The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.     

Cloning and characterisation of the abundant cytoplasmic 7S RNA from mouse cells.

A cDNA library has been prepared from mouse embryo small RNAs and screened for the presence of clones complementary to the highly abundant cytoplasmic 7S RNA. One clone (pA6) was selected which hybridized exclusively with 7S RNA on a Northern blot prepared from cytoplasmic RNA run on high resolution polyacrylamide/urea gels. Sequence analysis of this clone has shown that at least 65 nucleotides at the 5' end of 7S RNA are extensively homologous with the highly repeated mouse B1 family. Heterologous hybridisations between the cloned mouse 7S sequence and RNAs prepared from rat, human and chick cells have shown that the non-B1 part of the 7S RNA molecule has been highly conserved during recent eucaryotic evolution. There are multiple copies of 7S RNA genes in the genomes of mouse, human, rat and chick cells, but substantial differences exist in copy number and genomic organisation in these organisms.     

Regulation of three beta-tubulin mRNAs during rat brain development.

The nucleotide sequence of a complete rat brain beta-tubulin T beta 15 has been determined from three overlapping cDNA clones. The overall length of the T beta 15 sequence is 1589 bp and shows between 84.5% and 88.6% homology within the coding region as compared with chick and human beta-tubulin sequences. On the other hand, the 3'-non-coding region is highly divergent. Comparison of the derived amino acid sequences from different species demonstrates that the amino acid changes are not randomly distributed, but rather there are several conserved and two highly variable regions common to beta-tubulin polypeptides from various sources. The T beta 15 sequence encodes a dominant neuronal 1.8-kb beta-tubulin mRNA species. Two other minor beta-tubulin mRNA species of 2.6 and 2.9 kb are present in rat brain. By using two synthetic oligonucleotide probes complementary to the carboxyl-terminal divergent region and to the amino-terminal conserved region, we have shown that the three mRNAs are distinct species, which are developmentally regulated. The level of the 1.8-kb mRNA species increases till the age of 12 days thereafter its level decreases. The 2.9-kb mRNA is an early neuronal mRNA species, while the 2.6-kb mRNA is a late neuronal species which is detected at 30 days of rat brain development. The data illustrate that there is a differential expression of the beta-tubulin multigene family during rat brain development which may suggest different functions for the various beta-tubulin isotopes.     

Chick integrin alpha V subunit molecular analysis reveals high conservation of structural domains and association with multiple beta subunits in embryo fibroblasts.

We have cloned and characterized a chick homologue of the human vitronectin receptor alpha subunit (alpha v) whose primary sequence is 83% identical with its human counterpart but less than 40% identical with any other known integrin alpha subunit. Comparison of the chick and human sequences reveals several highly conserved regions, including the cytoplasmic domain. The putative ligand binding domain contains alpha v-specific residues that may contribute to ligand binding specificity. These are concentrated in three regions that are located before and between the first three Ca2+ binding domains. Polyclonal antibodies raised against two peptides deduced from the putative cytoplasmic and extracellular domains of the chick alpha v sequence recognize specifically integrin heterodimers in chick embryo fibroblasts. At least three putative beta subunits coimmunoprecipitate with the chick alpha v subunit. In addition to a protein with the same molecular weight as beta 3 (94K), protein bands of Mr 84K and 110K are also coprecipitated. By successive immunodepletions, we demonstrate that this latter Mr 110K subunit is beta 1, which appears to be one of the alpha v-associated subunits in chick embryo fibroblasts.     

Cloning and sequencing of the cDNA encoding tyrosinase of the Japanese pond frog, Rana nigromaculata.

We cloned and sequenced the cDNA encoding tyrosinase (TYN) of the Japanese pond frog, Rana nigromaculata. The 3511-bp cDNA contained a 54-bp 5'-noncoding region, a 1596-bp open reading frame encoding TYN of 532 amino acids (aa), and a 1861-bp 3'-noncoding region. The aa sequence of frog TYN predicted from the cDNA sequence was homologous to that of mouse and human TYNs. The aa sequence including the copper-binding domain, which is likely the active center of TYN, was highly conserved among these three species and Neurospora crassa, Streptomyces antibioticus, and S. glaucescens. The frog TYN also contains possible glycosylation sites and conserved Cys at sites similar to those in the mouse and human TYNs. There are two hydrophobic regions at the N-terminus and near the C-terminus, which are likely the signal (leader) peptide and a transmembrane domain, respectively.     

A uniquely conserved regulatory signal is found around the translation initiation site in three different collagen genes

We have determined the nucleotide sequence of the 5' untranslated region and the sequence encoding the signal peptide for mRNAs of the chick alpha 1 type I and alpha 1 type III collagen. These sequences were obtained by synthesizing the corresponding cDNAs using as primers either a synthetic oligonucleotide to prime alpha 1 type I cDNA or a DNA fragment isolated from a genomic clone coding for alpha 1 type III collagen to prime the cognate cDNA. Both primers were selected so that the resulting cDNAs would be short and would contain sequence information for the 5' untranslated region and the signal peptide of the proteins. The nucleotide sequences of these cDNAs were compared with the corresponding sequence of alpha 2 type I collagen. In each mRNA the 5' untranslated segment is approximately 130 nucleotides and contains two or more AUG triplets preceding the AUG which serves as a translation initiation codon. A sequence of about 50 nucleotides surrounding the translation initiation codon is remarkably conserved in all three mRNAs, whereas the sequences preceding and following this segment diverge markedly. This homologous sequence contains an almost identical inverted repeat sequence which could form a stable stem-loop structure. The initiation codon and the AUG which precedes it are found at the same place within this symmetrical sequence and the distance between them is invariant. The rest of the conserved sequence shows a less perfect symmetry. This conserved sequence has not been found in other genes. Our data suggest that these three and perhaps other collagen genes contain an identical regulatory signal that may play a role in determining the level of expression of these genes by modulating translational efficiency.     

The molecular cloning and characterization of potential chick DM-GRASP homologs in zebrafish and mouse

A full-length zebrafish cDNA clone and a partial mouse cDNA clone similar to chick DM-GRASPwere isolated and analyzed. The nucleotide sequence of the full-length zebrafish clone shares 54% identity, and predicts 39% amino acid identity, with chick DM-GRASP. The partial mouse clone shares 76% nucleotide identity, and predicts 76% amino acid identity, with chick DM-GRASP. The predicted proteins encoded by both of these clones exhibit conserved structural domains that are characteristic of the chick protein. These features may identify them as a distinct subfamily within the immunoglobulin superfamily of cell adhesion molecules. Express of the zebrafish DM-GRASP protein is similar to chick DM-GRASP and is principally restricted to a small subset of developing sensory and motor neurons during axonogenesis. Zebrafish DM-GRASP expression was temporally regulated and limited to specific axon domains. This regional expression correlated with fasciculated axon domains. These results suggest that the zebrafish and mouse cDNA clones represent the respective fish and mammalian homologs of thick DM-GRASP. The highly selective expression of zebrafish DM-GRASP suggests that it is involved in the selective fasciculation and guidance of axons along their normal pathways. 1994 John Wiley & Sons, Inc.     

cDNA cloning, characterization and chromosome mapping of Crtap encoding the mouse cartilage associated protein.

Recently we have isolated and characterized a cDNA coding for a novel developmentally regulated chick embryo protein, cartilage associated protein (CASP). Here we describe the isolation and characterization of the cDNAs coding for the mouse CASP. Comparison of the mammalian putative protein sequence with the chick sequence shows a very high identity overall (51%); in particular the chick protein is homologous to the half amino terminus of the mouse protein. Furthermore, the comparison of the CASP cDNA sequence with sequences of the genebank database confirms our hypothesis that the CASP genes belong to a novel family that also includes genes encoding for some nuclear antigens. In all mouse tissues examined three CASP mRNAs species are detected, whereas in chick tissues a single mRNA is present. Immunohistochemistry studies show that the protein is expressed in all mouse embryonic cartilages. The mouse cartilage associated protein gene (Crtap) was assigned to chromosome 9F3-F4 by fluorescence in situ hybridization.     

cDNA to chick lumican (corneal keratan sulfate proteoglycan) reveals homology to the small interstitial proteoglycan gene family and expression in muscle and intestine.

A 1.9-kb cDNA clone to chick lumican (keratan sulfate proteoglycan) was isolated by screening an expressing vector library made from chick corneal RNA with antiserum to chick corneal lumican. The cDNA clone contained an open reading frame coding for a 343-amino acid protein, Mr = 38,640. Structural features of the deduced sequence include: a 18-amino acid signal peptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 62% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I, where X represents any amino acid. Lumican contains three variations of this sequence that are tandemly linked to form a unit and three units tandemly linked to form the leucine-rich region. The sequential arrangement of these repeats and their spacing suggest that this region arose by duplication. The deduced sequence shows five potential N-linked glycosylation sites, four of which are in the leucine-rich region. These sites are also potential keratan sulfate attachment sites. The cDNA clone to lumican hybridizes to a 2.0-kb mRNA found in tissues other than cornea, predominantly muscle and intestine. Radiolabeling and immunoprecipitation studies show that lumican core protein is also synthesized by these tissues. The primary structure of lumican is similar to fibromodulin, decorin, and biglycan, which indicates it belongs to the small interstitial proteoglycan gene family. The expression of lumican in tissues other than cornea indicates a broader role for lumican besides contributing to corneal transparency.     

Glyceraldehyde 3-phosphate dehydrogenase protein and mRNA are both differentially expressed in adult chickens but not chick embryos

We have determined the 679 nucleotide sequence of a cDNA clone which, by hybridization-translation experiments, corresponds to a 36K chick brain protein. Our studies provide a partial amino acid sequence for this protein, identifying it as chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antisera raised against purified chicken GAPDH reacted with a 36K protein present in chick brain extracts and estimated to be the fourth most prevalent protein, as determined by either Coomassie Blue staining or by in vitro translation of chick brain mRNA. The amounts of GAPDH mRNA in chick brain, liver and muscle and adult chicken brain are similar, whereas the relative amount of adult chicken muscle GPDH mRNA is greatly elevated and that of adult liver lowered. The GAPDH protein levels showed a similar variation between tissues, suggesting that the levels of GAPDH protein are largely regulated by the amount of available GAPDH mRNA. The chicken GAPDH clone does not hybridize to rat mRNA, even though GAPDH is one of the most evolutionarily conserved proteins, indicating that selection pressures are heavier at the primary protein sequence level than at the nucleic acid sequence level for this gene, a situation contrasting to that of the tubulins.     

The complete sequence and expression patterns of the atrial myosin heavy chain in the developing chick

We have earlier reported partial cloning of a cDNA of a chick atrial myosin heavy chain (MHC) gene, CCSV2 and its expression pattern in embryonic chick hearts (Oana et al (1995) Eur J Cell Biol 67, 42-49). In this study, five overlapping cDNA clones (including CCSV2) which together encode the entire open reading frame of the chick atrial MHC gene were characterized, and both the entire nucleotide sequence consisting of 5825 bases and the deduced amino acid sequence consisting of 1931 amino acids determined. Reinvestigation of the nucleotide sequence of the previously reported and presumably different chick atrial specific MHC cDNA clone, AMHC1 (Yutzey et al (1994) Development 120, 871-883), revealed that our clone and AMHC1 encoded the same MHC. The chick atrial MHC gene was strongly expressed in developing chick atria from a very early stage (Hamburger and Hamilton stage 9, 29-33 h) to the adult stage. This gene was also expressed, although weakly, in the ventricle, somite (the precursor to skeletal muscle) and skeletal muscle during embryonic stages but not in adults.     

The structure and expression of a gene encoding chick claw keratin.

A cDNA library was constructed from embryonic chick claw mRNA and a claw keratin (cKer)-encoding clone was isolated and sequenced. Subsequently, a genomic clone, containing four cKer-encoding genes (cKer) was isolated and one of the genes (cKer1) was completely sequenced. The cKerl gene appears to be differentially expressed in the keratinizing tissue appendages of the embryonic chick, being abundantly expressed in the claw and at a low level in feather tissue. Comparison of the deduced amino acid (aa) sequence of the cKer to those of feather (fKer) and scale keratins (sKer) showed that the regions conserved between fKer and sKer are also found in the cKer. The glycine-rich as repeat region characteristic of sKer is also present in a shortened form in the cKer sequence. Like the fKer genes (fKer) and the feather histidine-rich protein-encoding gene (HRP), the cKer1 gene also contains one intron which interrupts the 5'-noncoding region at an equivalent position to that found in the fKer and HRP genes. Genomic Southern analysis using the cKer cDNA as a probe indicated the presence of several related genes in the chick genome.     

三种鳞翅目夜蛾科昆虫α-微管蛋白基因的克隆与mRNA表达水平

根据昆虫微管蛋白的分子特征筛选对昆虫微管有效的抑制剂来控制昆虫的生长发育或不同器官的有效功能的表达来达到控制害虫的目的,在未来的害虫综合治理中具有广泛的应用前景。以预蛹期甜菜夜蛾Spodoptera exigua、小地老虎Agrotis ypsilon和八字地老虎Agrotis c-nigrum为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),分别扩增得到以上3种夜蛾科昆虫的α-微管蛋白基因的cDNA序列,3种昆虫的该基因序列均包括1个1353个碱基的开放阅读框。这3个cDNA序列均编码1个含450个氨基酸的蛋白,分子量约为50kDa。氨基酸的142~148位存在1个微管蛋白信号片段GGGTGSG,在氨基酸序列的C-端都有1个酪氨酸残基,N-端存在1个对转录后调控非常重要的保守区MRECI序列,以上特点与其他昆虫α-微管蛋白氨基酸序列保守区序列相同。序列比对表明,克隆得到的α-微管蛋白基因的核苷酸序列是高度保守的,同源性为94.4%~97.0%,而氨基酸的序列同源性达到100%。利用RT-PCR技术在3种昆虫4龄、5龄、6龄幼虫、蛹期4个不同发育阶段和6龄期的肠道、体壁、脂肪体3种不同组织中都检测到了α-微管蛋白基因在mRNA水平的表达。     

Molecular cloning of chick UCH-6 which shares high similarity with human UCH-L3: its unusual substrate specificity and tissue distribution.

A full-length cDNA encoding ubiquitin C-terminal hydrolase-6 (UCH-6) was isolated from the chick skeletal muscle cDNA library. The sequence of two peptides generated from purified UCH-6 matched perfectly with the predicted amino acid sequence. Nucleotide sequence analysis of the cDNA containing an open reading frame of 690 base pairs revealed that the protease consists of 230 residues with a calculated molecular mass of 26,315 Da. UCH-6 belonged to members of the UCH family containing highly conserved Cys, His, and Asp domains and showed 86% amino acid identity to human UCH-L3. Interestingly, most tissues examined contained significant amounts of UCH-6 mRNA, while human UCH-L3 is expressed only in the brain, lungs, and red cells. Moreover, UCH-6, unlike other UCH family enzymes including UCH-L3, could release free ubiquitin from ubiquitin-beta-galactosidase fusion proteins both in vivo and in vitro. The ubiquitous expression pattern and unusual substrate specificity of UCH-6 suggest that the enzyme may represent a distinct subfamily of UCH-L3.