed from the maternal allele in the brain. Sgceand Zac1are broadly expressed in embryos, with Zac1being 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. Sgceis 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.Zac1maps 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 ZAChas been described for a number of breast and ovarian carcinomas, suggesting that ZACis a potential tumor suppressor gene.

In a complementary DNA (cDNA) screening of murine Th2-skewed lymphocytes with our recently developed signal sequence trap method termed SST-REX, a novel type 1 cytokine receptor, Delta1 (d1), was identified. Although d1 is ubiquitously expressed in multiple tissues, the expression level is higher in Th2-skewed lymphocytes than in Th1-skewed ones. The d1 cDNA encodes a 359–amino acid type 1 membrane protein. The extracellular domain of 206 amino acids showed 24% identity with the murine common?? receptor that is shared among the receptors for interleukin(IL)-2, IL-4, IL-7, IL-9, and IL-15. The membrane-proximal region of d1 includes a box1 motif, which is important for association with Janus kinases (JAKs), and showed a significant homology with that of the mouse erythropoietin receptor (EPOR). A box2 motif was also found in close proximity to the box1 region. Dimerization of the cytoplasmic region of d1 alone did not transduce proliferative signals in IL-3–dependent cell lines. However, the membrane-proximal region of d1 could substitute for that of human EPOR in transmitting proliferative signals and activating JAK2. These results suggest that d1 is a subunit of cytokine receptor that may be involved in multiple receptor systems and play a regulatory role in the immune system and hematopoiesis.

In a complementary DNA (cDNA) screening of murine Th2-skewed lymphocytes with our recently developed signal sequence trap method termed SST-REX, a novel type 1 cytokine receptor, Delta1 (δ1), was identified. Although δ1 is ubiquitously expressed in multiple tissues, the expression level is higher in Th2-skewed lymphocytes than in Th1-skewed ones. The δ1 cDNA encodes a 359–amino acid type 1 membrane protein. The extracellular domain of 206 amino acids showed 24% identity with the murine common γ receptor that is shared among the receptors for interleukin(IL)-2, IL-4, IL-7, IL-9, and IL-15. The membrane-proximal region of δ1 includes a box1 motif, which is important for association with Janus kinases (JAKs), and showed a significant homology with that of the mouse erythropoietin receptor (EPOR). A box2 motif was also found in close proximity to the box1 region. Dimerization of the cytoplasmic region of δ1 alone did not transduce proliferative signals in IL-3–dependent cell lines. However, the membrane-proximal region of δ1 could substitute for that of human EPOR in transmitting proliferative signals and activating JAK2. These results suggest that δ1 is a subunit of cytokine receptor that may be involved in multiple receptor systems and play a regulatory role in the immune system and hematopoiesis.

We have cloned and characterized LATS2,a novel mammalian homologue of the Drosophilatumor suppressor gene lats/warts.Northern blot analysis showed ubiquitous expression of mouse LATS2 (MmLATS2)mRNA, whereas expression of human LATS2 (HsLATS2)mRNA was enhanced in skeletal muscle and heart. Immunoblotting analysis of fractionated cell lysates showed HsLats2 to be a nuclear protein. We mapped the MmLATS2gene to mouse chromosome 14 by interspecific backcross analysis. We also mapped the HsLATS2gene (by fluorescence in situhybridization) to the 13q11–q12 region, in which a loss of heterozygosity has been frequently observed in many primary cancers and to which the tumor suppressor genes RBand BRCA2have also been mapped.

Calcium is an important second messenger in eukaryotic cells. Many of the effects of calcium are mediated via its interaction with calmodulin and the subsequent activation of Ca2+/calmodulin-dependent (CaM) kinases. CaM kinases are involved in a wide variety of cellular processes including muscle contraction, neurotransmitter release, cell cycle control, and transcriptional regulation. While CaMKII has been implicated in learning and memory, the biological role of the other multifunctional CaM kinases, CaMKI and CaMKIV, is largely unknown. In the course of a degenerate RT-PCR protein kinase screen, we identified a novel serine/threonine kinase, Pnck.In this report, we describe the cloning, chromosomal localization, and expression of Pnck,which encodes a 38-kDa protein kinase whose catalytic domain shares 45–70% identity with members of the CaM kinase family. The gene for Pncklocalizes to mouse chromosome X, in a region of conserved synteny with human chromosome Xq28 that is associated with multiple distinct mental retardation syndromes. Pnckis upregulated during intermediate and late stages of murine fetal development with highest levels of expression in developing brain, bone, and gut. Pnckis also expressed in a tissue-specific manner in adult mice with highest levels of expression detected in brain, uterus, ovary, and testis. Interestingly, Pnckexpression in these tissues is restricted to particular compartments and appears to be further restricted to subsets of cells within those compartments. The chromosomal localization of Pnck,along with its tissue-specific and restricted pattern of spatial expression during development, suggests that Pnckmay be involved in a variety of developmental processes including development of the central nervous system.

We previously identified a novel protein kinase, Hunk,by means of a degenerate PCR screen designed to isolate kinases expressed in the murine mammary gland. We now describe the molecular cloning, chromosomal localization, and activity of this kinase and characterize its spatial and temporal pattern of expression in the mouse. We have isolated a 5.0-kb full-length cDNA clone that contains the 714-amino-acid open reading frame encoding Hunk. Analysis of this cDNA reveals that Hunk is most closely related to the SNF1 family of serine/threonine kinases and contains a newly described SNF1 homology domain. Accordingly, antisera specific for Hunk detect an 80-kDa polypeptide with associated phosphotransferase activity. Hunkis located on distal mouse chromosome 16 in a region of conserved synteny with human chromosome 21q22. During fetal development and in the adult mouse, HunkmRNA expression is developmentally regulated and tissue-specific. Moreover, in situhybridization analysis reveals that Hunkexpression is restricted to subsets of cells within a variety of organs in the adult mouse. These findings suggest a role for Hunk in murine development.

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines that share the gp130 receptor subunit. Of these family members, leukemia inhibitory factor (LIF) is most closely related to OSM, and various overlapping biologic activities have been described between human LIF and OSM (hLIF and hOSM). Two types of functional hOSM receptors are known: the type I OSM receptor is identical to the LIF receptor that consists of gp130 and the LIF receptor ß subunit (LIFRß), and the type II OSM receptor consists of gp130 and the OSM receptor ß subunit (OSMRß). It is thus conceivable that common biologic activities between hLIF and hOSM are mediated by the shared type I receptor and OSM-specific activities are mediated by the type II receptor. However, in contrast to the human receptors, recent studies have demonstrated that mouse OSM (mOSM) does not activate the type I receptor and exhibits unique biologic activity. To elucidate the molecular structure of the functional mOSM receptor, we cloned a cDNA encoding mOSMRß, which is 55.5% identical to the hOSMRß at the amino acid level. mOSM-responsive cell lines express high-affinity mOSM receptors, as well as mOSMRß, whereas embryonic stem cells, which are responsive to LIF but not to mOSM, do not express mOSMRß. mOSMRß alone binds mOSM with low affinity (kd?=?13.0 nmol/L) and forms a high-affinity receptor (kd?=?606 pmol/L) with gp130. Ba/F3 transfectants expressing both mOSMRß and gp130 proliferated in response to mOSM, but failed to respond to LIF and human OSM. Thus, the cloned mOSMRß constitutes an essential and species-specific receptor component of the functional mOSM receptor. Reminiscent of the colocalization of the mOSM and mLIF genes, the mOSMRß gene was found to be located in the vicinity of the LIFRß locus in the proximal end of chromosome 15.

The mouse Gh/tissue transglutaminase gene (Tgm2), coding a dual-function protein that both binds guanosine triphosphate (GTP) and catalyzes the posttranslational modification of proteins by transamidation of glutamine residues, has been cloned. Sequence analysis ofTgm2and comparison with the TGase sequences of other species allowed correction of several apparent sequencing artifacts in theTgm2cDNA.Tgm2spans approximately 34 kb and has 13 exons and 12 introns. Although the structure ofTgm2shows similarity to that of other transglutaminase genes, with introns ranging from 921 bp to >5 kb, several introns differ considerably in size from those of the human Ghgene,TGM2. Tgm2maps to the distal region of mouse chromosome 2, a region syntenic to human chromosome 20q containing TGM2.Tgm2is in the vicinity of two uncloned mouse mutations, diminutive (dm) and blind-sterile (bs). Genomic DNA fromdmmice was unavailable; however, Southern blot analysis ofbsDNA showed no gross rearrangements ofTgm2.

Through the preparation of a subtracted cDNA library, we have extensively isolated genes whose expression is induced in mouse spermatogenic cells. One of the genes encoded a protein with a RING zinc-finger motif, which we termed sperizin (spermatid-specific ring zinc finger). Transcription of the sperizin gene was not observed in prepubertal testis, but became detectable at day 23. Northern andin situhybridization analyses indicated that the sperizin gene was exclusively expressed in the round spermatid. The sperizin gene is intronless, and GFP-tagged sperizin was found to be localized in the cytoplasm of ectopically expressed somatic cultured cells. We assigned the chromosomal localization of the sperizin gene to chromosome 17 using an interspecific backcross mapping panel. The data suggest that we have identified a new member of the RING-finger family of proteins that may be involved in spermatogenesis.

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines that share the gp130 receptor subunit. Of these family members, leukemia inhibitory factor (LIF) is most closely related to OSM, and various overlapping biologic activities have been described between human LIF and OSM (hLIF and hOSM). Two types of functional hOSM receptors are known: the type I OSM receptor is identical to the LIF receptor that consists of gp130 and the LIF receptor β subunit (LIFRβ), and the type II OSM receptor consists of gp130 and the OSM receptor β subunit (OSMRβ). It is thus conceivable that common biologic activities between hLIF and hOSM are mediated by the shared type I receptor and OSM-specific activities are mediated by the type II receptor. However, in contrast to the human receptors, recent studies have demonstrated that mouse OSM (mOSM) does not activate the type I receptor and exhibits unique biologic activity. To elucidate the molecular structure of the functional mOSM receptor, we cloned a cDNA encoding mOSMRβ, which is 55.5% identical to the hOSMRβ at the amino acid level. mOSM-responsive cell lines express high-affinity mOSM receptors, as well as mOSMRβ, whereas embryonic stem cells, which are responsive to LIF but not to mOSM, do not express mOSMRβ. mOSMRβ alone binds mOSM with low affinity (kd = 13.0 nmol/L) and forms a high-affinity receptor (kd = 606 pmol/L) with gp130. Ba/F3 transfectants expressing both mOSMRβ and gp130 proliferated in response to mOSM, but failed to respond to LIF and human OSM. Thus, the cloned mOSMRβ constitutes an essential and species-specific receptor component of the functional mOSM receptor. Reminiscent of the colocalization of the mOSM and mLIF genes, the mOSMRβ gene was found to be located in the vicinity of the LIFRβ locus in the proximal end of chromosome 15.

The binding of polypeptide growth factors to their appropriate cell surface transmembrane receptors triggers numerous biochemical responses, including the transcriptional activation of specific genes. We have used a differential display approach to identify fibroblast growth factor-1-inducible genes in murine NIH 3T3 cells. Here, we report that the fibroblast growth factor-inducible-14 (Fn14) gene is a growth factor-regulated, immediate-early response gene expressed in a developmental stage- and adult tissue-specific manner in vivo. This gene, located on mouse chromosome 17, is predicted to encode an 129-amino acid type Ia membrane protein with no significant sequence similarity to any known protein. We have used two experimental approaches, direct fluorescence microscopy and immunoprecipitation analysis of biotinylated cell surface proteins, to demonstrate that Fn14 is located on the plasma membrane. To examine the biological consequences of constitutive Fn14 expression, we isolated NIH 3T3 cell lines expressing variable levels of epitope-tagged Fn14 and analyzed their phenotypic propertiesin vitro. These experiments revealed that Fn14 expression decreased cellular adhesion to the extracellular matrix proteins fibronectin and vitronectin and also reduced serum-stimulated cell growth and migration. These results indicate that Fn14 is a novel plasma membrane-spanning molecule that may play a role in cell-matrix interactions.

Numerous proteins are cleaved or “shed” from their membrane-bound form. One such protein, tumour necrosis factor α (TNF-α), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-α converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-α. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.

Heterotrimeric G proteins, composed of α, β, and γ subunits, transduce signals from transmembrane receptors to a wide range of intracellular effectors. The G protein γ subunits, which play an indispensible role in this communication, constitute a large and diverse multigene family. Using an interspecific backcross panel, we have determined the mouse chromosomal locations of five γ subunit genes: γ2, γ8, γ10, γ12, and γCone. Combined with previous mapping studies, these data indicate that, with the possible exception of γ1 and γ11, the G protein γ subunit genes are well dispersed within the mouse and human genomes.

ABSTRACTRfv3is a host resistance gene that operates through an unknown mechanism to control the development of the virus-neutralizing antibody response required for recovery from infection with Friend retrovirus. The Rfv3gene was previously mapped to an approximately 20-centimorgan (cM) region of chromosome 15. More refined mapping was not possible, due to a lack of microsatellite markers and leakiness in the Rfv3phenotype, which prevented definitive phenotyping of individual recombinant mice. In the present study, we overcame these difficulties by taking advantage of seven new microsatellite markers in the Rfv3region and by using progeny tests to accurately determine the Rfv3phenotype of recombinant mice. Detailed linkage analysis of relevant crossovers narrowed the location of Rfv3to a 0.83-cM region. Mapping of closely linked genes in an interspecific backcross panel allowed us to exclude two previous candidate genes, Ly6andWnt7b. These studies also showed for the first time that the Hsf1gene maps to the Rfv3-linked cluster of genes including Il2rb, Il3rb, and Pdgfb. This localization of Rfv3to a region of less than 1 cM now makes it feasible to attempt the cloning of Rfv3by physical methods.

A novel gene product, GPR56, with homology to the seven transmembrane-domain receptor superfamily, has been cloned by PCR amplification using degenerate oligonucleotide primers and subsequent screening of a human heart cDNA library. The isolated 2.8-kb cDNA clone encodes a protein of 693 amino acids that shows highest identity (32%) to HE6, a member of a subclass of the class B secretin-like G-protein-coupled receptors. Northern analysis of various human tissues revealed a wide distribution of the transcript with highest levels found in thyroid gland, brain, and heart.In situhybridization analysis of human thyroid gland as well as rat heart and brain tissue confirms these results and identifies the hippocampus and hypothalamic nuclei as brain areas with particularly high expression of GPR56 mRNA. The high level of mRNA expression, its wide distribution, and the mucin-like extracellular domain of the receptor protein suggest a possible role for this receptor in cell–cell interaction processes. The human gene for GPR56 has been isolated and its exon–intron structure determined. The total length of the human GPR56 gene is approximately 15 kb, and it consists of 13 exons. Fluorescencein situhybridization, PCR analysis of somatic cell hybrids, and interspecific mouse backcross mapping have localized the genes to human chromosome 16q13 and mouse chromosome 8.

The NPM-MLF1 fusion protein is expressed in blasts from patients with myelodysplasia/acute myeloid leukemia (MDS/AML) containing the t(3;5) chromosomal rearrangement. Nucleophosmin (NPM), a previously characterized nucleolar phosphoprotein, contributes to two other fusion proteins found in lympho-hematopoietic malignancies, anaplastic large cell lymphoma (NPM-ALK) and acute promyelocytic leukemia (NPM-RARα). By contrast, the function of the carboxy-terminal fusion partner, myelodysplasia/myeloid leukemia factor 1 (MLF1), is unknown. To aid in understanding normal MLF1 function, we isolated the murine cDNA, determined the chromosomal localization of Mlf1,and defined its tissue expression by in situhybridization. Mlf1was highly similar to its human homologue (86% and 84% identical nucleotide and amino acid sequence, respectively) and mapped to the central region of chromosome 3, within a segment lacking known mouse mutations. Mlf1tissue distribution was restricted during both development and postnatal life, with high levels present only in skeletal, cardiac, and selected smooth muscle, gonadal tissues, and rare epithelial tissues including the nasal mucosa and the ependyma/choroid plexus in the brain. Mlf1transcripts were undetectable in the lympho-hematopoietic organs of both the embryonic and adult mouse, suggesting that NPM-MLF1 contributes to the genesis of MDS/AML in part by enforcing the ectopic overexpression of MLF1within hematopoietic tissues.

We have identified and characterized a 12th subfamily of vertebrate annexins by systematic analysis of the primary structure, chromosomal mapping, and molecular evolution of unique cDNA and protein sequences from human and mouse. Distinctive features included rare expression, a codon deletion in conserved repeat 3, and an unusual ablation of the type II calcium-binding sites in tetrad core repeats 1, 3, and 4. The paralogy of novel annexin A10 (following revised nomenclature) was confirmed by FISH-mapping human ANXA10to chromosome 4q33 and genetic linkage mapping mouse Anxa10to midchromosome 8. Phylogenetic analysis established that the 5′ and 3′ halves of the annexin A6 octad are more closely related to annexins A5 and A10, respectively, than they are to each other. Molecular date estimates, paralogy linkage maps between human chromosomes 4 and 5, and annexin structural considerations led to the proposal that annexins A5 and A10 may have been the direct progenitors of annexin A6 octad formation via chromosomal duplication during the genome expansion in early chordates.

Abstract.: LIM-only (LMO) proteins are transcription regulators that function by mediating protein–protein interaction and include the T cell oncogenes encoding LMO1 and LMO2. The oncogenic functions of LMO1 and LMO2 are thought to be mediated by interaction with LDB1 since they form a multimeric protein complex(es). A new member of the Lmo family, Lmo4, has also recently been identified via its interaction with Ldb1. Sequence analysis of the mouse Lmo4 gene shows that it spans about 18 kb and consists of at least six exons, including two alternatively spliced 5′ exons. Unlike Lmo1, the two 5′ exons of Lmo4 do not encode protein. Comparison of the Lmo4 gene structure with the other LMO family members shows the exon structure of Lmo4 differs in the position of exon junctions encoding the second LIM domain and in a novel exon-intron junction at the penultimate codon of the gene. Lmo4 is thus the least conserved known member of the LIM-only family in both nucleotide sequence and exon structure. Physical mapping of the Lmo4/LMO4 genes has shown mouse Lmo4 is located on Chromosome (Chr) 3 and human LMO4 on Chr 1p22.3. This chromosome location is of interest as it occurs in a region that is deleted in a number of human cancers, indicating a possible role of LMO4 in tumorigenesis, like its relatives LMO1 and LMO2.

Abstract.: Thrombospondins are a family of extracellular, adhesive proteins that are widely expressed in vertebrates. Five distinct gene products, designated thrombospondin-1 through -4 and cartilage oligomeric matrix protein (COMP), have been identified. With the exception of thrombospondin-4, the structure and location of thrombospondin genes have been determined in the human and/or mouse genomes. In this study, the structure and location of the murine thrombospondin-4 gene and the location of the human thrombospondin-4 gene are reported. The murine thrombospondin-4 gene is approximately 4.5 kb in length and includes 22 exons. Interspecific backcross analysis of progeny derived from matings of (C57BL/6J �Mus spretus)F1� C57BL/6J mice indicates that the thrombospondin-4 gene is tightly linked to the Dhfr locus on murine Chromosome (Chr) 13. The human gene maps to Chr 5 in band q13 by in situ hybridization to human metaphase chromosomes. The thrombospondin-4 promoter is similar to promoters of some housekeeping, growth control, and other thrombospondin genes in that it contains multiple GC box sequences and lacks a CAAT box. The presence of multiple E-box sequence motifs is consistent with thrombospondin-4 expression in muscle and bone tissue.

A cDNA encoding a novel protein has been previously isolated from two independent sources: melanoma cell cultures and chondrocytes. The protein from human melanoma cell lines and tumors is called melanoma inhibitory activity (MIA) (Blesch et al. [1994] Cancer Res. 54:5695–5701) and the protein from primary bovine chondrocytes and cartilaginous tissues is called cartilage-derived retinoic acid-sensitive protein (CD-RAP) (Dietz and Sandell [1996] J. Biol. Chem. 271:3311–3316). In order to investigate the gene regulation and function of CD-RAP/MIA, the mouse gene locus was isolated and analyzed. Developmental expression was determined by in situ hybridization to mouse embryos. Expression was limited to cartilaginous tissues and was initiated with the advent of chondrogenesis, remaining abundant throughout development. The mouse gene was isolated and sequenced from a 129Sv library and sequenced directly from an additional strain, B6C3Fe. The mouse CD-RAP/MIA gene is 1.5 kbp and consists of four exons. The promoter sequence of the gene contains many potential regulatory domains including 8 basic helix-loop-helix protein-binding domains and an AT-rich domain, both motifs shown to be present in the cartilage-specific enhancer of the type II procollagen gene. Other potential cis-acting motifs include binding sites for GATA-1, NF-IL6, PEA3, w-elements, NFκB, Zeste and Sp1. The gene, called cdrap, was localized to the end of an arm of chromosome 7 at the same site as the transforming growth factor β1 (Tgf-β1) and the glucose phosphate isomerase 1 (Gpi1) genes. Potential mouse mutants that mapped to the same region of chromosome 7 were identified. Two of the potential mutants with skeletal phenotypes were sequenced, pudgy (pu) and extra toes with spotting (Xs^j); however, no mutations were found in the coding sequence. To determine whether CD-RAP/MIA is associated with tumors of cartilage, mRNAs from a variety of rodent tissues and cell lines were screened. Expression was detected in a rodent tumor, the Swarm rat chondrosarcoma and a chondrosarcoma cell line derived from it, but not in other tissues or tumors of non-cartilage origin. Immunolocalization revealed CD-RAP/MIA protein localized in cartilage only. These results show that the normal expression of CD-RAP/MIA is limited to cartilage; however, pathologically, it is expressed both in melanoma and chondrosarcoma. The restricted expression of CD-RAP/MIA may provide an opportunity to monitor cartilage metabolic activity as well as the tumor activity of melanoma and chondrosarcoma. Dev. Dyn. 208:516–525, 1997. © 1997 Wiley-Liss, Inc.

Cloning of the Drosophila Shaker gene established that a neurological phenotype including locomotor dysfunction can be caused by a mutation in a voltage-gated potassium (K) channel gene. Shaker sequences have been used to isolate a large family of related K channel genes from both flies and mammals. Toward elucidating the evolutionary relationship between loci and the potential causal connection that K channels may have to mammalian genetic disorders, we report here the genetic mapping of 12-16 different murine, voltage-gated K channel genes. We find that multiple genes, in some cases from distantly related K channel subfamilies, occur in clusters in the mouse genome. These mapping results suggest that the K channel gene subfamilies arose through ancient localized gene duplication events, followed by chromosomal duplications and rearrangements as well as further gene duplication. We also note that several neurologic disorders of both mouse and human are associated with the chromosomal regions containing K channel genes. Copyright 1994, 1999 Academic Press

A mouse histamine H2 receptor (H2R) gene was isolated from a mouse ES cell genomic DNA library. The gene encoded a 358-amino-acid protein and displayed a 95% homology with the rat histamine H2 receptor at the amino acid level. It had features characteristic of other G-protein-coupled receptors. Reverse transcriptase–polymerase chain reaction analysis of total RNA prepared from mouse tissues showed that the gene was highly expressed in stomach and moderately in brain and heart. A weak expression was also detected in liver. An interspecific backcross analysis revealed that the mouse H2R gene, designatedHrh2,was located in the central region of chromosome 13.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are signal transducers for members of the TNF receptor superfamily. We previously identified murine TRAF5 (mTRAF5) and showed that it specifically interacts with the lymphotoxin-β receptor (LT-βR) and activates the transcription factor NF-κB. Here we have cloned the human TRAF5 homologue (hTRAF5) by cross hybridization with mTRAF5 cDNA. hTRAF5 cDNA is composed of 2894 nucleotides with a 557-amino-acid open reading frame that exhibits 77.5 and 80% identity to mTRAF5 at the nucleotide and amino acid levels, respectively. Northern blot analysis revealed that hTRAF5 mRNA is expressed in all visceral organs. Western blotting revealed that hTRAF5 protein was abundantly expressed in the human follicular dentritic cell line, FDC-1, and to a much lesser degree in several tumor cell lines. Interspecific backcross mapping revealed thatTraf5is located in the distal region of mouse chromosome 1, which shares a region of homology with human chromosome 1q. Fluorescencein situhybridization confirmed regional localization to human chromosome 1q32.

Myosins are molecular motors that move along filamentous actin. Seven classes of myosin are expressed in vertebrates: conventional myosin, or myosin-II, as well as the 6 unconventional myosin classes -I, -V, -VI, -VII, -IX, and -X. We have mapped in mouse 22 probes encompassing all known unconventional myosins and, as a result, have identified 16 potential unconventional myosin genes. These genes include 7 myosins-I, 2 myosins-V, 1 myosin-VI, 3 myosins-VII, 2 myosins-IX, and 1 myosin-X. The map location of 5 of these genes was identified in human chromosomes by fluorescencein situhybridization.

Atm, the mouse homolog of the human ATM gene defective in ataxia–telangiectasia (A–T), has been identified. The entire coding sequence of the Atm transcript was cloned and found to contain an open reading frame encoding a protein of 3066 amino acids with 84% overall identity and 91% similarity to the human ATM protein. Variable levels of expression of Atm were observed in different tissues. Fluorescencein situhybridization and linkage analysis located the Atm gene on mouse chromosome 9, band 9C, in a region homologous to the ATM region on human chromosome 11q22–q23.

Studies of transcriptional activation by interferons and a variety of cytokines have led to the identification of a family of proteins that serve as signal transducers and activators of transcription, Stats. Here, we report that the seven mouse Stat loci map in three clusters, with each cluster located on a different mouse autosome. The data suggest that the family has arisen via a tandem duplication of the ancestral locus, followed by dispersion of the linked loci to different mouse chromosomes. Copyright 1995, 1999 Academic Press

Type VII collagen is the major component of anchoring fibrils, critical attachment structures at the dermal-epidermal basement membrane zone. Genetic linkage analyses with recently cloned human type VII collagen cDNAs have indicated that the corresponding gene, COL7A1, is the candidate gene in the dystrophic forms of epidermolysis bullosa. To gain insight into the evolutionary conservation of COL7A1, in this study we have isolated mouse type VII collagen cDNAs by screening a mouse epidermal keratinocyte cDNA library with a human COL7A1 cDNA. Two overlapping mouse cDNAs were isolated, and Northern hybridization of mouse epidermal keratinocyte RNA with one of them revealed the presence of a mRNA transcript of ~9.5 kb, the approximate size of the human COL7A1 mRNA. Nucleotide sequencing of the mouse cDNAs revealed a 2760-bp open reading frame that encodes the 5' half of the collagenous domain and a segment of the NC-1, the noncollagenous amino-terminal domain of type VII collagen. Comparison of the mouse amino acid sequences with the corresponding human sequences deduced from cDNAs revealed 82.5% identity. The evolutionary divergence of the gene was relatively rapid in comparison to other collagen genes. Despite the high degree of sequence variation, several sequences, including the size and the position of noncollagenous imperfections and interruptions within the Gly-X-Y repeat sequence, were precisely conserved. Finally, the mouse Col7a1 gene was located by interspecific backcross mapping to mouse Chromosome 9, a region that corresponds to human chromosome 3p21, the position of human COL7A1. This assignment confirms and extends the relationship between the mouse and the human chromosomes in this region of the genome. Copyright 1993, 1999 Academic Press

Paralemmin is a newly identified protein that is associated with the plasma membrane and with intracellular membranes through a lipid anchor. It is abundant in brain, is expressed at intermediate levels in the kidney and in endocrine cells, and occurs at low levels in many other tissues. As it is a candidate for genetic disorders that affect membrane functions, we have determined the structure of the human paralemmin gene,PALM,showing that it is organized into nine exons. Moreover, we have performed chromosomal assignments of the human and mouse paralemmin genes, localizing them to regions of homology at human 19p13.3 and the central mouse chromosome 10. Finally, mutation analysis using RNA from mice homozygous for the mutant genesgrizzled(gr),mocha(mh),mocha 2J(mh2J),jittery(ji) andhesitant(jihes), which map to this area, excluded mutations in theirPalmcoding sequences.

Macrophage migration inhibitory factor, MIF, is a cytokine released by T-lymphocytes, macrophanges, and the pituitary gland that serves to integrate peripheral and central inflammatory responses. Ubiquitous expression and developmental regulation suggest that MIF may have additional roles outside of the immune system. Here we report the structure and chromosomal location of the mouse Mif gene and the partial characterization of five Mif pseudogenes. The mouse Mif gene spans less than 0.7 kb of chromosomal DNA and is composed of three exons. A comparison between the mouse and the human genes shows a similar gene structure and common regulatory elements in both promoter regions. The mouse Mif gene maps to the middle region of chromosome 10, between Bcr and S100b, which have been mapped to human chromosomes 22q11 and 21q22.3, respectively. The entire sequence of two pseudogenes demonstrates the absence of introns, the presence of the 5' untranslated region of the cDNA, a 3' poly(A) tail, and the lack of sequence similarity with untranscribed regions of the gene. The five pseudogenes are highly homologous to the cDNA, but contain a variable number of mutations that would produce mutated or truncated MIF-like proteins. Phylogenetic analyses of MIF genes and pseudogenes indicate several independent genetic events that can account for multiple genomic integrations. Three of the Mif pseudogenes were also mapped by interspecific backcross to chromosomes 1, 9, and 17. These results suggest that Mif pseudogenes originated by retrotransposition. Copyright 1995, 1999 Academic Press

We report here the molecular cloning of a mouse histamine H1 receptor gene. The protein deduced from the nucleotide sequence is composed of 488 amino acid residues with characteristic properties of GTP binding protein-coupled receptors. Our results suggest that the mouse histamine H1 receptor gene is a single locus, and no related sequences were detected. Interspecific backcross analysis indicated that the mouse histamine H1 receptor gene (Hrh1) is located in the central region of mouse Chromosome 6 linked to microphthalmia (Mitfmi), ras-related fibrosarcoma oncogene 1 (Raf1), and ret proto-oncogene (Ret) in a region of homology with human chromosome 3p.

We report the identification of a new recessive prenatal lethal insertional mutation,amnionless(amn).amnmutant embryos first appear abnormal during the Early Streak stage, between E6.5 and E7.0, when they initiate mesoderm production. Subsequently, theamnmutants become developmentally arrested between the Mid and Late Streak stages of gastrulation and they die and are resorbed between E9.5 and E10.5. While extraembryonic structures, including the chorion, yolk sac blood islands, and allantois appear to develop normally, the small embryonic ectoderm remains undifferentiated and generates no amnion. In addition, the embryonic mesoderm that is produced does not become organized into node, notochord, and somites and there is no morphological evidence of neural induction. Interspecific backcross and fluorescencein situhybridization analyses map the transgene insertion, and thus theamnmutation, to the distal region of mouse chromosome 12, which has synteny with human chromosome 14q32. A gene encoding a 7.5-kb transcript has been identified at a junction between the integrated transgene and host chromosome 12 sequences that meets three criteria expected of a candidateamngene. This gene maps to the site of transgene insertion; it is transcribed during gastrulation, and its expression is disrupted inamnmutant embryos. Nucleotide sequencing studies show that the 567 amino acid protein encoded by the 7.5-kb transcript is a member of the newly defined family of putative signal transducing proteins, TRAFs, that associate with the cytoplasmic domains of members of the tumor necrosis factor (TNF) receptor superfamily. Thus, we have named the gene encoding the 7.5-kb transcriptTRAFamn.TRAFamn is identical to a recently reported protein (CD40bp, CAP-1, CRAF1, LAP1) that can bind the cytoplasmic domains of CD40 and the lymphotoxin β receptor (LTβR), both of which are known members of the TNF receptor superfamily. The implications of these findings regarding a possible role for the TNF receptor superfamily during gastrulation are discussed.

Members of the aquaporin family of molecular water transporters are expressed in diverse epithelia and in complex developmental patterns. Using a cDNA for mouseAqp1,the structural gene was isolated and a restriction map was constructed. The 13-kbAqp1gene contains four exons with intronic boundaries corresponding to other known aquaporin genes. Transcription begins 67 bp 5′ to the translation initiation site and 20 bp 3′ from a TATAA consensus sequence.Aqp1was localized by interspecific mouse backcross mapping to the central region of mouse chromosome 6 syntenic with human chromosome 7p14, whereAQP1had previously been localized. These studies have revealed marked structural similarities between the mouseAqp1and the humanAQP1genes, suggesting that further comparative studies may provide molecular insight into genetic regulatory features shared by both species.