A new heparin binding protein regulated by retinoic acid from chick embryo

A 19 KDa heparin binding protein was previously purified from chicken embryos. Essentially localized within basement membranes in early embryonic tissues, this protein is very rich in basic and cystein residues. Its N-terminal fragment is similar to corresponding fragment of two other proteins expressed during embryogenesis and postnatal period. Its synthesis and secretion are induced by retinoic acid in chicken myoblasts and fibroblasts. This new retinoic acid induced heparin binding protein (RI-HB) does stimulate neurite outgrowth and proliferation on PC12 cells. These results suggest that retinoic acid could regulate some aspect of differentiation and development by inducing the synthesis of a new family of growth and neurotrophic factors.     

A new family of proteins with high activity for heparin and regulated by retinoic acid]

An heparin binding protein (RIHB) was purified from chick embryos. Essentially expressed during early embryogenesis it is mainly localized within basement membranes. Its synthesis and that of the RIHB mRNA are induced by retinoic acid in chicken myoblasts cell culture. This protein belongs to the same family that HBGAM or Pleiotropin and MK protein two other heparin binding proteins exhibiting growth and/or neurotrophic activities.     

The octamer binding factor Oct6: cDNA cloning and expression in early embryonic cells.

We have cloned a cDNA encoding a novel octamer binding factor Oct6 that is expressed in undifferentiated ES cells. Expression of the Oct6 gene is downregulated upon differentiation of these cells by aggregate formation. Furthermore the gene is transiently up regulated during retinoic acid induced differentiation of P19 EC cells, reaching maximum levels of expression one day after RA addition. Sequence analysis of the cDNA encoding the Oct6 protein indicated that the Oct6 gene is a member of the POU-HOMEO domain gene family. The gene expresses a 3 kb mRNA encoding a 449 amino acid protein with an apparent molecular weight of 45 kD. The sequence of the Oct6 POU domain is identical to that of the rat SCIP (Tst-1) gene. The Oct6 expression pattern suggests a role for this DNA binding protein in neurogenesis as well as early embryogenesis.     

The cellular retinoic acid binding proteins

The two cellular retinoic acid binding proteins, CRABP I and CRABP II, belong to a family of small cytosolic lipid binding proteins and are highly conserved during evolution. Both proteins are expressed during embryogenesis, particularly in the developing nervous system, craniofacial region and limb bud. CRABP I is also expressed in several adult tissues, however, in contrast, CRABP II expression appears to be limited to the skin. It is likely that these proteins serve as regulators in the transport and metabolism of retinoic acid in the developing embryo and throughout adult life. It has been proposed that CRABP I sequesters retinoic acid in the cytoplasm and prevents nuclear uptake of retinoic acid. A role in catabolism of retinoic acid has also been proposed. Recent gene targeting experiments have shown that neither of the two CRABPs are essential for normal embryonic development or adult life. Examination of CRABP I expression at subcellular resolution reveals a differential cytoplasmic and/or nuclear localization of the protein. A regulated nuclear uptake of CRABP I implies a role for this protein in the intracellular transport of retinoic acid. A protein mediated mechanism which controls the nuclear uptake of retinoic acid may play an important role in the transactivation of the nuclear retinoic acid receptors.     

A new family of heparin-binding factors: strong conservation of midkine (MK) sequences between the human and the mouse

A retinoic acid responsive gene, MK, specifies for a heparin binding factor termed midkine (MK), which is the initial member of a new protein family involved in regulation of growth and differentiation. A cDNA clone of human MK was isolated from a fetal kidney cDNA library. Human MK mRNA was expressed in PA1 teratocarcinoma cells as well as in the kidney. Sequence analysis of the cDNA clone and of a part of the genomic clone yielded the predicted protein sequence of human MK. Human and mouse MK sequences are highly conserved: 87% of amino acids are identical and all amino acid changes are conservative except for an insertion. Comparison of MK and HB-GAM/pleiotrophin (another member of the family) from various species revealed sequences conserved in the family and those specific for each protein.     

Characterization and expression of two chicken cDNAs encoding ubiquitin fused to ribosomal proteins of 52 and 80 amino acids

We have determined the complete nucleotide sequence of two chicken cDNAs, Ub-t52 and Ub-t80, encoding ubiquitin fused to ribosomal proteins of 52 and 80 amino acids. The deduced amino acid sequences of the ribosomal proteins are identical or very similar to the homologous human and rat proteins and to the corresponding proteins of other species. Unexpectedly, the ubiquitin moiety of the Ub-t52 protein showed two amino acid substitutions: serine-20 has been replaced by asparagine and serine-57 by alanine. Ubiquitin is a protein strongly conserved during evolution, with no changes in sequence previously reported in vertebrates. Ub-t52 and Ub-t80 are highly expressed in early embryogenesis and during postmitotic stages of spermatogenesis, in parallel with the expression of the polyubiquitin gene UbII. Whereas the 5′ untranslated regions (5′UTRs) of the chicken polyubiquitin mRNAs showed marked differences in mature testes in relation to somatic tissues, no differences were observed in the 5′UTRs of the ubiquitin-ribosomal protein mRNAs. These mRNAs possess a 5′-terminal oligopyrimidine tract that could be used as a mechanism to postpone translation during postmitotic stages of spermatogenesis, as has been proposed in quiescent cells.     

Craniofacial abnormalities induced by ectopic expression of the homeobox gene Hox-1.1 in transgenic mice

Hox-1.1 is a murine homeobox-containing gene expressed in a time- and cell-specific manner during embryogenesis. We have generated transgenic mice that ectopically express Hox-1.1 from the chicken beta-actin promoter. In these mice Hox-1.1 expression was changed to an almost ubiquitous pattern. Ectopic expression of Hox-1.1 leads to death of the transgenic animals shortly after birth and is associated with multiple craniofacial anomalies, such as cleft palate, open eyes at birth, and nonfused pinnae. This phenotype is similar to the effects seen after systemic administration of retinoic acid during gestation. This suggests that retinoic acid embryopathy and the specific developmental defects caused by ectopic expression of a potential developmental control gene share a common pathogenic mechanism.     

Unravelling of physiological functions of retinoids using a dominant-negative retinoic acid receptor

We have constructed dominant-negative retinoic acid receptors by substituting a single amino acid which has been found in a dominant-negative thyroid hormone receptor, and have expressed the dominant-negative retinoic acid receptors in the epidermis, a potential target organ of retinoic acid. The resultant transgenic mice exhibited dramatic suppression of epidermal development, demonstrating the absolute requirement of retionic acid in normal skin development. This novel method, targeted expression of the dominant-negative receptor, is theoretically applicable to any organ, thus opening the way to defining the physiological roles of retionic acid as well as other lipophilic hormones during embryogenesis as well as in adults     

鸡脂肪酸结合蛋白基因的克隆和测序分析

根据哺乳动物脂肪酸结合蛋白基因序列设计一对引物对鸡基因组进行PCR扩增,将163bp扩增片段进行克隆和测序,并与猪的脂肪酸结合蛋白（fatty acid binding protein,FABP）基因序列进行同源性比较。该基因因片段与猪的心脏脂肪酸结合蛋白（heart fatty acid binding protein,H-FABP）基因有68%的同源性,与猪的脂肪型脂肪酸结合蛋白（adipocyte fatty acid binding protein,A-FABP）基因有75%的同源性,演绎成氨基酸之后与猪的脂肪型脂肪酸结合蛋白相应的氨基酸有75%的同源性。Northern结果表明该基因只在脂肪组织中表达。     

Cloning and characterization of chicken adipocyte fatty acid binding protein gene

Fatty acid binding proteins (FABPs) are members of a superfamily of lipid-binding proteins and occur intracellularly in vertebrates and invertebrates. This study was designed to clone and characterize the adipocyte fatty acid binding protein (A-FABP) gene in the chicken. PCR primers were designed according to mammalian A-FABP gene sequence to amplify partial cDNA of A-FABP gene from chicken adipose tissues, and the full length of the gene was cloned by 5'RACE and 3'RACE. Analysis of sequence showed that the cDNA of the chicken A-FABP gene was 74 and 73% homologous with porcine and human A-FABP gene, respectively. The similarity was 77, 28, and 23% at the predicted amino acid level with human A-FABP, human L-FABP, and human I-FABP, respectively. RT-PCR and Northern blot analysis indicated that the chicken A-FABP gene, similar to that of the mammal, is only expressed in fat tissues. This is the first report to identify and characterize A-FABP gene in the chicken.     

Cloning and expression of the chicken immunoglobulin joining (J)-chain cDNA

 The J chain is a component of polymeric immunoglobulin (Ig) molecules and may play an important role in their polymerization and the transport of polymeric Ig across epithelial cells. In this study, the primary structure of the chicken J chain was determined by sequencing cDNA clones. The cDNA had an open reading frame of 476 nucleotides encoding a putative protein of 158 amino acid residues including the signal sequence. The 3′ untranslated region consisted of 1216 nucleotides and a poly(A) tail. The deduced amino acid sequence of the chicken J chain had a high degree of homology to that of human, cow, rabbit, mouse, frog, and earthworm, with eight conserved Cys residues identical to the mammalian J chains. Northern blot hybridization performed with total RNA from various chicken tissues revealed high levels of J-chain mRNA expression in spleen, intestine, Harderian gland, and bursa of Fabricius, and low levels in the thymus. The J chain was expressed in the bursa as early as day 15 of embryogenesis. These data indicated that the chicken J-chain gene displays a high degree of homology with that of other species, and is expressed at an early stage of development of the chicken immune system. Received: 21 May 1999 / Revised: 30 August 1999     

Mutating the charged residues in the binding pocket of cellular retinoic acid-binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability.

Three-dimensional modeling of the complex between retinoic acid-binding protein (CRABP) and retinoic acid suggests that binding of the ligand is mediated by interaction between the carboxyl group of retinoic acid and two charged amino acids (Arg-111 and Arg-131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein-ligand interaction, amino acid substitutions were made by oligonucleotide-directed, site-specific mutagenesis. The wild-type and mutant proteins were expressed in E. coli and subsequently purified. Like wild-type CRABP, the mutant proteins are composed mainly of beta-strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild-type protein. Mutants in which Arg-111 and Arg-131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild-type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild-type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP-retinoic acid complex. These data suggest a model for CRABP-retinoic acid interaction in which the repulsive forces between the positively-charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively-charged pair of amino acids produces a protein that is more thermostable than wild-type CRABP but less effective at ligand-binding.     

Purification and crystallization of a retinoic acid-binding protein from rat epididymis. Identity with the major androgen-dependent epididymal proteins

The retinoic acid binding activity in the lumen of the rat epididymis (Ong, D., and Chytil, F. (1988) Arch. Biochem. Biophys. 267, 474-478) has been purified to homogeneity. The protein exists in two forms, one form having an additional three amino acids at the amino terminus. The amino acid sequence of the protein was determined to 20 amino acids and proved to be identical to that of the major androgen-dependent proteins from rat epididymis as deduced from the cDNA sequence. These proteins are thought to play a role in sperm maturation, perhaps, it can be suggested now, by delivering retinoic acid to the sperm. The retinoic acid-binding protein has sequence homology to the serum retinol-binding protein and is predicted to have the same overall fold of the polypeptide chain. The epididymal retinoic acid-binding protein has been crystallized from 39 to 43% saturated ammonium sulfate, 10 mm Tris, pH 8.0. The crystals are space group P2(1), with a = 39.4, b = 58.9, c = 65.4 a, beta = 105 degrees 16 min.     

Preferential expression of cellular retinoic acid binding protein in a subpopulation of neural cells in the developing mouse embryo

The cellular retinoic acid binding protein is thought to be involved in the retinoic-acid-mediated signal transduction pathway. We have isolated the mouse cellular retinoic acid binding protein cDNA from an embryonal-carcinoma-derived cell line by using differential cDNA cloning strategies. In situ hybridization on sections of mouse embryos of various developmental stages indicated that the cellular retinoic acid binding protein gene, which we localized on mouse chromosome 9, is preferentially expressed in a subpopulation of neurectodermal cells. This restricted expression pattern suggests an important role for cellular retinoic acid binding protein in murine neurogenesis.     

The complete amino acid sequence of the cellular retinoic acid-binding protein from bovine retina

The complete amino acid sequence of cellular retinoic acid-binding protein from bovine retina was obtained by Edman degradation of peptides obtained from enzymatic and CNBr digests. The 136 residue sequence is identical to that reported recently for the protein from bovine adrenal tissue indicating that the same gene is expressed in both tissues.