Biochemical characterization of arylsulfatase E and functional analysis of mutations found in patients with X-linked chondrodysplasia punctata.

X-linked chondrodysplasia punctata (CDPX) is a congenital disorder characterized by abnormalities in cartilage and bone development. Mutations leading to amino acid substitutions were identified recently in CDPX patients, in the coding region of the arylsulfatase E (ARSE) gene, a novel member of the sulfatase gene family. Transfection of the ARSE full-length cDNA, in Cos7 cells, allowed us to establish that its protein product is a 60-kD precursor, which is subject to N-glycosylation, to give a mature 68-kD form that, unique among sulfatases, is localized to the Golgi apparatus. Five missense mutations found in CDPX patients were introduced into wild-type ARSE cDNA by site-directed mutagenesis. These mutants were transfected into Cos7 cells, and the arylsulfatase activity and biochemical properties were determined, to study the effect of these substitutions on the ARSE protein. One of the mutants behaves as the wild-type protein. All four of the other mutations resulted in a complete lack of arylsulfatase activity, although the substitutions do not appear to affect the stability and subcellular localization of the protein. The loss of activity due to these mutations confirms their involvement in the clinical phenotype and points to the importance of these residues in the correct folding of a catalytically active ARSE enzyme.     

Analysis of the pattern of QM expression during mouse development

QM, a novel gene that was originally identified as a putative tumor suppressor gene, has since been cloned from species encompassing members of the plant, animal, and fungal kingdoms. Sequence comparison indicates that QM has been highly conserved throughout eukaryotic evolution. QM is a member of a multigene family in both mouse and man, is expressed in a broad range of tissues, and is downregulated during adipocyte differentiation. Jif-1, a chicken homolog of QM, has been reported to interact with the protooncogene c-Jun, and to inhibit transactivation of AP-1 regulated promoters in vitro. Furthermore, disruption of the yeast QM homolog is lethal. Although these studies suggest that the QM gene product plays an important role within the normal cell, the precise role of QM has remained elusive. In this study, a thorough analysis of the pattern of QM expression during mouse development was undertaken, using the techniques of whole mount in situ hybridization and whole mount immunohistochemistry, in combination with conventional immunohistochemical analysis of tissue sections. QM is expressed in numerous embryonic tissues, and is differentially expressed throughout the embryo. The cytoplasmic localization of QM is consistent with its reported association with ribosomes, and inconsistent with its previously hypothesized function as a direct modulator of the nuclear protooncogene c-Jun. QM is expressed in the developing epidermis, and is particularly strong within developing limbs. Analysis of embryos of various stages of gestation indicate that QM is downregulated in the surface ectoderm of the embryo as development proceeds. QM protein is not detectable within either nucleated or enucleated red blood cell precursors. QM is strongly expressed within chondrocytes within the transition zone of developing limb cartilage, as well as within differentiated keratinocytes of the suprabasal regions of the epidermis. Furthermore, within both cartilage and skin, there is an inverse relationship between QM expression and proliferative capacity. This pattern of QM expression suggests that this novel gene product may be involved in processes such as posttranslational protein processing which are essential for differentiation of specific tissues during embryogenesis.     

Differential expression of the cellular src gene during vertebrate development

Cellular genes that are homologous to the transforming genes of certain RNA tumor viruses are suspected to play a functional role during normal developmental processes. To investigate this further, we are studying the expression of the cellular homolog of the Rous sarcoma virus transforming gene (c-src) during embryogenesis of fish, frog, and chicken by quantitative determination of the activity of the c-src encoded protein kinase (pp60^c-src). The kinase activity from embryos of fish, frog, and chicken displays the same enzymatic characteristics as the kinase from adult animals: It phosphorylates only tyrosine residues in protein substrates, and its activity is relatively insensitive to inhibition by the diadenosine nucleotide Ap4A. During the course of development, the varying kinase activity level reflects differential expression of the c-src gene product. The kinase activity is low during early development, increases dramatically during organogenesis, and decreases thereafter to the level found in adult animals. The kinase activity displays an organ specificity, with brain showing the highest activity in embryos as well as in adults. Muscle, however, shows high activities during organogenesis, but no or barely detectable activity in adult animals. Our data suggest, therefore, that the c-src gene product plays more of a role in differentiation than in proliferation processes during embryogenesis, and that it may act as a pleiotropic effector.     

Cloning and sequencing of the CRABP-I locus from chicken and pufferfish: analysis of the promoter regions in transgenic mice

Retinoic acid (RA), a derivative of vitamin A, is an important molecule for development and homeostasis of vertebrate organisms. The intracellular retinoic acid binding protein CRABP-I has a high affinity for RA, and is thought to be involved in the mechanism of RA signalling. CRABP-I is well conserved in evolution and shows a specific expression pattern during development, but mice made deficient for the protein by gene targeting appear normal. However, the high degree of homology with CRABP-I from other species indicates that the protein has been subject to strong selective conservation, indicative of an important biological function. In this paper we have compared the conservation in the expression pattern of the mouse, chicken and pufferfish CRABP-I genes to substantiate this argument further. First we cloned and sequenced genes and promoter regions of the CRABP-I genes from chicken and the Japanese pufferfish, Fugu rubripes. Sequence comparison with the mouse gene did not show any large blocks of homology in the promoter regions. Nevertheless, the promoter of the chicken gene directed expression to a subset of the tissues that show expression with the promoter from the mouse gene. The pattern observed with the pufferfish promoter is even more restricted, essentially to rhombomere 4 only, indicating that this region may be functionally the most important for CRABP-I expression in the developing embryo     

Genetic and physical mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) in chicken

The chicken natural resistance-associated macrophage protein 1 (NRAMP1) gene has been mapped by linkage analysis by use of a reference panel to develop the chicken molecular genetic linkage map and by fluorescence in situ hybridization. The chicken homolog of the murine Nramp1 gene was mapped to a linkage group located on Chromosome (Chr) 7q13, which includes three genes (CD28, NDUSF1, and EF1B) that have previously been mapped either to mouse Chr 1 or to human Chr 2q. Physical mapping by pulsed-field gel electrophoresis revealed that NRAMP1 is tightly linked to the villin gene and that the genomic organization (gene order and presence of CpG islands) of the chromosomal region carrying NRAMP1 is well conserved between the chicken and mammalian genomes. The regions on mouse Chr 1, human Chr 2q, and chicken Chr 7q that encompass NRAMP1 represent large conserved chromosomal segments between the mammalian and avian genomes. The chromosome mapping of the chicken NRAMP1 gene is a first step in determining its possible role in differential susceptibility to salmonellosis in this species.     

Chicken keratin-19: Cloning of cDNA and analysis of expression in the chicken embryonic gut

From many recent studies, it has been argued that keratins (cytokeratins) play important roles in the morphogenesis and differentiation of organ development. To learn the role of keratin in digestive tract development, a cDNA of the chicken homolog of keratin-19 ( GK-19 ) was cloned and its expression pattern was analyzed in the digestive tract of chicken embryos. The GK-19 full-length sequence was approximately 1.6 kb and showed more than 80% similarity to human and mouse keratin-19. The result of in situ hybridization with the proventriculus (glandular stomach) of different developmental stages showed that GK-19 expression disappeared specifically in the glandular epithelium from day 6 to day 9 of incubation. Furthermore, GK-19 was localized in the notochord, floor plate, anterior lobe of the pituitary gland and mesonephros. These results suggest the possibility that GK-19 may have multiple roles in organogenesis during embryogenesis.     

String of Pearls Encodes Drosophila Ribosomal Protein S2, Has Minute-like Characteristics, and Is Required during Oogenesis

The first allele of string of pearls (sop) was isolated as a recessive female sterile mutant in a P element enhancer trap screen. Oogenesis in homozygous sop females arrests at approximately stage 5. In addition, homozygous flies of both sexes have Minute-like characteristics that include reduced bristles, delayed development and larval lethality. sop maps to 30D/E on chromosome 2L and encodes the Drosophila homolog of eukaryotic ribosomal protein S2. The gene is present in a single copy in the Drosophila genome and the level of mRNA present in mutant animals is reduced. The identification of a mutant allele that blocks development at a mid-stage of oogenesis may indicate that sop has a specific developmental role during oogenesis in addition to its general role in protein synthesis as a component of the small ribosomal subunit.     

Regulated expression of the X. tropicalis connexin43 promoter

The spatio-temporal expression pattern of the connexin43 gene during Xenopus development has been described (Van der Heyden et al. 2001). To further investigate the regulation and function of connexin43 (Cx43) in amphibians, we have isolated the gene from Xenopus tropicalis (Xt) and determined its structure. The X. tropicalis Cx43 gene displays the typical two exon-one intron connexin configuration, where the first exon is non-coding. The predicted amino acid sequence of the XtCx43 protein is highly homologous to that of X. laevis, chicken and mammals. Expression of XtCx43 cDNA in N2A cells results in gap-junction plaque formation. Promoter activity of a 3.5 kb upstream region of the X. tropicalis Cx43 gene, including exon 1, mimics endogenous timing of expression after injection of reporter constructs in X. laevis embryos.     

Characterization of a Highly Conserved Human Homolog to the Chicken Neural Cell Surface Protein Bravo/Nr-CAM That Maps to Chromosome Band 7q31

The neuronal cell adhesion molecule Bravo/Nr-CAM is a cell surface protein of the immunoglobulin (Ig) superfamily and is closely related to the L1/NgCAM and neurofascin molecules, all of which contain six immunoglobulin domains, five fibronectin repeats, a transmembrane region, and an intracellular domain. Chicken Bravo/Nr-CAM has been shown to interact with other cell surface molecules of the Ig superfamily and has been implicated in specific pathfinding roles of axonal growth cones in the developing nervous system. We now report the characterization of cDNA clones encoding the human Bravo/Nr-CAM protein, which, like its chicken homolog, is composed of six V-like Ig domains and five fibronectin type III repeats. The human Bravo/Nr-CAM homolog also contains a transmembrane and intracellular domain, both of which are 100% conserved at the amino acid level compared to its chicken homolog. Overall, the human Bravo/Nr-CAM homolog is 82% identical to the chicken Bravo/Nr-CAM amino acid sequence. Independent cDNAs encoding four different isoforms were also identified, all of which contain alternatively spliced variants around the fifth fibronectin type III repeat, including one isoform that had been previously identified for chicken Bravo/Nr-CAM. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult human brain tissue. Fluorescencein situhybridization maps the gene for human Bravo/Nr-CAM to human chromosome 7q31.1–q31.2. This chromosomal locus has been previously identified as containing a tumor suppressor candidate gene commonly deleted in certain human cancer tissues.     

Structure and developmental expression of the chicken CDC2 kinase.

The cdc2 protein kinase plays a key role in controlling the eukaryotic cell cycle. We have isolated a cDNA clone for the chicken homolog of the cdc2 gene, raised antibodies against the corresponding protein, and studied the expression of cdc2 mRNA and protein during chicken embryonic development. The protein encoded by the chicken cdc2 cDNA shares extensive structural homology with cdc2 gene products from other species. Moreover, when expressed in fission yeast, the chicken cdc2 kinase is able to rescue a temperature-sensitive (ts) cdc2 mutant, demonstrating that it is functional as a cell cycle regulator. By Northern analysis and immunoblotting, we found that in total embryos both cdc2 mRNA and protein levels decreased substantially between day 3 and day 11 after egg laying, and no significant amounts of either cdc2 mRNA or protein were detected in adult liver, brain, heart or skeletal muscle. These data indicate the existence of a coarse correlation between the abundance of cdc2 mRNA and the proliferative state of a given tissue. Interestingly, however, when examining individual embryonic tissues, no correlation was observed between levels of cdc2 mRNA and protein, suggesting that cdc2 expression in developing chicken may be regulated at multiple levels.     

The nucleotide sequence and the tissue-specific expression of Drosophila c-src

We have examined the coding capability and expression of the Drosophila homolog of the vertebrate proto-oncogene c-src. Sequence analysis of a cDNA clone representing the Drosophila c-src locus suggests that the gene encodes a 62 kd protein that is remarkably similar to the protein product of chicken c-src. The Drosophila c-src locus is transcribed into three mRNAs that are each regulated independently during development. Drosophila c-src RNA is abundant in embryos and pupae but rare in larvae and adults. In situ hybridization reveals that after the first 8 hr of development, c-src RNA accumulates almost exclusively in neural tissues such as the brain, ventral nerve chord, and eye-antennal discs, and in differentiating smooth muscle. We conclude that c-src may not be a mitotic signal but instead may play a role in the development of neural tissue and smooth muscle.     

Enzymatic activity and in vivo distribution of 5'-nucleotidase, an extracellular matrix binding glycoprotein, during the development of chicken striated muscle.

The ecto-enzyme 5'-nucleotidase isolated from chicken gizzard has previously been shown to be a potent ligand of two glycoproteins of the extracellular matrix, namely fibronectin and laminin. Using immunofluorescent labeling techniques we observed that 5'-nucleotidase codistributed with laminin during the development of chicken striated muscle. In contrast, ecto-5'-nucleotidase was only faintly detectable on cells surrounded by a matrix expressing high levels of fibronectin. This distribution pattern distinguished 5'-nucleotidase from the pluripotent extracellular matrix receptors, chicken beta 1-integrins, which are expressed equally well in muscle and connective tissue. In addition, the specific activity of striated muscle ecto-5'-nucleotidase was stable during development and increased markedly posthatching. At each age considered, this specific activity corresponded to an 80-kDa enzyme which was inhibited by alpha,beta-methyleneadenosine diphosphate or by a monoclonal antibody directed against the smooth muscle isoform of the enzyme. Previous in vitro studies have revealed that 5'-nucleotidase is involved in the spreading of various mesenchyme-derived cells, such as chicken embryonic fibroblasts and myoblasts, on a laminin substrate. A prerequisite to examining a potential in vivo role for 5'-nucleotidase as an extracellular matrix ligand was to study its distribution. In adult muscle, 5'-nucleotidase displayed a more restricted distribution than in embryo. Results show that, in vivo, 5'-nucleotidase is revealed by immunofluorescent labeling using poly- and monoclonal antibodies to chicken gizzard 5'-nucleotidase in two structures, the costameres and myotendinous junctions, which are closely related to the focal adhesion sites observed in cell culture.     

Characterization of chicken Mda5 activity: regulation of IFN-β in the absence of RIG-I functionality

In mammals, Mda5 and RIG-I are members of the evolutionary conserved RIG-like helicase family that play critical roles in the outcome of RNA virus infections. Resolving influenza infection in mammals has been shown to require RIG-I; however, the apparent absence of a RIG-I homolog in chickens raises intriguing questions regarding how this species deals with influenza virus infection. Although chickens are able to resolve certain strains of influenza, they are highly susceptible to others, such as highly pathogenic avian influenza H5N1. Understanding RIG-like helicases in the chicken is of critical importance, especially for developing new therapeutics that may use these systems. With this in mind, we investigated the RIG-like helicase Mda5 in the chicken. We have identified a chicken Mda5 homolog (ChMda5) and assessed its functional activities that relate to antiviral responses. Like mammalian Mda5, ChMda5 expression is upregulated in response to dsRNA stimulation and following IFN activation of cells. Furthermore, RNA interference-mediated knockdown of ChMda5 showed that ChMda5 plays an important role in the IFN response of chicken cells to dsRNA. Intriguingly, although ChMda5 levels are highly upregulated during influenza infection, knockdown of ChMda5 expression does not appear to impact influenza proliferation. Collectively, although Mda5 is functionally active in the chicken, the absence of an apparent RIG-I-like function may contribute to the chicken's susceptibility to highly pathogenic influenza.     

FIP-2, an IkappaB-kinase-gamma-related protein,is associated with the Golgi apparatus and translocates to the marginal band during chicken erythroblast differentiation

Using an antiserum directed against marginal band associated proteins of chicken erythrocytes we isolated clones encoding the chicken homolog of 14.7K-interacting protein 2 (FIP-2), a protein potentially involved in tumor necrosis factor-alpha/nuclear factor-kappaB signaling, from a chicken erythroblast cDNA library. We found that chicken FIP-2 was expressed in a variety of tissues and cell types, but unlike its human counterpart, alternative splicing does not appear to take place. Analysis of intracellular localization revealed that FIP-2 was concentrated at the Golgi apparatus in most cells. Perturbation of the Golgi structure without loss of Golgi function (by treatment with nocodazole) resulted in a retention of FIP-2 at the dispersed Golgi fragments. In contrast, disruption of both Golgi structure and function (by brefeldin A) led to a loss of FIP-2 from Golgi membranes. Remarkably, during erythroblast differentiation FIP-2 was found to translocate from the Golgi to the marginal band. Our results support the hypothesis of a function of the Golgi apparatus in signal transduction. Moreover, our results raise the possibility that the marginal band of chicken erythrocytes, in addition to its role in morphogenesis, has a function in signal transduction and that FIP-2 is in some way involved in its formation.     

Molecular characterization of NbCHMP1 encoding a homolog of human CHMP1 in Nicotiana benthamiana

In humans, CHMP1 encodes a protein of dual function that plays a role in both modification of chromatin structure and endosomal vesicle trafficking. Recently, it was found that sal1, a CHMP1 homolog in maize, is important for the development of the aleurone cell layers in maize endosperm. In this study, we investigated the structure and function of a Nicotiana benthamiana CHMP1 homolog designated NbCHMP1. NbCHMP1 encodes a small protein with a bipartite nuclear localization signal at its N-terminus, and good homology with the corresponding genes from diverse plants and animals. NbCHMP1 mRNA was present at comparable levels in stems, roots, flowers, and leaves. A GFP fusion of the full length NbCHMP1 protein was localized to the cytosol in distinct structures, while a GFP fusion of its N-terminal 80 aa was targeted to the nucleus, suggesting dual-targeting of NbCHMP1 in plants. Overexpression of NbCHMP1 in yeast did not affect its growth and the expressed protein was present in the cytosol in particulate form. Virus-induced gene silencing of NbCHMP1 resulted in subtle alterations of leaf morphology and color, without significantly affecting plant viability or development. Thus the CHMP1 homolog apparently does not play an essential role in the development of the vegetative tissues of N. benthamiana, in contrast to the essential role of sail in formation of the aleurone cell layers during maize endosperm development.     

EGF-R as a hemopoietic growth factor receptor: the c-erbB product is present in chicken erythrocytic progenitors and controls their self-renewal.

c-erbB, encoding the EGF receptor (EGF-R), was originally identified as the cellular homolog of a chicken leukemia oncogene. In humans, EGF-R is distributed widely except in hemopoietic tissues, and its amplification is associated with epidermal and glial malignancies. Here we show that c-erbB is present in normal chicken erythrocytic progenitors and transmits the mitogenic signal induced by TGF alpha. Cells that contain high affinity EGF-R are at approximately the BFU-E stage, and their long-term renewal can be induced by TGF alpha. Upon addition of insulin and erythropoietin, they can be induced to terminally differentiate into red cells. We previously demonstrated that v-erbA blocks differentiation of chicken erythrocytic progenitors but does not abrogate their growth factor dependence for proliferation. These data indicate that proliferation and differentiation are not necessarily coupled in these cells. They also demonstrate a direct role of c-erbB in the control of self-renewal of normal chicken erythrocytic progenitors and could account for the predominant leukemogenic potential of the chicken erbB gene.