Structure and expression of the chicken calmodulin I gene

The chicken calmodulin I (CaMI) gene has been isolated and characterized on the level of cDNA and genomic DNA. The deduced amino acid (aa) sequence is identical to the one of chicken CaMII which consists of 148 aa. The CaMI gene contains six exons. Its intron/exon organization is identical to that of the chicken CaMII and the CaMI and CaMIII genes of rat and human. Expression of the CaMI gene was detected in all chicken tissues examined, although at varying levels. The gene is transcribed into four mRNAs of 0.8, 1.4, 1.7 and 4.4 kb as determined by Northern blot analysis. Our results demonstrate that the “multigene-one-protein” principle of CaM synthesis is not only applicable to mammals whose CaM is encoded by three different genes, but also to chickens.     

Structure and expression of the chicken beta nerve growth factor gene.

The 3' exon of the chicken beta nerve growth factor (NGF) gene was isolated by the use of a murine cDNA probe. DNA sequence analysis of the clone suggests a mature chicken NGF protein of 118 amino acids, showing approximately 85% homology to mouse and human NGF. In addition to this conservation of the mature NGF, parts of the propeptide and the untranslated 3' end of the NGF gene are also highly homologous in chicken, human and mouse. Therefore, these sequences probably subserve important functions. Expression of NGF mRNA in various chicken tissues was examined by RNA blot analysis with a chicken NGF probe. A single mRNA of 1.3 kb was detected at high levels in heart and brain of 10-week-old roosters, and, at lower levels in spleen, liver and skeletal muscle. These data suggest a correlation between NGF expression and the density of sympathetic innervation in peripheral organs, in analogy with findings for mammalian tissues. In the adult avian brain, NGF mRNA is found at higher concentration in the optic tectum and cerebellum than in the cortex and hippocampus. This pattern of NGF expression differs from that previously described for the rat brain. During late stages of development (day 18), NGF mRNA was expressed both in heart and brain of embryos but at lower levels than in the adult.     

Organization and expression of the chicken N-myc gene.

We cloned the chicken N-myc gene and analyzed its structure and expression. We found that it consisted of three exons with coding regions in exons 2 and 3. Comparison to mammalian N-myc genomic sequence indicated that nucleotide sequences of the 5'-flanking region, noncoding exon 1, and introns were not conserved, but coding and 3' noncoding sequences showed significant homology to mammalian N-myc. Alignment of deduced amino acid sequences of chicken and mammalian N-myc proteins revealed nine conserved domains interrupted by different lengths of nonhomologous sequences. Two of the domains were specific to N-myc proteins, and the other seven were common to c-myc proteins. Northern blot (immunoblot) and in situ hybridization analyses of 3.5-day-old chicken embryos revealed that high-level expression of the N-myc gene was confirmed to certain tissues, e.g., the central nervous system, neural crest derivatives, and mesenchyme of limb buds. In the beak and limb primordia, N-myc expression in the mesenchyme was higher toward the distal end, suggesting possible involvement in positional assignment of the tissue within the rudimentary structures.     

Structure and expression of a chicken gene coding for U1 RNA

We have isolated and sequenced a genomic fragment containing sequences complementary to chicken U1 RNA. The sequence of this genomic U1 gene is completely homologous and colinear with that of chicken U1 RNA. This U1 gene is part of a multigene family (6--10 copies per haploid genome), and these loci do not appear to be closely clustered. Sequences complementary to other snRNAs are not present within the 2.5 kb genomic fragment containing the U1 gene. We have determined that U1 RNA is synthesized by polymerase II; however, a "Hogness box" is not present upstream from its cap site at the position usually observed for mRNA genes. The synthesis of U1 RNA in oviduct nuclei during different states of hormonal induction also appears to be constitutive.     

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.     

Sequence and expression of the chicken calcitonin gene

The avian calcitonin gene was isolated and sequenced; two mRNAs are expressed by tissue-specific alternate splicing. The peptides encoded by the mRNAs are the protein precursors of either calcitonin or calcitonin gene-related peptide (CGRP). Calcitonin is expressed predominantly in ultimobranchial bodies and CGRP in brain.     

The chicken stathmin gene and its expression in the embryo.

Stathmin, which functions as an intracellular relay in signal transduction pathways, has been suggested as a potential indicator of pluripotent cells in the early mouse embryo. In this study, chicken stathmin cDNA and genomic DNA were analyzed. In mammals stathmin consists of five exons and four introns; exons 3, 4, and 5 in the mammalian stathmin gene are equivalent to one relatively large exon in the chicken stathmin gene. Introns equivalent to introns 3 and 4 in the mammalian stathmin gene are not present in the counterpart gene in chickens and, although intron 2 was shown to be present in both mammals and birds, it is smaller in the chicken stathmin gene. Despite differences in the genomic organization of the gene and its smaller size in chickens compared with that in humans and mice, similarities in the coding sequences and in the expression of the chicken and mouse stathmin genes at certain stages of embryo development, as determined by whole-mount in situ hybridization experiments, suggest that their products are functional homologues. The argument is thus substantiated for further investigations into the use of regulatory regions of the stathmin gene in a system for the establishment of long-term cultures of germline competent chicken embryonic stem (ES) cells by the selective ablation of differentiated cells in culture using drug selection.     

Structure and developmental expression of the chicken NGF receptor

The nucleotide and deduced amino acid sequence of a cDNA clone of the chicken NGF receptor (NGFR) is reported and is compared with sequences of mammalian NGF receptors. A model is presented in which monodentate or bidentate binding of NGF dimers to repeated cysteine-rich sequence elements of the receptor yields low- or high-affinity NGF binding, respectively. In situ hybridization is used to characterize expression of NGFR in developing chick from 40 hr to 10 days of embryogenesis. NGFR mRNA expression is detected in premigratory neural crest cells, in epibranchial placode cells, and in all sensory, sympathetic and parasympathetic derivatives of these structures. In the embryonic CNS, NGFR mRNA is detected in the mantle zone but not the periventricular germinal zone throughout most of the neural tube. By Embryonic Day 8, NGFR mRNA is detected in a substantial fraction of cells in every brain region, with highest levels present in developing motor neurons. NGFR mRNA also is transiently expressed in many mesenchymal cell populations including cells in branchial arch, sclerotome, muscle anlagen, and feather follicles. The functional significance of wide-spread embryonic expression of the NGF receptor is discussed.