Identification and characterization of a RING zinc finger gene (C-RZF) expressed in chicken embryo cells.

To identify changes in gene expression that occur in chicken embryo brain (CEB) cells as a consequence of their binding to the extracellular matrix molecule cytotactin/tenascin (CT/TN), a subtractive hybridization cloning strategy was employed. One of the cDNA clones identified was predicted to encode 381 amino acids and although it did not resemble any known sequences in the nucleic acid or protein data bases, it did contain the sequence motif for the cysteine-rich C3HC4 type of zinc finger, also known as a RING-finger. This sequence was therefore designated the chicken-RING zinc finger (C-RZF). In addition to the RING-finger, the C-RZF sequence also contained motifs for a leucine zipper, a nuclear localization signal, and a stretch of acidic amino acids similar to the activation domains of some transcription factors. Southern analysis suggested that C-RZF is encoded by a single gene. Northern and in situ hybridization analyses of E8 chicken embryo tissues indicated that expression of the C-RZF gene was restricted primarily to brain and heart. Western analysis of the nuclear and cytoplasmic fractions of chicken embryo heart cells and immunofluorescent staining of chicken embryo cardiocytes with anti-C-RZF antibodies demonstrated that the C-RZF protein was present in the nucleus. The data suggest that we have identified another member of the RING-finger family of proteins whose expression in CEB cells may be affected by CT/TN and whose nuclear localization and sequence motifs predict a DNA-binding function in the nucleus.

The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds.

The putative actin-binding role of hydrophobic residues Trp546 and Phe547 in chicken gizzard heavy meromyosin.

In the course of myosin-catalyzed ATP hydrolysis, certain amino acid residues in myosin interact with counterparts in actin to produce the relational changes that underlie muscle contraction; some of these interactions are ionic, but the stronger interactions are hydrophobic. In an effort to identify myosin residues participating in hydrophobic interactions, myosin (from smooth muscle) fragments with mutations at suspected sites were engineered and compared with wild-type fragments. It was found that the ATPase of doubly mutated (Trp546Ser and Phe547His) fragments was minimally activated by actin and did not decorate actin well to form the regular arrowhead pattern characteristic of myosin binding to actin filaments. Thus, we suggest that Trp546 and Phe547 are important participants in the hydrophobic actin-myosin interaction.

The genetic link between the Chinese bamboo partridge (Bambusicola thoracica) and the chicken and junglefowls of the genus Gallus.

Further comparison of mitochondrial control-region DNA base sequences of 16 avian species belonging to the subfamily Phasianinae revealed the following: (i) Generalized perdicine birds (quails and partridges) are of ancient lineages. Even the closest pair, the common quail (Coturnix coturnix japonica) and the Chinese bamboo partridge (Bambusicola thoracica), maintained only 85.71% identity. (ii) The 12 species of phasianine birds previously and presently studied belonged to three distinct branches. The first branch was made exclusively of members of the genus Gallus, while the second branch was made of pheasants of the genera Phasianus, Chrysolophus, and Syrmaticus. Gallopheasants of the genus Lophura were distant cousins to these pheasants. The great argus (Argusianus argus) and peafowls of the genus Pavo constituted the third branch. The position of peacock-pheasants of the genus Polyplectron in the third branch was similar to that of the genus Lophura in the second branch. Members of the fourth phasianine branch, such as tragopans and monals, were not included in the present study. (iii) The one perdicine species, Bambusicola thoracica, was more closely related to phasianine genera Gallus and Pavo than to members of other perdicine genera. The above might indicate that Bambusicola belong to one-stem perdicine lineage that later splits into two sublineages that yielded phasianine birds, one evolving to Gallus, and the other differentiating toward Pavo and its allies.

Periodicity of strong nucleosome positioning sites around the chicken adult beta-globin gene may encode regularly spaced chromatin.

Positioned nucleosomes contribute to both the structure and the function of the chromatin fiber and can play a decisive role in controlling gene expression. We have mapped, at high resolution, the translational positions adopted by limiting amounts of core histone octamers reconstituted onto 4.4 kb of DNA comprising the entire chicken adult beta-globin gene, its enhancer, and flanking sequences. The octamer displays extensive variation in its affinity for different positioning sites, the range exhibited being about 2 orders of magnitude greater than that of the initial binding of the octamer. Strong positioning sites are located 5' and 3' of the globin gene and in the second intron but are absent from the coding regions. These sites exhibit a periodicity (approximately 200 bp) similar to the average spacing of nucleosomes on the inactive beta-globin gene in vivo, which could indicate their involvement in packaging the gene into higher-order chromatin structure. Overlapping, alternative octamer positioning sites commonly exhibit spacings of 20 and 40 bp, but not of 10 bp. These short-range periodicities could reflect features of the core particle structure contributing to the pronounced sequence-dependent manner in which the core histone octamer interacts with DNA.

The rotated hypoblast of the chicken embryo does not initiate an ectopic axis in the epiblast.

In the amniotes, two unique layers of cells, the epiblast and the hypoblast, constitute the embryo at the blastula stage. All the tissues of the adult will derive from the epiblast, whereas hypoblast cells will form extraembryonic yolk sac endoderm. During gastrulation, the endoderm and the mesoderm of the embryo arise from the primitive streak, which is an epiblast structure through which cells enter the interior. Previous investigations by others have led to the conclusion that the avian hypoblast, when rotated with regard to the epiblast, has inductive properties that can change the fate of competent cells in the epiblast to form an ectopic embryonic axis. Thus, it has been suggested that the hypoblast normally induces the epiblast to form a primitive streak at a specific locus. In the work reported here, an attempt was made to reexamine the issue of induction. In contrast to previous reports, it was found that the rotated hypoblast of the chicken embryo does not initiate formation of an ectopic axis in the epiblast. The embryonic axis always initiates and develops according to the basic polarity of the epiblast layer. These results provoke a reinterpretation of the issues of mesoderm induction and primitive streak initiation in the avian embryo.

Cloning and expression of the cDNA of chicken cation-independent mannose-6-phosphate receptor.

We cloned and sequenced the 8767-bp full-length cDNA for the chicken cation-independent mannose-6-phosphate receptor (CI-MPR), of interest because, unlike its mammalian homologs, it does not bind insulin-like growth factor II (IGF-II). The cDNA encodes a protein of 2470 aa that includes a putative signal sequence, an extracytoplasmic domain consisting of 15 homologous repeat sequences, a 23-residue transmembrane sequence, and a 161-residue cytoplasmic sequence. Overall, it shows 60% sequence identity with human and bovine CI-MPR homologs, and all but two of 122 cysteine residues are conserved. However, it shows much less homology in the N-terminal signal sequence, in repeat 11, which is proposed to contain the IGF-II-binding site in mammalian CI-MPR homologs, and in the 14-aa residue segment in the cytoplasmic sequence that has been proposed to mediate G-protein-coupled signal transduction in response to IGF-II binding by the human CI-MPR. Transient expression in COS-7 cells produced a functional CI-MPR which exhibited mannose-6-phosphate-inhibitable binding and mediated endocytosis of recombinant human beta-glucuronidase. Expression of the functional chicken CI-MPR in mice lacking the mammalian CI-MPR should clarify the controversy over the physiological role of the IGF-II-binding site in mammalian CI-MPR homologs.

Nonsteroidal antiinflammatory drugs cause apoptosis and induce cyclooxygenases in chicken embryo fibroblasts.

Programmed cell death (apoptosis) is an intrinsic part of organismal development and aging. Here we report that many nonsteroidal antiinflammatory drugs (NSAIDs) cause apoptosis when applied to v-src-transformed chicken embryo fibroblasts (CEFs). Cell death was characterized by morphological changes, the induction of tissue transglutaminase, and autodigestion of DNA. Dexamethasone, a repressor of cyclooxygenase (COX) 2, neither induced apoptosis nor altered the NSAID effect. Prostaglandin E2, the primary eicosanoid made by CEFs, also failed to inhibit apoptosis. Expression of the protooncogene bcl-2 is very low in CEFs and is not altered by NSAID treatment. In contrast, p20, a protein that may protect against apoptosis when fibroblasts enter G0 phase, was strongly repressed. The NSAID concentrations used here transiently inhibit COXs. Nevertheless, COX-1 and COX-2 mRNAs and COX-2 protein were induced. In some cell types, then, chronic NSAID treatment may lead to increased, rather than decreased, COX activity and, thus, exacerbate prostaglandin-mediated inflammatory effects. The COX-2 transcript is a partially spliced and nonfunctional form previously described. Thus, these findings suggest that COXs and their products play key roles in preventing apoptosis in CEFs and perhaps other cell types.

Transdifferentiation of chicken embryonic cells into muscle cells by the 3' untranslated region of muscle tropomyosin.

Transfection with a plasmid encoding the 3' untranslated region (3' UTR) of skeletal muscle tropomyosin induces chicken embryonic fibroblasts to express skeletal tropomyosin. Such cells become spindle shaped, fuse, and express titin, a marker of striated muscle differentiation. Skeletal muscle tropomyosin and titin organize in sarcomeric arrays. When the tropomyosin 3' UTR is expressed in osteoblasts, less skeletal muscle tropomyosin is expressed, and titin expression is delayed. Some transfected osteoblasts become spindle shaped but do not fuse nor organize these proteins into sarcomeres. Transfected cells expressing muscle tropomyosin organize muscle and nonmuscle isoforms into the same structures. Thus, the skeletal muscle tropomyosin 3' UTR induces transdifferentiation into a striated muscle phenotype in a cell-type-specific context.

Chicken double-stranded RNA adenosine deaminase has apparent specificity for Z-DNA.

A M(r) 140,000 protein has been purified from chicken lungs to apparent homogeneity. The protein binds with high affinity to a non-BNA conformation, which is most likely to the Z-DNA. The protein also has a binding site for double-stranded RNA (dsRNA). Peptide sequences from this protein show similarity to dsRNA adenosine deaminase, an enzyme that deaminates adenosine in dsRNA to form inosine. Assays for this enzyme confirm that dsRNA adenosine deaminase activity and Z-DNA binding are properties of the same molecule. The coupling of these two activities in a single molecule may indicate a distinctive mechanism of gene regulation that is, in part, dependent on DNA topology. As such, DNA topology, through its effects on the efficiency and extent of RNA editing may be important in the generation of new phenotypes during evolution.

Pax-6 and lens-specific transcription of the chicken delta 1-crystallin gene.

The abundance of delta-crystallin in the chicken eye lens provides an advantageous marker for tissue-specific gene expression during cellular differentiation. The lens-specific expression of the delta 1-crystallin gene is governed by an enhancer in the third intron, which binds a positive (delta EF2) and negative (delta EF1) factor in its core region. Here we show by DNase I footprinting, electrophoretic mobility-shift assays, and cotransfection experiments with the delta 1-promoter/enhancer fused to the chloramphenicol acetyltransferase reporter gene that the delta 1-crystallin enhancer has two adjacent functional Pax-6 binding sites. We also demonstrate by DNase I footprinting that the delta EF1 site can bind the transcription factor USF, raising the possibility that USF may cooperate with Pax-6 in activation of the chicken delta 1- and alpha A-crystallin genes. These data, coupled with our recent demonstration that Pax-6 activates the alpha A-crystallin gene, suggest that Pax-6 may have been used extensively throughout evolution to recruit and express crystallin genes in the lens.

Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family.

Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs.

In Vivo screening and discovery of novel candidate thalidomide analogs in the zebrafish embryo and chicken embryo model systems

This study was supported by a Wellcome Trust-NIH PhD Studentship to SB, WDF and NV. Grant number 098252/Z/12/Z. SB, CHC and WDF are supported by the Intramural Research Program, NCI, NIH. NHG and WL are supported by the Intramural Research Program, NIA, NIH.